Factors affecting outcomes of embryo transfer in dromedary camels: A retrospective study.

The present study aimed to use a comprehensive approach for evaluating 12 factors related to embryo quality (shape, size and transparency), donors (age, ET type, number of recovered embryos and day of uterine flushing), recipients (age), males (age and individual variations) and environment (season and year) which could affect the outcome of ET in terms of pregnancy (PR) and pregnancy losses in dromedary camels. During three breeding seasons, 116 donor females were mated repeatedly at 12- to 14-day intervals by fertile male camels (n = 33) without stimulation of the ovaries (WSPO). Superovulation (SPO ET) regimen was applied for each donor female twice or thrice per season. In the occasions of applying superovulation regimen, donor females having an ovulatory follicle were mated instead of GnRH administration and superovulation regimen was applied 4 days post-mating (MIX ET). The uteri of all donor females were flushed at Day 8 or 9 post-mating, and a total of 2,095 embryos were recovered and transferred individually to 924 recipient females. Pregnancy diagnoses were conducted at Day 10 after ET (Days 18-19 of gestation) by using progesterone assay and by transrectal ultrasonography (TRU) at Days 30 and 60 of gestation. By using logistic regression analysis, transparency of embryos and age of recipient females had significant effects on PR at Days 18-19 (p < .01), 30 (p < .01) and 60 (p < .01; p < .05, respectively) of gestation. The shape of embryos had significant effects on the PR at Days 30 (p < .05) and 60 (p < .01) of gestation. Type of ET and the breeding season (year) had significant (p < .05) effects on the PR at Day 30, while day of flushing had the same effect on PR at Day 60. Regarding the pregnancy losses, transparency and shape of the embryo, type of ET, breeding season had significant (p < .05) effect on the late embryonic mortalities (LEM) and shape and season of year had significant (p < .01 and p < .05, respectively) effect on LEM/early foetal mortalities (EFM). Regarding male individual factor, there was a tendency for a significant (p = .055) effect of male camels on the PR at Days 18-19 and rate of LEM. In conclusion, transferring a spherical, transparent or a large-sized embryo (>750 µm) into recipient females ageing between 8 and 11 y could greatly improve the PR from Days 18 to 60 of gestation. Also, embryo recovered from donor females with Mix ET type or embryos sired by certain male camel or at Day 8 post-mating of the donor could improve the 2-month PR. In addition, transferring a transparent or spherical-shaped embryo or embryos recovered from donor females with SPO or Mix ET could reduce the pregnancy losses during the first 2 months of pregnancy.

Preservation of dromedary camel embryos at 4 °C for up to 5 days: Factors affecting the pregnancy and pregnancy loss rates.

The aims of the present study were to evaluate the effect of cooling of the dromedary camel embryos on the pregnancy and pregnancy loss rates, and to investigate the factors which might affect the outcomes of the transfer of cooled embryos. After the donors (n = 56) had been super-ovulated and mated, they were flushed at Day 8 or 9 post-mating. Of 487 collected embryos, 110 were refrigerated at 4°C for up to 5 days in holding medium (HM), flushing medium supplemented with 10% fetal calf serum (FM + FCS) or TCM199 supplemented with 50% FCS and HEPES (TCM + FCS + HEPES). Both fresh (n = 377) and cooled embryos were transferred individually into synchronized recipients. Pregnancy diagnoses were carried out at Days 18-19, 30 and 60 post-mating of the donors. Transferring of fresh embryos into the recipients resulted in significantly higher pregnancy rates at Days 18-19 (53.1% vs. 38.2%, P < 0.01), Day 30 (46.4% vs. 31.8%, P < 0.01) and Day 60 (42.4% vs. 26.4%, P < 0.005) compared with those of cooled embryos, respectively. Pregnancy rates after transferring cooled embryos progressively decreased with the prolongation of the storage period. A significant difference in the pregnancy rate (56% vs. 13%, respectively, P < 0.05) was recorded only at Days 18-19 between cooled embryos held for one day and those held for 5 days. The pregnancy rates at Days 18-19, Day 30 and Day 60 were non-significantly higher when TCM + HEPES and FCS medium used for cooling of embryos compared to those of FM + FCS or HM medium. Cooling of spherical embryos resulted in significantly higher pregnancy rates at Days 30 (45.6% vs. 17.0%, respectively, P < 0.005) and 60 (42.1 vs. 9.4%, respectively, P < 0.005) and a significantly lower pregnancy loss rate (11.1% vs. 66.6%, respectively, P < 0.005) compared to those resulting from cooling of folded embryos. Neither the size of embryo nor the day of flushing had a significant effect on the pregnancy and pregnancy loss rates after the transfer of cooled embryos. In conclusion, pregnancy could be obtained after the transfer of camel embryos refrigerated for up to 5 days. In addition, higher pregnancy rates could be obtained if only spherical embryos are selected for cooling.

Protective effect of quercetin on nicotine-induced prooxidant and antioxidant imbalance and DNA damage in Wistar rats.

We have investigated the protective effect of quercetin (QN) against nicotine-induced prooxidant and antioxidant imbalance in circulation, lung, liver and kidney of experimental rats. The protective effect of QN was compared with N-acetylcysteine (NAC), a well-known antioxidant. Male albino rats of Wistar stain were used for the experimental study. Lung toxicity was induced by subcutaneous injection of nicotine at a dose of 2.5mg/kg body weight (5 days a week, for 22 weeks) and QN was given simultaneously by intragastric intubations for 22 weeks. The body weight gain of rats during experimental period was significantly decreased in nicotine treated group, whereas QN co-treated rats significantly increased in their body weight. The levels of lipid peroxidative indices viz., thiobarbituric acid reactive substances and hydroperoxides, and nitric oxide in circulation, lung, liver and kidney of nicotine-treated rats were increased significantly when compared to normal, which were brought down to near normal in QN co-treated group. Endogenous antioxidant status viz., superoxide dismutase, catalase, glutathione peroxidase and reduced glutathione were found to be significantly decreased in circulation, lung, liver and kidney of nicotine-treated group, which were significantly increased in QN-administered groups. The extent of DNA damage (evaluated by comet assay) was significantly increased in circulatory blood of nicotine-treated rats, which was effectively brought down by QN treatment. The protective effect of QN against nicotine toxicity was comparable to that of NAC. Our data suggest that QN exerts its protective effect by modulating the extent of lipid peroxidation and augmenting antioxidant defense system and thus protects the DNA in experimental animals.

Ellagic acid, a natural polyphenol protects rat peripheral blood lymphocytes against nicotine-induced cellular and DNA damage in vitro: with the comparison of N-acetylcysteine.

The present work is aimed at evaluating the protective effect of ellagic acid (EA), a natural polyphenolic compound that is widely distributed in fruits and nuts against nicotine-induced toxicity in rat peripheral blood lymphocytes. The effect of EA against nicotine toxicity was compared with N-acetylcysteine (NAC), a well-known antioxidant. Lymphocytes were exposed to nicotine at the doses of 0.125, 0.25, 0.5, 1, 2, 3 and 4 mM for 1h in culture media. Thiobarbituric acid reactive substances (TBARS), a lipid peroxidative marker and reduced glutathione (GSH), as indicative of endogenous antioxidant status were analyzed to fix the optimum dose. The lowest concentration eliciting significant damage was 1 mM nicotine and maximum damage was observed with 3 mM concentration, as evidenced by increased levels of TBARS and decreased levels of GSH. Hence, the test concentration was fixed at 3 mM nicotine. To establish most effective protective support we used five different concentrations of EA (10, 50, 100, 150 and 300 microM) against 3 mM nicotine. A dose-dependent inhibitory effect was observed with all doses of EA. Maximum protection was observed at the dose of 100 microM EA. So, 100 microM dose was used for further studies. We have tested five different concentrations of NAC-0.25, 0.5, 1, 2 and 4 mM to elucidate the optimum protective dose against nicotine toxicity. One millimolar NAC showed a significant protection against nicotine toxicity. Protective effect of EA against nicotine toxicity was elucidated by analyzing the lipid peroxidative index, viz., TBARS, hydroperoxides (HP) and endogenous antioxidant status, viz., superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), reduced glutathione (GSH), Vitamins A, E and C. DNA damage and repair were assessed by using alkaline single-cell microgel electrophoresis (Comet assay) and micronucleus assay. There was a significant increase in the levels of lipid peroxidative index, severity in DNA damage and micronuclei number in nicotine-treated group, which was positively modulated by EA treatment. Antioxidant status was significantly depleted in nicotine-treated group, which was effectively restored by EA treatment. The protection of EA against nicotine toxicity was equally effective to that of NAC. EA and NAC treatment alone did not produce any damage to the normal lymphocytes at their effective doses. These findings suggest the potential use and benefit of EA as a modifier of nicotine-induced genotoxicity.

Protective effect of ferulic acid on nicotine-induced DNA damage and cellular changes in cultured rat peripheral blood lymphocytes: a comparison with N-acetylcysteine.

Nicotine is the major pharmacologically active substance in cigarette smoke and plays an important etiological role in the development of lung cancer. Incidence of cancer may be related to oxidative damage to host genome by nicotine. These oxidative actions may be modified by the phytochemicals present in food. The present study describes the protective effect of ferulic acid (FA), a naturally occurring nutritional compound on nicotine-induced DNA damage and cellular changes in cultured rat peripheral blood lymphocytes in comparison with N-acetylcysteine (NAC), a well-known antioxidant. One-hour exposure of lymphocytes to nicotine at the doses of 0.125, 0.25, 0.5, 1, 2, 3 and 4 mM induced a statistically significant dose-dependent increase in the levels of thiobarbituric acid reactive substances (TBARS), a lipid peroxidative marker and decrease in the levels of reduced glutathione (GSH), an important endogenous antioxidant. The lowest concentration eliciting significant damage was 1 mM nicotine and maximum damage was observed with 3 mM concentration. Hence, the test concentration was fixed at 3 mM nicotine. We have used 5 different doses of FA (10, 50, 100, 150 and 300 microM) and NAC (0.25, 0.5, 1, 2 and 4 mM) to test their protective effects. In all the groups, FA and NAC showed a dose-dependent inhibitory effect. Maximum protection was observed at the dose of 150 microM FA and 1mM NAC. So, 150 microM FA and 1mM NAC were used for further studies. There was a significant increase in the levels of lipid peroxidative index (TBARS and hydroperoxides (HP)), severity of DNA damage (evaluated by comet assay) in nicotine-treated group, which were significantly decreased in FA and NAC-treated groups. Nicotine treatment significantly decreased the endogenous antioxidant status viz., superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), GSH, vitamin A, E and C. Co-administration of FA and NAC to nicotine-treated lymphocytes showed a significant increase in the antioxidant status. The protective effect of FA was merely equal to that of NAC effect. FA and NAC treatment alone did not produce any toxicity to the normal lymphocytes at their effective doses. On the whole, there is overwhelming evidence that FA has the ability to modulate DNA damage and a variety of cellular changes that occur during nicotine-induced toxicity in rat peripheral blood lymphocytes.

Effect of quercetin on nicotine-induced biochemical changes and DNA damage in rat peripheral blood lymphocytes.

We elucidated the protective effect of quercetin, a polyphenolic flavonoid, on lipid peroxidation, endogenous antioxidant status and DNA damage during nicotine-induced toxicity in cultured rat peripheral blood lymphocytes as compared to N-acetylcysteine (NAC), a well-known antioxidant. Lymphocytes were exposed to nicotine (3 mM) with and without quercetin and NAC (1 mM) in RPMI-1640 medium for 1 h. In preliminary experiments to fix the effective dose of quercetin, different doses of quercetin (25, 50, 75, 100 and 200 microM) were administered to lymphocytes with nicotine, and lipid peroxidation markers (thiobarbituric acid reactive substances and hydroperoxides) were analysed. A 75 microM dose of quercetin was found to be effective as evidenced by decreased lipid peroxidation. To evaluate the protective potential of quercetin against genotoxic effects of nicotine we used comet and micronucleus assays, which are valid parameters to assess genetic damage. In addition, biochemical changes including lipid peroxidation and antioxidant status were assessed. There were significant increases in the levels of lipid peroxidation, comet parameters and micronuclei frequencies, followed by decrease in the endogenous antioxidant status, in nicotine-treated lymphocytes, which were brought back to near normal by quercetin or NAC treatment. The protective effect of quercetin against nicotine toxicity was comparable to that of NAC. These findings suggest that quercetin can be as effective as NAC in protecting rat peripheral lymphocytes against nicotine-induced cellular and DNA damage.