The Combined Effect of Melatonin Implant and Removal of Buck Seminal Plasma on Cryopreservation During the Nonbreeding Season.

This study aimed to determine how melatonin (MT) and seminal plasma affected the freezability of buck sperm during the nonbreeding season. Semen was collected from eight bucks before (pre-MT) and after (post-MT) MT application in the nonbreeding season. Individual ejaculates were collected from the bucks, split into two equal groups according to the removal of seminal plasma (SP) (-) or nonremoval of SP (+). For washing, the groups of ejaculates were centrifuged, and the supernatant was separated, SP (-) and SP (+) ejaculates were diluted, then frozen. Semen samples were examined for sperm motility, plasma membrane integrity, defective acrosomes, DNA fragmentation, and mitochondrial membrane function at the native and post-thaw stages. When the general average post-thaw motility (p < 0.01), plasma membrane (p < 0.05), acrosome (p < 0.05), and DNA integrity rates (p < 0.05) and mitochondrial membrane potential (MMP) (p < 0.01) were evaluated, it was seen that MT administration caused a statistically significant improvement. The dramatic effect of nonremoval of seminal plasma on motility and plasma membrane integrity is more clearly observed in individual semen samples frozen in the pre-MT group (p < 0.05). Also, it was observed that removing seminal plasma in the post-MT group caused even milder post-thaw acrosome damage compared with the SP (+) group (p < 0.05). The effect of removing seminal plasma was not observed in terms of DNA integrity and MMP rates in pre- and post-MT groups. As a result, it was concluded that MT application and removal of seminal plasma in the nonbreeding season result in improvement in the freezability of buck semen.

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.

Royal jelly supplemented soybean lecithin-based extenders improve post-thaw quality and incubation resilience of goat spermatozoa.

The aim of the present study was to evaluate different concentrations of royal jelly (RJ) supplemented extenders for post-thaw quality and incubation resilience of goat spermatozoa. Semen samples were collected from five goats. Pooled semen were diluted with soybean lecithin-based extender without RJ (control) or supplemented with different concentrations (0.25, 0.5 and 0.75%) of RJ (RJ0.25, RJ0.5, RJ0.75 respectively), at a final concentration of 150 × 106 spermatozoon/mL. Semen samples were assessed for sperm motility, plasma membrane 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). The addition of RJ (0.5%, 0.75%) led to higher percentages of subjective motilities (55.33 ± 2.29%, 57.67 ± 2.58%) compared to control and RJ0.25 groups (49.00 ± 2,80%, 51.67 ± 3.09%) (P < 0.05) following the freeze-thawing process. RJ0.5 and RJ0.75 groups had higher plasma membrane functional integrities (66.40 ± 1.34%, 68.20 ± 2.05%) and lower defected acrosome rates (24.60 ± 3.36%, 23.80 ± 2.27%) compared to the other groups (P < 0.05). DNA damaged spermatozoa in all groups were not significant (P > 0.05). In the end of incubation, motility and HOST rates of RJ0.5 (14.00 ± 3.87%, 31.20 ± 3.70%) and RJ0.75 (15.00 ± 3.27%, 29.20 ± 2.59%) groups were higher than control (8.00 ± 2.54%, 18.20 ± 3.11%) and RJ0.25 (9.00 ± 2.07%, 20.60 ± 2.88%) groups (P < 0.05). Also defected acrosome and DNA fragmation rates of RJ0.5 (32.20 ± 1.30%, 5.4 ± 0.55%) and RJ0.75 (29.20 ± 1.30%, 5.80 ± 0.45%) groups were significantly lower than control (38.80 ± 0.84%, 7.40 ± 1.34%) and RJ0.25 (39.80 ± 2.05%, 7.00 ± 1.58) groups. This study shows that RJ supplemented extenders have beneficial effect on goat sperm parameters at 0 h and 6 h of incubation.

Cryopreservation of ram semen with antioxidant supplemented soybean lecithin-based extenders and impacts on incubation resilience.

The scope of this study was investigation the affects of various antioxidants on 1% soybean lecithin-based semen extenders for ram semen cryopreservation. Ejaculates, collected via electrically stimulated ejaculation, that have a thick consistency, rapid wave motion (3-5 on a 0-5 scale) and >75% initial motility were pooled. The pooled samples were split into four equal aliquots as 5 mM Methionine, 5 mM Cysteamine, 1 mM Cysteine and a sample of antioxidant-free control group. Each sample group was diluted to a ratio of 1/5 (semen/extender, v/v) as final concentration and two step dilution method was used for cryopreservation. Extender groups 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). Semen samples also incubated for 6 h in humidified air with 5% CO2 at 39 °C to evaluate post-thaw incubation resilience of semen characteristics. The results showed that freezing and thawing procedures had negative effects on motility (P < 0.05), plasma membrane integrity (P < 0.05) and acrosomal integrity (P < 0.05). After 6 h of incubation time, the Cysteine supplemented extender group yielded significantly higher results than other extender groups in terms of spermatological parameters. Furthermore MDA levels in the antioxidant groups were lower than control group (P < 0.05). Nevertheless, there were no significant differences among antioxidant groups.

Freeze-dried egg yolk based extenders containing various antioxidants improve post-thawing quality and incubation resilience of goat spermatozoa.

The aim of this study was to evaluate different antioxidants-supplemented freeze-dried egg yolk based extenders for the post-thawing quality and incubation resilience of goat spermatozoa. Pooled semen were diluted in a two-step dilution method to a final concentration of 1/5 (semen/extender) in control and antoxidant supplemented freeze-dried extenders (methionine, cysteamine and butylated hydroxytoluene). 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). Membrane lipid peroxidation status was also analyzed using the malondialdehyde (MDA) concentration. In the study, antioxidant supplemented freeze-dried egg yolk based extenders have beneficial effect on goat sperm parameters. In addition, we achieved a higher quality in post thawed goat semen even after 6 h incubation when the extender was supplemented by 5 mM BHT or cysteamine.

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.