Effects of triton X-100 pretreatment of lyophilized and frozen-thawed ram sperm on preimplantation embryo developmental competence.

In this study, it was aimed to determine the effect of destruction of lyophilized and frozen-thawed ram sperm plasma and acrosomal membrane on development of embryos produced by intracytoplasmic sperm injection (ICSI). Semen samples were divided into two groups for lyophilization (L) and freezing (F). For the removal of the plasma membrane, L and F groups were incubated with Triton X-100 (LTX-100 and FTX-100, respectively). Integrities of the plasma membrane, acrosome and chromatin structure were evaluated. Oocytes were injected with these sperm groups. Although no plasma membrane and acrosome integrities of the L (0.0%) group were detected, the plasma membrane integrity of the F group (69.4%) was significantly higher than the FTX-100 group (23.6%) (p < 0.05). The acrosome integrity of the FTX-100 group (3.80%) was significantly lower than the F group (55.6%) (p < 0.05). The chromatin integrities of L and F groups were higher than the Triton X-100 treated groups (p < 0.05). ICSIs with L, LTX-100, F and FTX-100 sperm were produced similar cleavage and blastocyst rates. In conclusion, data presented here confirm that ram spermatozoa can effectively be lyophilized and injected into oocytes for initiation of embryonic development and Triton X-100 pretreatment is not necessary while using lyophilized and frozen semen.

Effects of alpha-lipoic acid on ram semen cryopreservation and post-thaw life span.

The aim of the present study was to evaluate the effects of an alpha-lipoic acid-supplemented extender on ram semen at a post-thaw stage and after incubation (6 hr). Semen samples were collected from five Kivircik Rams. Pooled semen was diluted with an egg yolk-based extender with different concentrations of alpha-lipoic acid (0.25 mmol L-1 , 0.5 mmol L-1 and 1 mmol L-1 ) and without alpha-lipoic acid. Motility, plasma membrane functional integrity (HOST), acrosome integrity (FITC-Pisum sativum agglutinin) and DNA integrity (TUNEL) were assessed at post-thaw and 6 hr after incubation of the frozen-thawed semen. At the post-thaw stage, 0.25 mmol L-1 alpha-lipoic acid had a positive effect on sperm motility and plasma membrane functional integrity compared to the control group (p < .05). At the post-incubation stage (6 hr), it was determined that the motility and plasma membrane functional integrity of the antioxidant groups were adversely affected compared to the control group (p < .05) and 1 mmol L-1 dose of alpha-lipoic acid had a harmful effect on DNA integrity compared to the control group (p < .05). The results of the study demonstrated that alpha-lipoic acid has positive effects at post-thaw but have harmful effects on long term to ram semen.

Toxicity assessment of chronic exposure to common insecticides and bee medications on colony development and drones sperm parameters.

The effect of agrochemicals and beekeeping treatments on drones have not been widely investigated compared to workers or queens. In the present study, we investigated the chronic exposure of chemicals set (deltamethrin, acetamiprid, oxalic acid, fumagillin, and amitraz) on some sperm parameters and on the histomorphology of seminal vesicles. We also assessed the colony development and nosema load before and after the exposure. Thirty native Apis mellifera anatolica honeybee colonies with sister queens equalized with brood and total frame of bees were used for this experiment. Five colonies were used for each group. Deltamethrin, acetamiprid and fumagillin were given as oral chronic exposure at final concentrations of 25.10-6 mg L-1, 0.01 m L-1 and 50 mg L-1 respectively in syrup solution (50/50). Colonies were exposed to oxalic acid by spraying 5 mL per frame space of 3% (w/v) of oxalic acid dihydrate. Finally, the amitraz was applied based on the manufacturer's instructions. The concentrations chosen represented the field-realistic concentrations and those used by beekeepers in the region. Results showed that deltamethrin reduced brood production. In the same group, we found a high increase in nosema load. All treatments decreased sperm count except for fumagillin but this compound increased sperm mortality and increased the percentage of sperm with defected acrosome integrity. The amitraz exhibited a high sperm mortality and high percentage of sperm with defected membrane integrity function. The sperm parameters such as the count, the motility, the acrosome integrity, the membrane function of sperm, and the histomorphology of seminal vesicles of drones exposed to oxalic acid were the most affected. Bee medications commonly used such as oxalic acid and fumagillin should be more investigated and should be considered by beekeepers and particularly queen breeders.

Successful cryopreservation of honey bee drone spermatozoa with royal jelly supplemented extenders.

The aim of the present study was to evaluate different concentrations of royal jelly (RJ) supplemented extenders for post-thawing quality of drone sperm. Semen samples were collected from sexually mature drones. Pooled semen was diluted with extender without RJ (control) or supplemented with different concentrations of RJ (1, 2, 4 or 8%). Sperm motility, plasma membrane functional integrity, and acrosomal integrity were evaluated. At post thaw, the highest sperm motility and acrosomal integrity rates were obtained in the RJ1 group. Functional integrity of sperm membrane was better preserved in the RJ1 and RJ2 groups compare to the other groups. The study shows that RJ supplemented extenders have beneficial effects on drone semen parameters. The results of the present study demonstrated advantage of using 1% RJ supplemented extender.

Administration time of misoprostol affects fertility rate in artificially inseminated Kivircik ewes with frozen-thawed ram semen.

The aim of this study was to determine the effects of the administration time of misoprostol (11 h (Miso11) and 6 h (Miso6) before artificial insemination) on fertility rates in Kivircik ewes (control: n = 41, Miso11: n = 32 and Miso6: n = 33) during breeding season. Artificial insemination (AI) was performed 48 h after sponge removal using frozen-thawed semen (150 million sperm per dose in 0.25 ml straws). Estrus synchronization parameters (onset and duration) and lambing rate were evaluated. No significant difference was observed among groups for the estrus onset and duration hours (P > 0.05). The lambing rates in the control, Miso11 and Miso6 groups were 39.0, 62.5 and 54.5%, respectively. There were significant differences among the control, Miso11 and Miso6 groups according to lambing rates (P < 0.05). In conclusion, misoprostol treatment significantly improved fertility in ewes when using frozen-thawed semen in AI. Administration of misoprostol 11 h before AI resulted in a higher lambing rate than that at 6 h before AI; therefore, treatment of misoprostol 11 h before AI can effectively be used.

Effect of rainbow trout (Oncorhynchus mykiss) seminal plasma on the post-thaw quality of ram semen cryopreserved in a soybean lecithin-based or egg yolk-based extender.

The aim of the current study was to evaluate the effects of different concentrations of rainbow trout seminal plasma (RTSP) (0.1%, 1% and 10%) in extenders containing either egg yolk or lecithin for use in Awassi ram semen cryopreservation. Pooled sperm were diluted in a two-step dilution method to a final concentration of 1/5 (semen/extender) in egg yolk or lecithin extender containing no RTSP, 0.1%, 1% or 10% RTSP (v/v). Semen samples were assessed for sperm motility, plasma membrane integrity [hypoosmotic swelling test (HOST) and Hoechst 33258] and defective acrosomes [FITC-conjugated Pisum sativum agglutinin (PSA-FITC)] at the following five time points: after dilution with extender A; after equilibration; and post-thaw at 0h, 3h and 5h. Malondialdehyde (MDA) was examined only after thawing. Freezing and thawing procedures (dilution, equilibration and post-thaw incubation at 0h, 3h and 5h) negatively affected the motility (P<0.001) and acrosome integrity (P<0.001). Additionally, freezing and thawing negatively affected the plasma membrane integrity, as determined by the HOST and Hoechst 33258 (P<0.001). The extender group affected the motility (P<0.001) and the HOST results (P<0.001). Levels of MDA in the egg yolk extender with 1% RTSP group were significantly lower than in the lecithin control group (P<0.05). In conclusion, the egg yolk extender groups that were supplemented with 10% and 1% RTSP provided greater cryoprotective effects for semen survivability during 5h incubation than the other extender groups.

Successful ram semen cryopreservation with lyophilized egg yolk-based extender.

The aim of this study was to evaluate the effects of lyophilized egg yolk extender on ram semen cryopreservation. Ejaculates with a thick consistency, rapid wave motion (3-5 on a 0-5 scale) and >75% initial motility were pooled. Sperm were diluted to final concentration of 1/5 (semen/extender) in lyophilized egg yolk or fresh egg yolk extenders using two-step dilution method. The equilibrated semen was frozen in 0.25 mL straws. Semen samples were assessed for sperm motility, plasma membrane functional integrity using hypoosmotic swelling test (HOST), damaged acrosome using FITC-Pisum sativum agglutinin (PSA-FITC) and DNA integrity using terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) at three time points: after dilution with extender A, equilibration and post-thaw. The results showed that freezing and thawing procedures (dilution, equilibration and thawing) had negative effects on motility (P<0.001), plasma membrane integrity (P<0.001), acrosome integrity (P<0.001) and DNA integrity (P<0.001). In the study, there were no significant differences between lyophilized and fresh egg yolk extenders when comparing motility, plasma membrane integrity, acrosome integrity and DNA integrity between groups. In conclusion, lyophilized egg yolk extender provided similar cryoprotective effects with fresh egg yolk extender to cryopreserve ram semen.