Relationship between toxicity of cryoprotectants, osmotic and oxidative stresses in awassi ram sperm.

BACKGROUND: The relationship between the toxicity of cryoprotectants and their osmotic and/or oxidative stresses remains to be further investigated. OBJECTIVE: To investigate the toxic effects of different cryoprotectants and osmotic stress on Awassi ram sperm and to determine the relationship between oxidative and antioxidative status of the sperm. MATERIALS AND METHODS: Pooled sperm samples were exposed to sucrose solutions of different concentrations (75 to 900 mOsm) and isosmotic condition (290-325 mOsm) was re-established by adding HEPES buffered Tyrode's lactate. Sperm samples were mixed with 0.5, 1.0 and 1.5 M of glycerol, methanol, 2-methoxyethanol, dimethylacetamide or 1,2-propanediol for 5 min and returned to isosmotic condition. Sperm samples were exposed to cryoprotectants at 4 degree C for 2 hours and isosmotic conditions were re-established. Motility, viability, acrosome integrity and oxidative or antioxidative parameters were determined. RESULTS: Treatment with hypo- or hyperosmotic sucrose solution reduced motility and viability without affecting acrosome integrity. The addition and removal of glycerol and dimethylacetamide (1.0 or 1.5 M) decreased sperm motility, while cryoprotectants had no effect on viability except for 1.5 M glycerol. Chilling significantly reduced the motility and viability of the sperm, but not the acrosome integrity. Rapid addition or removal of cryoprotectants also did not affect the acrosome integrity. Cryoprotectants changed only the ceruloplasmin level, while there were significant post-chilling differences in lipid hydroperoxide, paraoxonase and ceruloplasmin levels. CONCLUSION: Cryoprotectants without other additives have limited protection and glycerol can be toxic to spermatozoa. The oxidative stress plays a role in cryoprotectant toxicity and chilling stress. doi.org/10.54680/fr22210110612.

Impacts of Specific Cryoprotectants on Sperm Freezing and Relationships Between Cryodamage and Oxidation Stress Parameters in Awassi Ram Sperm.

BACKGROUND: The role of oxidative stress during cryoprotectant treatment has received little attention. OBJECTIVE: To assess the effects of different cryoprotectants and discover relationships between cryodamage and oxidative stress parameters on Awassi ram sperm. MATERIALS AND METHODS: The sperm samples diluted with Salamon's tris-citrate (TRIS) containing 20% centrifuged egg yolk and 0.5, 1.0 or 1.5 M Glycerol (Gly), methanol (M), 2-methoxyethanol (2-ME), dimethylacetamide (DMA) and 1.2 propanediol (PR). After 2 h of equilibration at +4 ºC, the sperm samples were frozen in liquid nitrogen vapour and stored. RESULTS: The best post-thaw motility (43.3%, 41.7%) of sperm was achieved when protected with 0.5 and 1.0 M glycerol. Arylesterase and ceruloplasmin parameters were significantly different after equilibration, whereas sulfhydryl groups were significantly different after freezing in their respective groups (P < 0.05). CONCLUSION: The increased use of glycerol caused greater loss of motility. The role of oxidative stress in freezing was also found to be limited.

Effects of long-term heat stress and dietary restriction on the expression of genes of steroidogenic pathway and small heat-shock proteins in rat testicular tissue.

The aim was to investigate the effects of long-term heat stress and dietary restriction on the expression of certain genes involving in steroidogenic pathway and small heat-shock proteins (sHSPs) in rat testis. Sprague Dawley rats (n = 24) were equally divided into four groups. Group I and II were kept at an ambient temperature of 22°C, while Groups III and IV were reared at 38°C for 9 weeks. Feed was freely available for Group I and Group III, while Group II and Group IV were fed 60% of the diet consumed by their ad libitum counterparts. At the end of 9 weeks, testicles were collected under euthanasia. Total RNA was isolated from testis tissue samples. Expression profiles of the genes encoding androgen-binding protein, follicle-stimulating hormone receptor, androgen receptor, luteinising hormone receptor, steroidogenic acute regulatory protein (StAR), cyclooxygenase-2 and sHSP genes were assessed at mRNA levels using qPCR. Long-term heat stress decreased the expression of StAR and HspB10 genes while dietary restriction upregulated StAR gene expression. The results suggested that long-term heat stress negatively affected the expression of StAR and HspB10 genes and the dietary restriction was able to reverse negative effect of heat stress on the expression of StAR gene in rat testis.

Expression profile of interferon tau-stimulated genes in ovine peripheral blood leukocytes during embryonic death.

Early and efficient detection of embryonic death (ED) has a valuable impact as important as early pregnancy diagnosis in ruminants. Among early pregnancy diagnosis methods, detection of the expression of interferon tau-stimulated genes (ISGs) in peripheral blood leukocytes (PBLs) is well documented in cows and ewes. Therefore, we hypothesized that the expression profile of ISGs in PBLs might also be useful for detecting ED in these animals. For this purpose, pregnant ewes were used as an experimental model. Pregnancy was detected on Day 18 after mating by transrectal ultrasonography. Pregnant ewes were divided into a control group (sham injection on Day 18, n = 10) and ED group (treated with 75 µg synthetic PGF2α on Day 18, n = 12). PBLs and plasma were collected on Days 0 (mating day), 15, 18, 19, 20, 21, 23, and 25 by jugular venipuncture. Total RNA was isolated from PBLs. ISGs expression levels were determined by real-time polymerase chain reaction in triplicate. Electrochemiluminescence immunoassay was used to measure progesterone (P4) levels in plasma. In the ED group, the P4 level declined to less than 1 ng/mL on Day 19 and remained at a low level until the end of the study. Compared with that on Day 0, receptor transporter protein 4 (RTP4) and ISG15 expression was upregulated on Day 15 and remained high until Day 21 in both groups, and RTP4 and ISG15 mRNA levels were attenuated on Days 23 and 25 only in the ED group (P < 0.001). Myxovirus resistance 1 expression was upregulated on Day 15 and remained high until Day 23 in both groups, but was attenuated on Day 25 in the ED group (P < 0.05). The B2-microglobulin mRNA level did not change significantly during the study in either group. These results indicate that the decline in P4 concentration was an immediate response to PGF2α and that the embryo may have survived longer than the CL on the basis of the extended period of ISGs expression. This suggests that the absence of P4 could be the reason for ED rather than a direct effect of PGF2α. In conclusion, the expression of ISGs, including ISG15, RTP4, and myxovirus resistance 1, but not B2-microglobulin, in PBLs may serve as a marker of ED.

Effect of dietary restriction on sperm characteristic and oxidative status on testicular tissue in young rats exposed to long-term heat stress.

This study was conducted to evaluate the effects of dietary restriction on oxidative status and sperm parameters in rats exposed to long-term heat stress. Forty healthy Sprague-Dawley rats, aged 2.5 month, were divided into four groups of 10 with respect to feeding and temperature regimen (room temperature (22 °C)-ad libitum, room temperature-dietary restriction (40%), high temperature (38 °C)-ad libitum, high temperature-dietary restriction). At the end of the 9th week, some oxidants (lipid hydroperoxide, total oxidant status, oxidative stress index) and antioxidants (total antioxidant status, sulfhydryl groups, ceruloplasmin, paraoxonase and arylesterase activities) were measured in the testis tissue. The concentration, motility, volume, abnormal sperm count, acrosome and membrane integrity of epididymal spermatozoon and intratesticular testosterone levels were evaluated. High temperature did not change oxidative and antioxidative parameters except for sulfhydryl groups and ceruloplasmin, yet it impaired all sperm values. Neither sperm values nor oxidative status apart from sulfhydryl groups, ceruloplasmin and arylesterase was affected by dietary restriction in the testis tissue. These results suggest that long-term heat stress does not have a significant effect on testicular oxidative status, while the spermatozoa are sensitive to heat stress in young rats. Dietary restriction failed to improve the sperm quality and oxidative status except some individual antioxidant parameters; conversely, it decreased intratesticular testosterone level in the young rats exposed to long-term heat stress.

Isolation of DNA from embryo and chorio-allantoic membranes and sexing by PCR in Japanese quail.

1. The aim of this study was to evaluate the amount and quality of genomic DNA isolated from embryos and their chorioallantoic membranes (CAM) and to investigate the utility of different PCR methods for identifying the sex of Japanese quail embryos. 2. Fertilised eggs were incubated at 37°C for 120 h and DNA was isolated from samples of embryos and CAM. Target regions of the CHD-W gene or XhoI repeat sequence were amplified by PCR and examined on agarose gels or by using a capillary electrophoresis system. 3. DNA samples from embryos had significantly higher OD260 values than those from CAM, while OD260/280 values were not significantly different between embryos and CAM. 4. Gender identification was not possible by PCR amplification of the CHD gene region or XhoI repeat sequences examined on agarose gels, whereas males and females of Japanese quail were distinguishable when PCR products of the CHD gene were separated by capillary electrophoresis. 5. The results showed that high molecular weight DNA could be isolated from both embryo and CAM of Japanese quail. DNA isolated from CAM could be used for molecular genetic studies where embryos would be used for other purposes, such as in situ hybridisation. A capillary electrophoresis system could be used for identifying the gender of Japanese quail embryos.

Effects of tiletamine-zolazepam anaesthesia on plasma antioxidative status and some haematological parameters in sheep.

It is not clear whether the anaesthetic agents tiletamine and zolazepam have antioxidant or pro-oxidant effects. Therefore, this study was carried out to investigate the effects of tiletamine-zolazepam anaesthesia on oxidant/antioxidant status in blood plasma and on haematological parameters in 10 healthy Awassi ewes. The tiletamine-zolazepam combination was administrated in a dose of 7.5 mg/kg intramuscularly. The animals were spontaneously breathing air during the procedure. Blood samples were collected by jugular venipuncture before induction and at 30, 60, 120 min, 24 h and 3 days after anaesthesia. Malondialdehyde concentration, an index of lipid peroxidation, was higher at 30, 60, 120 min and 24 h (P < 0.05) than the baseline value in the plasma. The level of glutathione decreased (P < 0.05) at 30, 60 and 120 min, then returned to the baseline level. Beta-carotene concentration was lower (P < 0.05) than the baseline value during anaesthesia with the exception of its level at 120 min. Glutathione peroxidase and catalase activities decreased (P < 0.05) at the onset of anaesthesia, then returned to baseline values. There was no significant change in vitamin A level. Red blood cell count, haematocrit and haemoglobin concentration significantly decreased (P < 0.05) only at 30 min and thereafter they gradually returned to the baseline values. Based on the results tiletamine-zolazepam anaesthesia seems to accelerate lipid peroxidation and to impair the enzymatic antioxidant defence in the blood plasma.

Effects of testosterone on lipid peroxidation, lipid profiles and some coagulation parameters in rabbits.

The purpose of this study was to investigate the effects of testosterone on some risk factors of atherosclerosis. Twenty-four male New Zealand white rabbits were randomly divided into three groups of eight. The first group was used as control. Second group was injected with 10 mg of testosterone propionate. Third group was castrated bilaterally. At the end of 6 weeks, lipid peroxidation (LPO), lipid profile, fibrinogen (FBN) level and coagulation parameters were evaluated. Testosterone administration decreased the level of high-density lipoprotein cholesterol (HDL-C), while castration increased this level (P < 0.05). Triglyceride (TG) and total cholesterol (TC) levels in the castration group were significantly higher (P < 0.05) than those in the testosterone group. The ratio of HDL-C:low-density lipoprotein cholesterol (LDL-C) decreased, while TC:HDL-C ratio increased (P < 0.05) in the testosterone group. No significant differences were found in the LDL-C and FBN levels among groups. However, there was a tendency for higher FBN level in the testosterone group. Testosterone administration resulted in an increase in the level of LPO (P < 0.05). Clotting time and prothrombin time prolonged in the castration group compared with testosterone group (P < 0.05). As a result, testosterone has exacerbating effect on atherosclerosis risk factors including lipid profile, LPO, FBN and coagulation system.

Effects of vitamin C on liver enzymes and biochemical parameters in rats anesthetized with halothane.

Halothane is an important human and veterinary anesthetic, which produces free radicals during biotransformation. Occasionally, these free radicals may cause hepatic injury, especially in case of multiple halothane exposures over short periods. Vitamin C may protect cellular lipids and lipoproteins against oxidative damage by the free radicals. This study investigated the effects of vitamin C on liver enzymes and other biochemical parameters in rats anesthetized with halothane. One group of rats was used as a control, and saline (0.9% NaCl) was injected intraperitoneally into these animals as a placebo. The second group of rats was used as an anesthesia control group and was only anesthetized by halothane for 2 h. The third group was anesthetized by halothane and injected vitamin C intraperitoneally. Activities of aspartate aminotransferase, alanine aminotransferase and alkaline phosphatase enzymes were significantly increased (p < 0.05, p < 0.01, p < 0.05, respectively) by halothane anesthesia, but decreased (p < 0.05, p < 0.05, p < 0.05, respectively) with administration of vitamin C. Concentrations of triglycerides, cholesterol, total bilirubin and creatinine were statistically affected (p < 0.05, p < 0.01, p < 0.05, and p < 0.01, respectively) by injection of vitamin C. Values of erythrocyte counts, packet cell volumes, hemoglobin concentration, leukocyte counts, rates of neutrophils and lymphocytes were significantly affected (p < 0.01, p < 0.05, p < 0.05, p < 0.01, p < 0.001 and p < 0.01, respectively) by halothane anesthesia. The values of erythrocyte counts, leukocyte counts, neutrophil and lymphocyte rates were significantly decreased (p < 0.05, p < 0.05, p < 0.05, p < 0.01 and p < 0.01, respectively) with administration of vitamin C. Based upon these results, vitamin C may play an important role in the prevention of hepatic cellular injury inflicted by halothane anesthesia.

Effects of testosterone and vitamin E on the antioxidant system in rabbit testis.

The aim of the study was to investigate the effects of testosterone propionate and vitamin E on the antioxidant system in the testis. Thirty-two male New Zealand White rabbits were randomly divided into four groups. The first group was used as control. The second group was injected with testosterone propionate, the third group vitamin E and the fourth group vitamin E and testosterone propionate combination. All treatments were carried out during 6 weeks and oxidative parameters were evaluated in homogenized testicular tissue. The levels of vitamin E and the activity of glutathione peroxidase were lower (P < 0.05) in the testosterone group than in controls. However, vitamin C and malondialdehyde levels were higher (P < 0.05) in this group than in controls. The levels of reduced glutathione, beta-carotene, vitamin C and E increased, but malondialdehyde levels decreased in the vitamin E group, when compared with controls (P < 0.05). Vitamin E and beta-carotene levels were significantly higher (P < 0.05) in the combination group than in testosterone group. However, MDA levels were lower (P < 0.05) in combination group than in the testosterone group. In conclusion, administration of testosterone propionate led to a significant elevation of oxidative stress. Vitamin E is quite an effective antioxidant which protects rabbit testis against lipid peroxidation, and, testosterone-induced lipid peroxidation could be improved by additional vitamin E treatment.

Effects of palm oil on lipid peroxidation, reduced glutathione, glutathione peroxidase, and vitamin A levels in the corpus uteri, cornu uteri and corpus luteum of young and adult female sheep.

The aim of the current study was to determine whether a rumen protected palm oil based diet affect malondialdehyde (MDA), glutathione peroxidase (GSH-Px), reduced glutathione (rGSH) and vitamin A levels in the tissues of cornu uteri, corpus uteri and corpus luteum over the barley based isoenergetic and isonitrogenous diet, and whether the response is different between ewes and ewe-lambs. During the breeding season, half of Morkaraman ewes (2-4-year-old, n = 10) and ewe-lambs (7-8-months-old, n = 10) was offered a barley based diet and the other half was offered a protected palm oil based diet for 42 +/- 0.7 days. At the end of the experiment all animals were slaughtered and measurements carried out in the tissues collected. In all animals tested, cornu uteri had the highest MDA levels followed by corpus uteri and corpus luteum (P < 0.01) but no differences were between the tissues observed in GSH-Px and rGSH levels (P > 0.05). Vitamin A levels were, however, higher in corpus luteum than in cornu uteri and corpus uteri (P < 0.05). Corpus uteri MDA levels were not different (P > 0.05) but rGSH levels were higher for the palm oil fed groups (P < 0.05). GSH-Px and rGSH levels were higher for ewe-lambs than ewes (P < 0.05). In conclusion, it appears that MDA, rGSH, GSH-Px, and vitamin A work in a different fashion for corpus uteri, cornu uteri and corpus luteum, and for ewes and ewe-lambs. Dietary palm oil did not significantly affect the parameters studied except higher rGSH levels in corpus uteri. Levels of antioxidatively active substances, such as rGSH and GSH-Px were lower in ewes compared with those in ewe-lambs.

Effects of intraperitoneally administered vitamin C on antioxidative defense mechanism in rats with diabetes induced by streptozotocin.

We determined the effects of intraperitoneally administered vitamin C on the lipid peroxidation (as thiobarbituric acid-reactive substances, TBARS) and vitamin C and E levels and reduced glutathione (GSH) and glutathione peroxidase (GSH-Px) activity in the plasma, red blood cells (RBC), liver, and muscle of rats in relation to oxidative damage associated with diabetes induced by streptozotocin (STZ). One group was used as control and a second as diabetic. A third group received 30 mg vitamin C i.p. every other day. On day 4 after the injection of vitamin C, animals in the second and third groups were made diabetic by i.p. injection of STZ and administered vitamin C for 21 consecutive days, and we determined TBARS, vitamin E, and GSH levels and GSH-Px activities in plasma, RBC, liver, and muscle samples. Vitamin E levels in the plasma and liver were significantly higher (P<0.05) in the control group than in the diabetic group. Also, TBARS levels in the plasma, RBC, liver, and muscle samples were significantly lower (P<0.05) in controls than in the diabetic group. The TBARS levels in the RBC, liver, and muscle samples of the vitamin C group were significantly lower (P<0.05, P<0.01, and P<0.001, respectively). However, GSH-Px and GSH activities in RBC, liver, and muscle and vitamin C levels in liver were not significantly different between control and diabetic groups. Vitamin E levels in plasma (P<0.05, P<0.01) and liver (P<0.001), vitamin C levels in liver (P<0.001), and GSH (P<0.01) and GSH-Px activities in RBC (P<0.05, P<0.01) were significantly higher in the vitamin C group than both the control and diabetic groups. These results indicate that vitamin C has significant protective effects on the blood, liver, and muscle of rats against oxidative damage in diabetes.

The effects of food withdrawal and darkening on lipid peroxidation of laying hens in high ambient temperatures.

The aim of this study was to investigate the effects of feed withdrawal and darkening on the antioxidative status of laying hens in high ambient temperatures between 30-40 degrees C. We determined vitamins A and E, lipid peroxidation as thiobarbituric-acid reactive substances (TBARS), glutathione peroxidase (GSH-Px) and reduced glutathione (GSH) in the serum, liver and muscle. Sixteen week old hens (Ross Brown) were divided into 3 groups of 30 hens each. The first group was used as a control. Hens in the second (feed withdrawal) group were subjected to feed removal between 14-18 h during the summer. Hens in the third (darkening) group were subjected to darkening of the hens house with black curtains between 14-18 h. At the end of 16 weeks, blood serum, liver and breast muscle samples were taken from all animals and analysed. Vitamins A and E levels in serum and liver were significantly (P < 0.05, P < 0.01) higher in feed withdrawal and darkened groups than in control group whereas the serum levels of the darkening group were highest. GSH-Px activity and GSH level in liver and muscle were also significantly (P < 0.05, P < 0.01) higher in feed withdrawal and the darkening group than in control. However, TBARS levels in liver and muscle were significantly (P < 0.05, P < 0.01) lower in feed withdrawal and darkening groups compared with control group, whereas the level of the darkening group was the lowest. In conclusion, we observed that feed withdrawal and darkening of hens in high temperatures during summertime resulted in an increase of antioxidant enzymes and vitamins levels and in a decrease of lipid peroxidation.