The aim of this study was to determine the freezability of ram semen with extenders containing mitoquinone or caffeic acid. Collected ram semen samples were pooled and divided into seven equal aliquots. Each aliquot was diluted with Tris-based extender according to control and antioxidant groups (Control, Mitoquinone (MitoQ) 100 nM, 150 nM, 200 nM; Caffeic Acid (CA) 50 µM, 100 µM, 150 µM). The post-thaw spermatological, antioxidant parameters and OGG1/ROMO1 gene expression and methylation were evaluated. It was determined that the CASA post-thaw motility of MitoQ200, CA100 and CA150 antioxidant groups were statistically higher than the control group (P < 0.05). The post-thaw plasma membrane integrity of MitoQ groups was compared with CA groups, CA100 group, which had the lowest plasma membrane integrity among the antioxidant groups. The post-thaw acrosome membrane damage of all the antioxidant groups were statistically lower than control group, except CA50 and CA100 groups (P < 0.05). For the post-thaw mitochondrial membrane potential, all groups except the CA50 group were determined to be superior to the control group (P < 0.05). No statistical difference was observed among all groups including control group in terms of post-thaw DNA fragmentation, MDA and TAC values. MitoQ significantly altered the expression of the OGG1 genes, an effect mediated through DNA methylation (P < 0.05). Furthermore, caffeic acid altered the ROMO1 gene expression (P < 0.05). Based on the spermatological parameters and gene expression and methylation levels obtained in the current study, it was determined that mitoquinone 200 nM and caffeic acid 150 µM doses had a positive effect on the freezability of ram semen.

The aim of this study was to elucidate the toxicity effect of widely used penetrating cryoprotective agent dimethyl sulfoxide (DMSO) on frozen drone semen and fertility. DMSO was replaced totally or partially with either dextran, polyvinylpyrrolidone (PVP), or polyethylene glycol (PEG), which are non-penetrating cryoprotectants. The freezing extender with 12%, 8%, 4% and 0% DMSO were supplemented with 0%, 4%, 8% and 12% of dextran, PVP, or PEG, respectively. For all treatment groups, the final cryoprotectant concentration was 12%. Dextran4, PVP4, and DMSO12 groups with good post-thaw motility, plasma membrane functional integrity that evaluated throughout the study and fresh semen were used for queen instrumental insemination (n=46). Two queen bees (4.4%) died due to manipulation. Alive queens [24 (55%) of 44] had been observed to survive after a week, and 17 (71%) of these queens continued to survive after three months. At the end of three months, 15 (88%) of the alive queens continued to produce worker bees. There were no differences among insemination groups (P>0.05) for the rate of survival for three months and presence of worker broods. After overwintering, there were two queens from the PVP4 and three queens from the Dextran4 group that survived and one queen from PVP4 and two queens from Dextran4 group that continued to produce worker bees. In conclusion, addition of 4% Dextran or PVP as an extender containing 8% DMSO solution improves queen bees' worker egg-laying period, but the same results were not observed for the PEG containing groups.

This study aimed to determine the influence of different doses of B-glucan on post-thaw spermatological parameters, lipid peroxidation, and total antioxidant activity of buck semen. In the non-breeding season, semen was collected from bucks twice weekly. After then, ejaculates were pooled and divided into four equal aliquots: B-glucan concentrations of 1 mM (BG1), 2 mM (BG2), and 4 mM (BG4), and a control group without antioxidants. Each sample group was diluted for cryopreservation using a dilution method involving two steps. The experimental groups were then evaluated for several parameters including sperm motility, plasma membrane functional integrity [hypoosmotic swelling test (HOST)], damaged acrosome rate [FITC-Pisum sativum agglutinin (FITC-PSA)], DNA integrity [terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL)], mitochondrial membrane potential using JC-1, evaluation of lipid peroxidation (Malondialdehyde; MDA) and determination of total antioxidant capacity; TAC). The post-thaw motility and plasma functional integrity of the control group were significantly lower than those values in the BG groups (P < 0.05). Although the numerically greatest acrosome damage was detected in the control group, it was only statistically different from BG1 and BG4 (P<0.05). While the DNA fragmentation rate of the control group was higher than BG4 group (P<0.05), it was similar to BG1 and BG2 groups (P>0.05). There was no statistical difference among all the groups regarding low mitochondrial membrane potential, MDA, and TAC rates. In line with our results, supplementation of 1mM, 2 mM and 4 mM B-glucan to freezing extender improves the post-thaw spermatological characteristics of goat semen.

This study was carried out to determine the effects of Equex on ram semen freezability in three different extenders. Semen samples were collected from five rams and divided into six equal aliquots. At first, three non-commercial base extenders (E1: Tris-based extender, E2: Tris based extender+ Cystein and E3: Skim Milk Powder (SMP)) were prepared. Each base extender was divided into two parts; a base extender (E1, E2 and E3) or fortified with Equex (E1E, E2E and E3E). The pooled semen was diluted according to the two-step dilution method, and cryopreserved using a programmable freezing machine. Sperm motility parameters were examined by CASA. The morphological properties were evaluated by flow cytometer. It was observed that the addition of Equex had a positive effect on post-thaw motility and live spermatozoa (P<0.001), but had no effect on the LIN and Polarized mitochondria. While the Equex addition decreased post-thaw VCL, VSL, VAP and WOB values, it didn"t have effect on the post-thaw intact acrosome rates (P<0.05). Furthermore, it was observed that Equex had a cryoprotective effect on post-thaw progressive motility in E1E and E3E groups (P<0.05), but was not observed in E2E group. In conclusion there was an interaction between Equex and extender ingredient. The cryoprotective effect of Equex was more evident in skim milkbased extender than that in tris-based extender.

This study aims to evaluate the effects of nano-ozone solution (NZS) on canine oocyte nuclear maturation, associated with the alterations of antioxidant and oxidant status and cyclin-dependent kinase 1 (CDK1), cyclin B1 gene expressions. Oocytes were cultured in four distinct concentrations of NZS (0.5, 1, 2, and 5 µg/mL) and parthenogenetically activated. The rates of oocytes arrested at the Germinal Vesicle (GV), Germinal Vesicle Breakdown (GVBD), Metaphase I (MI), and Metaphase II (MII) stages were statistically different among groups (P < 0.05). The oocytes cultured in 1 µg/mL NZS yielded the best oocyte maturation rate at the MI and MII stages; however, the lowest maturation and high degeneration rates were observed in Group E. The measurements of Malondialdehyde (MDA), reduced Glutathione (GSH), Superoxide Dismutase (SOD), and Ferric Reducing/Antioxidant Power assay (FRAP) were performed from IVM culture media. No statistical difference was observed in SOD and MDA results (P > 0.05). GSH levels were statistically significant between Group A-Group E (p = 0.003), Group B-Group E (p = 0.045), and Group E-Group D (p = 0.021). The culture media in Group D and Group E had high FRAP concentrations and significantly differed between groups (P < 0.05). CDK1, and cyclin B1 genes, which are subunits of maturation-promoting factor (MPF), are upregulated in Group B and Group C, while are downregulated in oocytes of Group E.This study showed that low, controlled doses of NZS (1 µg/mL) supplementation could improve the meiotic competence of canine oocytes and lead to positive response in expressions of CDK1 and cyclin B1 on the gene level.

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.

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.

Pet hayvanlarında biyoteknolojik çalışmalar son yıllarda hız kazanmaya başlamıştır. Köpeklerde başarısız yardımcı üreme teknikleriyle ilgili oluşan sorular, muhtemelen köpek türlerinin reproduktif fizyolojisine ait yetersiz bilgiden kaynaklanmaktadır. Fakat diğer taraftan pet biyolojisindeki uygulamalar, insan hastalıkları için model oluşturmaktadır. Bunun ötesinde gamet kriyopreservasyonunun gelişmesi, nesli tükenmekte olan türlerin korunması ve genetik banka oluşturulması için önemlidir. Bu çalışmada, köpek oositlerindeki düşük maturasyon oranlarına rağmen, partenogenetik aktivasyonun etkileri vitrifiye oositlerde test edildi. Köpek oositleri, Yıldırım Belediyesi Sokak Hayvanları Bakım ve Rehabilitasyon merkezinden alınan, 20 adet sağlıklı köpekten toplandı. Ovaryumların tekrarlı parçalanmasından sonra, seçilen COCs (kumulus oosit kompleksleri), 5% CO2 inkübatörde, mineral yağla kaplanmış 500 μl TCM-199 içeren dört-gözlü petrilerde, 39°C’de, 72 saat boyunca maturasyona bırakıldı. Maturasyondan sonra oositler, 0%, 10%, 20% etilen glikol içeren 50 ml PBl içinde sırasıyla, 10, 10 dakika ve 30 saniye muamele edildi. Oositler, 30 μl VS3 içeren kriyoviallere yerleştirilerek sıvı nitrojende donduruldu. Bu grubun oositleri (n=257) ‘vitrifiye oosit-VO’ olarak gruplandı. Çözdürme sonrasında, oositler ionomisinle 5 dakika ve sikloheksimid ile 3 saat muamele ederek partenogenetik aktivasyona bırakıldı. Sonrasında oositler 72 saat kültüre edilerek nükleer maturasyon değerlendirildi. Kontrol grubu olarak kullanılan oositler (n=257), ‘non vitrifiye oosit-FO’ olarak gruplandırıldı. Maturasyondan sonra, oositler direkt olarak ionomisin ve sikloheksimid ile muamele edilerek aktivasyona bırakıldı ve 72 saat kültüre edildi. Tüm oositler Hoechst33342 ile 30 dakika boyandıktan sonra nükleer maturasyon oranları mikroskopta değerlendirildi. Maturasyon oranları (MI+MII) gruplar arasında istatistiksel olarak anlamlı bulunmadı. (p>0,05). Gruplar arasında GV, GVBD, MI, ve MII oranlarında da istatistiksel fark bulunmadı (p>0,05). Maturasyon sonrasında, vitrifiye köpek oositlerinde partenogenetik aktivasyona bağlı nükleer değerlendirmeye çalışması bulunmamaktadır. Fakat bu uygulamada elde edilen düşük maturasyon oranlarının, ileri moleküler çalışmalarla açıklanması gerektiği kanısındayız.

The aim of this study is to evaluate the effects of scheduled vincristine sulfate therapy on canine oocyte quality and nuclear oocyte maturation, associated with total antioxidant and oxidant status of ovaries and Anti-Mullerian Hormone (AMH) concentrations in dogs with Canine Transmissible Venereal Tumor (CTVT). Six bitches suffering from CTVT and six healthy bitches were included in the study. Hemogram was carried out weekly. AMH measurements and ovariohysterectomy operations were performed after the termination of vincristine sulfate therapies. Tissue samples from ovaries were utilized for Malondialdehyde (MDA), reduced Glutathione (GSH), Superoxide Dismutase (SOD), Total Anti-oxidative Status (TAS), Total Oxidative Status (TOS) measurements, and Oxidative Stress Index (OSI) was calculated. Collected oocytes were evaluated for meiotic competence, after In Vitro Maturation (IVM) and parthenogenetic activation. No difference between the two groups was observed in hematologic parameters (P > 0.05). Meiotic stages of Germinal Vesicle Break Down (GVBD), Metaphase I (MI), and Metaphase II (MII) were significantly different between groups (P < 0.05). The number of oocytes reaching MII and meiotic resumption was lower in the CTVT group. Furthermore, AMH concentrations, oxidant parameters (OSI, TOS, and MDA), and antioxidant parameters (GSH, SOD, and TAS) were also statistically different between groups (P < 0.05).The results of this study show that vincristine sulfate application in the treatment of CTVT could alter oxidant/antioxidant status in ovaries. Apart from these, oocyte quality and IVM rates seem to decline related to gonadotoxicity. Moreover, AMH could be an important marker in the evaluation of oocyte qualities in bitches, as it is in women.

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.

The present study was conducted to observe the effect of different dilution rates on ram sperm motion characters (motility, progressivity, velocity and average of speed), head diameter, mitochondrial membrane potential (MMP) and live intact acrosome rate during liquid storage for 96 h at 5 degrees C. Normospermic semen samples that obtained from 4 adult rams were pooled. Pooled semen was split into two aliquots; Low Dilution Rate (LDR) (400 million) and High Dilution Rate (HDR) (20 million) motile spermatozoa per insemination dose/0.25 mL straw and were diluted with a skim milk-egg yolk-based extender. It was observed that the Total Motility (TM) and total progressivity of the LDR group decreased more rapidly than HDR group over time, in general. From the 12th h to the 96th h; the rapid progressivity (P<0.01), MMP (P<0.05), live sperm rates with intact acrosome (P<0.01) and straightness (STR) (P<0.001) of the HDR group results were statistically better than LDR. It was concluded that the spermatological properties examined during storage were damaged at different times and were affected by sperm concentration and also storage time.

Bu çalışma, gökkuşağı alabalığı seminal plazmasının (RTSP) bal arısı drone spermatozoonu üzerindeki etkilerini eritme sonrası araştırmayı amaçladı. Cinsel açıdan olgun erkek arılardan sperm örnekleri toplandı ve havuzlandı. Daha sonra havuzlanmış semen, farklı konsantrasyonlarda RTSP (% 2.5 /% 5 /% 10) ile ve RTSP (kontrol) takviyeli sulandırıcılar olmadan seyreltildi. Motilite, plazma membranı fonksiyonel bütünlüğü, akrozom bütünlüğü ve mitokondriyal fonksiyon kriyoprezervasyon sürecinden olumsuz etkilenmiştir, ancak DNA bütünlüğü etkilenmemiştir. Membran lipid peroksidasyon durumu da çözüldükten sonra malondialdehit (MDA) konsantrasyonu kullanılarak analiz edildi. RTSP10 grupları, kontrol grubuna kıyasla sperm motilitesi, plazma membran bütünlüğü, akrozomal bütünlük ve mitokondriyal fonksiyon üzerinde olumlu bir etkiye sahipti (P <0.05). Çalışma, RTSP takviyeli sulandırıcıların eritme sonrası drone sperm parametreleri üzerinde faydalı etkilere sahip olduğunu göstermektedir. Bu çalışmanın sonuçları,% 10 RTSP takviyeli bir sulandırıcı kullanmanın avantajını göstermiştir.

Th is study aimed to investigate the eff ect of the Rainbow trout seminal plasma (RTSP) supplemented (control, 10% or 15%) lyophilized extender on freezability of ram semen.Collected semen was pooled and split into two aliquots, and each of the ejaculates was diluted with fresh or lyophilized extenders with RTSP (0%, FC and LC; 10% F10 and L10or 15% F15 and L15) using two-step dilution method.Semen was frozen using the programmable freezing machine.Semen samples were examined for sperm motility, defective acrosomes, plasma membrane integrity and DNA fragmentation at native and post-thaw stage.Th e highest percentages of post-thaw motility and plasma integrity were observed in the F10, F15, and L15 (P<0.05).Th ere was no significant diff erence in the rate of postthaw defective acrosomes when the other extender groups were compared with the control.Th e highest percentage of post-thaw DNA fragmentation values were observed in the FC group, while the lowest DNA fragmentation was obtained in the F15 but only significant diff erent from FC and LC groups.In conclusion, the findings of this study show that the lyophilized extender with 15% RTSP added can be used successfully for freezing ram semen.

The present study was conducted to observe the effect of different dilution rates on ram sperm motion characters (motility, progressivity, velocity and average of speed), head diameter, mitochondrial membrane potential (MMP) and live intact acrosome rate during liquid storage for 96 h at 5ºC.Normospermic semen samples that obtained from 4 adult rams were pooled.Pooled semen was split into two aliquots; Low Dilution Rate (LDR) (400 million) and High Dilution Rate (HDR) (20 million) motile spermatozoa per insemination dose/0.25 mL straw and were diluted with a skim milk-egg yolk-based extender.It was observed that the Total Motility (TM) and total progressivity of the LDR group decreased more rapidly than HDR group over time, in general.From the 12 th h to the 96 th h; the rapid progressivity (P<0.01),MMP (P<0.05),live sperm rates with intact acrosome (P<0.01) and straightness (STR) (P<0.001) of the HDR group results were statistically better than LDR.It was concluded that the spermatological properties examined during storage were damaged at different times and were affected by sperm concentration and also storage time.

: Th is study aimed to investigate the eff ect of the Rainbow trout seminal plasma (RTSP) supplemented (control, 10% or 15%) lyophilized extender on freezability of ram semen. Collected semen was pooled and split into two aliquots, and each of the ejaculates was diluted with fresh or lyophilized extenders with RTSP (0%, FC and LC; 10% F10 and L10or 15% F15 and L15) using two-step dilution method. Semen was frozen using the programmable freezing machine. Semen samples were examined for sperm motility, defective acrosomes, plasma membrane integrity and DNA fragmentation at native and post-thaw stage. Th e highest percentages of post-thaw motility and plasma integrity were observed in the F10, F15, and L15 (P<0.05). Th ere was no significant diff erence in the rate of post-thaw defective acrosomes when the other extender groups were compared with the control. Th e highest percentage of post-thaw DNA fragmentation values were observed in the FC group, while the lowest DNA fragmentation was obtained in the F15 but only significant diff erent from FC and LC groups. In conclusion, the findings of this study show that the lyophilized extender with 15% RTSP added can be used successfully for freezing ram semen.

The study aimed to evaluate the eff ects of L-carnitine on honey bee (Apis mellifera) drone spermatozoon at post-thaw.Semen samples obtained from sexually mature drones were pooled.Then, pooled semen was diluted with diff erent concentrations of L-carnitine (2.5 mM/ 5 mM/ 10 mM) and without L-carnitine (control) supplemented extenders.Motility, plasma membrane functional integrity, acrosomal integrity and mitochondrial function were negatively aff ected by the cryopreservation process but DNA integrity was not aff ected.Malondialdehyde (MDA) concentration was used for analyzing the membrane lipid peroxidation status and a better result was obtained in the LC5 group compared with the control group at post-thaw.L-carnitine groups had a positive eff ect on sperm motility, plasma membrane integrity, and mitochondrial function compared to the control group (P<0.05).Acrosomal integrity was better preserved in the LC5 group compared to the control group.The study shows that LC supplemented extenders have beneficial eff ects on honey bee drone sperm parameters.The results of the present study demonstrated the beneficial eff ect of using a 5 mMol LC supplemented extender.

The study aimed to evaluate the eff ects of L-carnitine on honey bee (Apis mellifera) drone spermatozoon at post-thaw. Semen samples obtained from sexually mature drones were pooled. Then, pooled semen was diluted with diff erent concentrations of L-carnitine (2.5 mM/ 5 mM/ 10 mM) and without L-carnitine (control) supplemented extenders. Motility, plasma membrane functional integrity, acrosomal integrity and mitochondrial function were negatively aff ected by the cryopreservation process but DNA integrity was not aff ected. Malondialdehyde (MDA) concentration was used for analyzing the membrane lipid peroxidation status and a better result was obtained in the LC5 group compared with the control group at post-thaw. L-carnitine groups had a positive eff ect on sperm motility, plasma membrane integrity, and mitochondrial function compared to the control group (P<0.05). Acrosomal integrity was better preserved in the LC5 group compared to the control group. The study shows that LC supplemented extenders have beneficial eff ects on honey bee drone sperm parameters. The results of the present study demonstrated the beneficial eff ect of using a 5 mMol LC supplemented extender.