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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.

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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.

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The aim of this study was to evaluate the effect of both pure rainbow trout seminal plasma (RTSP) supplementation and RTSP-cysteine combination on cryopreservation success and post-thaw incubation resilience of ram semen in the nonbreeding season. For this purpose, different doses of RTSP (0%, 1%, 10%, and 15%) with or without cysteine supplementation were used for experiments. Ejaculates chosen for experiments were pooled and then divided into eight equal volumes for grouping (Control-ControlC, RTSP1-RTSP1C, RTSP10-RTSP10C, and RTSP15-RTSP15C). After cryopreservation, frozen-thawed semen samples were incubated for 5 hours at 37°C for determination of post-thaw incubation resistance. Motility, HOST, TUNEL, Rh123-PI, and CTC tests were performed at 0 hour and 3rd and 5th hours of post-thaw incubation to evaluate the efficacy of all experimental groups. The RTSP10 and RTSP10C groups were noted to provide the best protection on motility, plasma membrane integrity, DNA integrity, and mitochondrial function of cryopreserved ram semen. On the other hand, the best protection against cryo-capacitation was observed in RTSP15 and RTSP15C groups. The addition of cysteine was found to be effective when the higher (15%) or lower (1%) doses of RTSP were used, as well as for no use of RTSP.

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The objective of this study was to determine the optimum concentrations of rainbow trout seminal plasma (RTS) supplemented extenders for goat semen quality at post-thaw and after incubation. Five sexually mature Saanen goat (Capra aegagrus hircus) were used for semen collection. Pooled semen was diluted with soybean lecithin-based extender without RTS (control) or supplemented with different concentrations of RTS (1%, 2%, 4% or 8%), at a final concentration of 150 × 106 spermatozoon/ml. Sperm motility, plasma membrane functional integrity (HOST), damaged acrosome (PSA-FITC), mitochondrial activity (rhodamine123) and DNA integrity (TUNEL) were evaluated. Spermatological parameters were evaluated at post-thaw and after 6 hr incubation. RTS8 group preserved sperm motility, acrosomal integrity, plasma membrane functional integrity and mitochondrial function better than the control group (p < .05). The study demonstrated that RTS supplemented lecithin-based extenders have useful effects on goat spermatozoa. In addition, the results of the current study represented the positive effect of using 8% RTS supplemented extender.

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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.

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Brain histamine holds a key position in the regulation of behavioral states, biological rhythms, body weight, energy metabolism, thermoregulation, fluid balance, stress and reproduction in female animals. However, it is not clear whether central histamine exerts any effect on hypothalamic-pituitary-testicular in male rats and if so, the involvement of type of central histamine receptors. The current study was designed to determine the effect of centrally administrated histamine on plasma gonadotropin hormone-releasing hormone (GnRH), luteinizing hormone (LH), follicle stimulating hormone (FSH) and testosterone level, and sperm parameters, and to show the mediation of the central histaminergic H1, H2 and H3/H4 receptors on histamine-evoked hormonal and sperm parameters' effects. Studies were performed in male Sprague-Dawley rats. A total of 50 or 100 nmol doses of histamine were injected intracerebroventricularly (icv). 100 nmol dose of histamine significantly caused increases in plasma GnRH, LH, FSH and testosterone levels of animals, but not 50 nmol dose of histamine. Moreover, central pretreatment with chlorpheniramine, histaminergic H1 receptor antagonist (100 nmol), ranitidine and histaminergic H2 receptor antagonist (100 nmol) completely prevented histamine evoked increase in plasma GnRH, LH, FSH and testosterone levels, while thioperamide, histaminergic H3/H4 receptor antagonist (100 nmol) pretreatment failed to reverse sex hormones responses to histamine. Both central histamine treatment alone and central histamine treatment after central histaminergic receptors antagonists' pretreatments did not alter any sperm parameters in rats. In conclusion, our findings show that centrally administered histamine increases plasma GnRH, LH, FSH and testosterone levels of conscious male rats without change any sperm parameters. Moreover, according to our findings, central histaminergic H1, and H2 receptors mediate these histamine-induced effects.

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The aim of this study was to demonstrate the possible individual and/or synergistic effects of ß-glucan and dietary restrictions on the reproductive parameters of rats. For this purpose, forty male Sprague-Dawley rats were randomly divided into four equal groups (n = 10 per group). The first group was the control, the second group was kept under dietary restriction (DR), the third group was kept under a dietary restriction and given ß-glucan (DR + ßG) and the fourth group was supplemented only with ß-glucan (ßG; 20 mg/kg) intragastrically for 14 days. Motility, vitality and morphology of spermatozoa, reproductive organ weights (testis, vesicula seminalis and epididymis) and seminiferous tubule diameters were evaluated in experimental rats. ß-glucan had excellent effects on motility, live spermatozoa rate and the acrosome integrity when compared to the control group (p < 0.05). We also observed that ß-glucan administration to rats having dietary restriction could improve sperm motility and acrosome integrity (p < 0.05). While the ß-glucan improved seminiferous tubule diameter (p < 0.05), weights of the reproductive organs did not change positively as a result. This study demonstrated that ß-glucan treatment significantly improved some spermatological characteristics in rats. Therefore, treatment with ß-glucan could be used for its positive effects on motility, spermatozoa vitality rate and acrosome integrity for infertile men.

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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.

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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.

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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.

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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.

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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.

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The current study was designed to determine the effect of centrally administrated arachidonic acid (AA) on plasma gonadotropin hormone-releasing hormone (GnRH), follicle stimulating hormone (FSH), luteinizing hormone (LH) and testosterone level, and sperm parameters, and to show the mediation of the central cyclooxygenase (COX) to thromboxane A2 (TXA2) signaling pathway in AA-induced hormonal and sperm parameter effects. Studies were performed in male Sprague-Dawley rats. A total of 150 or 300 µl/5 µl doses of AA were injected intracerebroventricularly (icv). AA significantly caused dose- and time-dependent increases in plasma FSH, LH and testosterone levels of animals, but not plasma GnRH level. AA also significantly increased sperm motility of the rats without change sperm number. Pretreated with ibuprofen, a nonselective COX inhibitor (250 µg/5 µl; icv), and furegrelate, a TXA2 synthesis inhibitor (250 µg/5 µl; icv), prevented AA-evoked increase in plasma FSH, LH and testosterone levels, and sperm motility. In conclusion, our findings show that centrally administered AA increases plasma FSH, LH and testosterone levels and sperm motility of conscious male rats. Moreover, according to our findings, central COX-TXA2 signaling pathway mediates these AA-induced effects.

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Vitrification is becoming a preferred method for pre-implantation embryo cryopreservation. The objective of this study was to determine the differentially expressed genes of in vivo- and in vitro-produced bovine embryos after vitrification. In vitro- (IVF) and in vivo-derived (IVV) bovine blastocysts were identified as follows: in vitro-produced fresh (IVF-F), in vitro-produced vitrified (IVF-V), in vivo-derived fresh (IVV-F), in vivo-derived vitrified (IVV-V). The microarray results showed that 53 genes were differentially regulated between IVF and IVV, and 121 genes were differentially regulated between fresh and vitrified blastocysts (P < 0.05). There were 6, 268, 962, and 17 differentially regulated genes between IVF-F × IVV-F, IVF-V × IVV-V, IVF-F × IVF-V, and IVV-F × IVV-V, respectively (P < 0.05). While gene expression was significantly different between fresh and vitrified IVF blastocysts (P < 0.05), it was similar between fresh and vitrified IVV blastocysts. Significantly up-regulated KEGG pathways included ribosome, oxidative phosphorylation, spliceosome, and oocyte meiosis in the fresh IVF blastocyst samples, while sphingolipid and purine metabolisms were up-regulated in the vitrified IVF blastocyst. The results showed that in vitro bovine blastocyst production protocols used in this study caused no major gene expression differences compared to those of in vivo-produced blastocysts. After vitrification, however, in vitro-produced blastocysts showed major gene expression differences compared to in vivo blastocysts. This study suggests that in vitro-produced embryos are of comparable quality to their in vivo counterparts. Vitrification of in vitro blastocysts, on the other hand, causes significant up-regulation of genes that are involved in stress responses.

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