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Öğe Comparison of cryosurvival and spermatogenesis efficiency of cryopreserved neonatal mouse testicular tissue between three vitrification protocols and controlled-rate freezing(Academic Press Inc Elsevier Science, 2018) Yildiz, Cengiz; Mullen, Brendan; Jarvi, Keith; McKerlie, Colin; Lo, Kirk C.Grafting of cryopreserved testicular tissue is a promising tool for fertility and testicular function preservation in endangered species, mutant animals, or cancer patients for future use. In this study, we aimed to improve the whole neonatal mouse testicular tissue cryopreservation protocols by comparing cryosurvival, spermatogenesis, and androgen production of grafted testicular tissue after cryopreservation with three different vitrification protocols and an automated computed controlled-rate freezing. Whole neonatal mouse testes were vitrified with various vitrification solutions (V1) 40% EG + 18% Ficoll + 0.35 M Sucrose, (V2) DAP 213 (2 M DMSO + 1 M Acetamid + 3 M PG), or (V3) 15% EG + 15% PG + 0.5 M Sucrose (total solute concentration V1:74.34%, V2:44.0%, and V3:49.22% wt/vol). Alternatively, neonatal testicular tissue was also frozen in 0.7 M DMSO + 5% fetal bovine serum using controlled-rate freezing and compared to fresh grafted testicular tissue, sham grafted controls, and the vitrification protocol groups. Fresh (n = 4) and frozen-thawed (n = 4) testes tissues were grafted onto the flank of castrated male NCr Nude recipient mouse. The grafts were harvested after three months. Fresh or frozen-thawed grafts with controlled-rate freezing had the highest rate of tissue survival compared to other vitrified protocols after harvesting (p < 0.05). Both controlled-rate freezing and V1 protocol groups displayed the most advanced stages of spermatogenesis with elongated spermatids and spermatozoa in 17.6 +/- 1.3% and 16.3 +/- 1.9% of seminiferous tubules based on histopathological evaluation, respectively. Hosts of the testicular graft from controlled-rate freezing had higher levels of serum testosterone compared to all other vitrified-thawed graft groups (p < 0.05). This study shows that completed spermatogenesis from whole neonatal mouse testes were obtained when frozen with controlled-rate freezing and V1 vitrification solution and that testicular cryopreservation efficacy vary with the protocol and vitrification technique.Öğe Comparison of sperm quality and DNA integrity in mouse sperm exposed to various cooling velocities and osmotic stress(Elsevier Science Inc, 2010) Yildiz, Cengiz; Law, Napoleon; Ottaviani, Palma; Jarvi, Keith; McKerlie, ColinThe first objective was to compare sperm quality following conventional manual sperm freezing (cryovials held 1, 2, 3, and 4 cm, respectively, above liquid nitrogen (LN2) for 10 min, resulting in cooling velocities of approximately -14.9, -10.1, -6.6, and -5.1 degrees C/min, respectively), and cooling velocities of -5, -20, -40, and -100 degrees C/min in a programmed automated freezer, for sperm recovered from CD-1, B6129SF1, and C57BL/6NCrlBR mice. Furthermore, using these strains, as well as 129S/SvPaslco, and DBA/2NCrlBR mice, the second objective was to determine the effects on DNA integrity of sperm exposed to hyposmotic (1 mOsm/L) and hyperosmotic (2400 mOsm/L) solutions, compared to an isosmotic control (300 mOsm/L). For freezing above LN2 or in an automated freezer, 2 cm above LN2 and -100 degrees C/min, respectively, were optimal (P < 0.05-0.01), with no significant differences between these two approaches for post-thaw progressive motility, DNA integrity, and in vitro rates of fertilization and blastocyst formation. Both manual and automated freezing techniques increased post-thaw sperm DNA fragmentation (P < 0.01); the DNA integrity of post-thaw sperm was significantly affected by cooling velocity and strain background. Relative to isosmotic controls, a hyposmotic solution was more deleterious (P < 0.05-0.01) to sperm DNA integrity than a hyperosmotic solution for CD-1, B6129SF1, C57BL/6, and DBA mice (there were strain-dependent differences). In conclusion, optimization of freezing distance and cooling velocity (manual and automated freezing, respectively) were significant factors for efficient cryopreservation and re-derivation of mice from frozen-thawed sperm. Additionally, osmotically-driven volume changes in mouse sperm increased DNA fragmentation, with susceptibility affected by background strain. Crown Copyright (C) 2010 Published by Elsevier Inc. All rights reserved.Öğe Cryopreservation of scaly carp (Cyprinus carpio) sperm: effect of different cryoprotectant concentrations on post-thaw motility, fertilization and hatching success of embryos(Springer, 2014) Yavas, Ilker; Bozkurt, Yusuf; Yildiz, CengizThe aim of the present study was to determine the effect of various cryoprotectants on post-thaw sperm quality and fertilizing capacity of cryopreserved scaly carp (Cyprinus carpio) semen. The present study focused on freezing of scaly carp sperm utilizing a practical and inexpensive protocol for aquaculture. Semen was diluted with Kurokura's extender composing 3.6 g/l NaCl, 10 g/l KCl, 0.22 g/l CaCl2, 0.08 g/l MgCl2 and 0.2 g/l NaHCO3. The extender contained three different cryoprotectants (DMSO, DMA and egg yolk) at ratios of 5, 10 and 15 %. Semen was placed into 0.25-ml straws and exposed to liquid nitrogen vapor (-120 A degrees C) using an insulated box with an adjustable tray for 10 min and then plunged into liquid nitrogen (-196 A degrees C) tank. The thawing process was performed in a water bath at 40 A degrees C for 10 s. The results indicated that type of cryoprotectants and their concentrations are rather effective in scaly carp sperm cryopreservation on post-thaw sperm quality, while they are very important in order to obtain high fertilization rates. The highest fertilization rate was determined as 96.4 +/- A 0.15 % with 15 % egg yolk, while the highest hatching rate was determined as 99.3 +/- A 0.80 with 15 % DMA. In conclusion, the applied cryopreservation method for scaly carp sperm is suitable to fertilize high amounts of eggs.Öğe Effect of Ascorbic Acid and Proline Amino Acid Supplementations on Cryosurvival and Fertility Rates of Cryopreserved Honey Bee (Apis mellifera) Semen(Mary Ann Liebert, Inc, 2022) Guel, Aziz; Yildiz, Cengiz; Cetin, Nurdan Coskun; Yalcin, Oguz KaanThis study investigated the effect of ascorbic acid (vitamin C) and proline amino acid alone or together on the quality and fertility of frozen/thawed honey bee spermatozoa. The experiments were designed to compare a single ascorbic acid, a single proline amino acid, and different combinations of ascorbic acid with proline amino acid on the cryopreservation of honey bee semen based on sperm motility, viability, intact membrane (hypo-osmotic swelling test), and fertility rates. Eight cryopreserved study groups comprised Control II with no supplement, along with groups with ascorbic acid (2 mg), proline 25 mM, proline 50 mM, proline 100 mM, and combination groups of both ascorbic acid (2 mg) and proline 25 mM, proline 50 mM, and lastly proline 100 mM groups, respectively. Using 50 mM proline in the tested groups had the greatest impact on sperm motility, viability, the percentage of spermatozoa with intact membrane, and fertility. The cryopreservation process caused a gradual decrease in motility, viability, intact membrane (p < 0.05), and fertility rates (p < 0.01) in all the tested research groups as against the fresh semen control group. Successful honey bee sperm cryopreservation and fertility are achievable when using an appropriate sperm freezing protocol and antioxidant. Proline amino acid as an antioxidant in semen extender had a more beneficial influence on sperm quality parameters and fertility. The success of cryopreservation with antioxidants is related to the chosen antioxidant in a dose-dependent manner.Öğe Effect of cholesterol-loaded cyclodextrin on cryosurvival and fertility of cryopreserved carp (Cyprinus carpio) sperm(Academic Press Inc Elsevier Science, 2015) Yildiz, Cengiz; Yavas, Ilker; Bozkurt, Yusuf; Aksoy, MelihAddition of cholesterol-loaded cyclodextrin (CLC) to the diluents of mammalian semen increased stability and rigidity of phospholipid hydrocarbon chains of plasma membrane during sperm cryopreservation process. CLC has been tested successfully as ayoprotectant in various livestock sperm cryopreservation protocols but its efficacy for cryopreserving of fish sperm has not previously been tested. In the present study, different cholesterol loaded cyclodextrin concentrations were evaluated for the cryopreservation of carp (Cyprinus carpio) sperm. Sexually mature fish were induced to spermiation and ovulation with Ovopel. The extenders were prepared by using 300 mM glucose and 10% DMSO supplemented with different concentrations of CLC (0.5, 1.0, 1.5, 2.0, 2.5, and 3.0 mg per 120 x 10(6) spermatozoa) and without CLC (control). The pooled semen was diluted separately at a ratio of 1:3 (v/v) by using CLC extenders. Diluted semen placed into 0.25 ml straws were equilibrated at 4 degrees C for 15 min and frozen in liquid nitrogen vapor. Fertilization was conducted using a ratio of 1 x 105 spermatozoa/egg. Fresh sperm with no treatment showed the greatest sperm motility, duration of motility, viability, and fertilization results compared to the other tested cryopreserved and control groups (p < 0.05). Supplementation of 1.5 mg CLC to the extender showed the best cryoprotective effect for sperm motility, duration of motility, and viability against freezing damage in comparison to extenders containing 2.5 mg, 3.0 mg CLC, and control group (p < 0.05). Cryopreserved sperm containing 1.5 mg CLC provided greater result in term of fertilization success when compared to other extenders containing 0.5, 2.5, and 3.0 mg CLC or control (p <0.05). The amount of CLC effected post-thaw sperm quality and fertility as a dose-dependent manner. It is concluded that treatment of cholesterol-loaded cyclodextrin for carp sperm cryopreservation significantly improves cell cryosurvival and fertilization. (C) 2015 Elsevier Inc. All rights reserved.Öğe Effect of different antioxidants on motility, viability and fertilizing capacity of cryopreserved scaly carp (Cyprinus carpio) semen(Israeli Journal Of Aquaculture-Bamidgeh, 2014) Yavas, Ilker; Bozkurt, Yusuf; Yildiz, CengizThe present study investigated effect of different antioxidants such as taurine (25 mM, 50 mM, 75 mM and 100 mM), bovine serum albumine (BSA) (1%, 2.5%, 5%, 7.5%) and vitamin C (ascorbic acid) (2.8 mM, 5.6 mM, 8.4 mM and 11.2mM) on motility and fertilizing ability of cryopreserved scaly carp (Cyprinus carpio) semen. The results demonstrated that addition of taurine at doses of 50 mM and 75 mM and also BSA at doses of 2.5% and 5% improved fertilization ability of cryopreserved scaly carp semen. The highest post-thaw motility (42.4 +/- 1.5%), viability (67.8 +/- 2.6%), fertilization (59.3 +/- 0.5%) and also hatching rates (68.2 +/- 3.4%) were determined by supplementation of 75 mM taurine (p<0.05). These results suggest that supplementation of ionic extender with taurine is suitable for scaly carp semen cryopreservation.Öğe Effect of different avian egg yolk types on fertilization ability of cryopreserved common carp (Cyprinus carpio) spermatozoa(Springer, 2014) Bozkurt, Yusuf; Yavas, Ilker; Yildiz, CengizIn spite of the fact that egg yolk from different avian species has successfully been used as an additive for the cryopreservation of sperm in mammalian species, its efficacy for cryopreserving fish sperm has not previously been tested comparatively. Therefore, the present study was carried out to determine the effect of egg yolks from different avian species, namely domestic chicken (Gallus gallus domesticus), turkey (Meleagris gallopavo) and quail (Coturnix coturnix), on post-thaw motility and fertilization ability of cryopreserved common carp spermatozoa. Egg yolks from chicken, turkey and quail were analysed for moisture, total fat, protein, cholesterol and phospholipid profile. Total fat and cholesterol contents of the turkey egg yolk were higher than chicken and quail egg yolks (p < 0.05). Semen was frozen according to conventional slow freezing procedure. The extender contained 350 mM glucose, 30 mM Tris and 5 % glycerol supplemented with different ratios of avian egg yolk (10, 15 and 20 %). Semen was equilibrated at 4 A degrees C for 15 min and placed into 0.25-ml straws and frozen in liquid nitrogen vapour (for 10 min at -120 A degrees C) and finally stored in liquid nitrogen (-196 A degrees C) tank. The frozen spermatozoa were thawed in a water bath at 35 A degrees C for 30 s. Fertilization was conducted using a ratio of 1 x 10(5) spermatozoa/egg. Cryopreservation experiments resulted in higher post-thaw motility and fertilization rates. Mean post-thaw motility of cryopreserved spermatozoa was between 45 and 80 %, and fertilization rates, expressed as the percentage of eyed embryos, ranged from 70 to 95 %. In conclusion, the present study showed that turkey and quail egg yolks are suitable alternatives to the chicken egg yolk for the cryopreservation of common carp spermatozoa.Öğe Effect of different cryoprotectant agents on spermatogenesis efficiency in cryopreserved and grafted neonatal mouse testicular tissue(Academic Press Inc Elsevier Science, 2013) Yildiz, Cengiz; Mullen, Brendan; Jarvi, Keith; McKerlie, Colin; Lo, Kirk C.Restoration of male fertility associated with use of the cryopreserved testicular tissue would be a significant advance in human and animal assisted reproductive technology. The purpose of this study was to test the effects of four different cryoprotectant agents (CPA) on spermatogenesis and steroidogenesis in cryopreserved and allotransplanted neonatal mouse testicular tissue. Hank's balanced salt solution (HBSS) with 5% fetal bovine serum including either 0.7 M dimethyl sulfoxide (DMSO), 0.7 M propylene glycol (PrOH), 0.7 M ethylene glycol (EG), or glycerol was used as the cryoprotectant solution. Donor testes were collected and dissected from neonatal pups of CD-1 mice (one day old). Freezing and seeding of the testicular whole tissues was performed using an automated controlled-rate freezer. Four fresh (non-frozen) or frozen-thawed pieces of testes were subcutaneously grafted onto the hind flank of each castrated male NCr nude recipient mouse and harvested after 3 months. Fresh neonatal testes grafts recovered from transplant sites had the most advanced rate of spermatogenesis with elongated spermatid and spermatozoa in 46.6% of seminiferous tubules and had higher levels of serum testosterone compared to all other frozen-thawed-graft groups (p < 0.05). Fresh grafts and frozen-thawed grafts in the DMSO group had the highest rate of tissue survival compared to PrOH, EG, and glycerol after harvesting (p > 0.05). The most effective CPA for the freezing and thawing of neonatal mouse testes was DMSO in comparison with EG (p < 0.05) in both pre-grafted and post-grafted tissues based on histopathological evaluation. Likewise, the highest level of serum testosterone was obtained from the DMSO CPA group compared to all other cryoprotectants evaluated (p < 0.05). The typical damage observed in the frozen-thawed grafts included disruption of the interstitial stroma, intercellular connection ruptures, and detachment of spermatogonia from the basement membrane. These findings indicate that neonatal mouse testes were most effectively preserved when frozen with HBSS medium with DMSO and that the type of CPA is a significant factor to obtain the most advanced stages of spermatogenesis and steroidogenesis after cryopreservation, thawing, and transplantation of neonatal mouse testes. (C) 2013 Elsevier Inc. All rights reserved.Öğe THE EFFECT OF DIFFERENT PRESERVATION MEDIA AND TEMPERATURES ON SPERM QUALITY AND DNA INTEGRITY IN MOUSE CAUDA SPERMATOZOA(Cryo Letters, 2022) Yildiz, Cengiz; Coskun Cetin, Nurdan; Yavas, Ilker; Yalcin, Oguz Kaan; Yilmaz, Firdevs; Karaca, FikretBACKGROUND: Mouse sperm can be stored for long or short-time periods. Nevertheless long-term storage leds to significantly reduced sperm quality and fertility because of cryodamage. Thus, in the storage of semen in mice, it is necessary to focus on media and temperatures that gives good results in short-term storage. OBJECTIVE: To determine favorable media for short-term storage of mice spermatozoa by evaluating progressive motility, viability, membrane function integrity, acrosome integrity and fragmented DNA rates at various storage temperatures. MATERIALS AND METHODS: Mouse spermatozoa were collected from epididymides of mature CD1 males and samples were stored at 24 degrees C and 4 degrees C for 60 h. RESULTS: Motility, viability and membrane function of mice spermatozoa were greatest when stored in KSOM media. Motility and viability were not different when stored at refrigerator or room temperature in KSOM compared to HTF or PBS mediums for 48 h, but were after 60 h. There wasn't any significant variation in terms of acrosome integrity in different preservation conditions. Fragmented DNA rates were similar in fresh sperm with KSOM and HTF media, while there was higher damage in PBS medium at 60 h. Overall, sperm parameters were affected significantly by the time of storage and type of preservation medium, and PBS extender was not suitable for mice spermatozoa at room and refrigerated temperatures as it caused the lowest progressive motility, viability, membrane function integrity and the highest DNA damage. CONCLUSION: Mice spermatozoa stored in KSOM retained the best sperm quality parameters both 24 degrees C and 4 degrees C for the first 48 h.Öğe Effect of Extender Supplemented with Different Sugar Types on Post-thaw Motility, Viability and Fertilizing Ability of Cryopreserved Common Carp (Cyprinus carpio) Spermatozoa(Israeli Journal Of Aquaculture-Bamidgeh, 2016) Bozkurt, Yusuf; Yavas, Ilker; Yildiz, CengizThe influence of various sugar types supplemented to the extender on post-thaw motility, viability, and fertilizing capacity of cryopreserved common carp (Cyprinus carpio) semen were investigated. The results indicated that types of sugar significantly influenced motility, motility duration, and viability rates (P<0.05). Glucose, maltose, sucrose, and trehalose provided higher motility compared to the sugar-free control in post-thaw samples. Trehalose provided highest progressive motility duration, and higher viable sperm rates were obtained with all sugar types except xylose. Xylose exhibited the lowest post-thaw progressive motility duration (35.2 +/- 1.4s). The mean highest fertilization (78.2 +/- 1.4%) and eyed egg rates (94.3 +/- 1.5%) were determined using trehalose in the extender, and differences between the treatments in the fertilization and eyeing rates were significant (P<0.05). Finally, the present study showed that sugars, especially maltose and trehalose (disaccharide), improved post-thaw spermatozoa motility and fertility in common carp semen.Öğe The Effects of Ferulic Acid, Tryptophan, and L-Glutamine on the Cryopreservation of Mouse Spermatozoa(Mary Ann Liebert, Inc, 2024) Kocak, Gokhan; Yildiz, CengizIn this study, the effects of ferulic acid (0.1, 1, ve 10 mM), tryptophan (5, 25, ve 50 mM), and L-glutamine (10, 50, ve 100 mM) at different doses added to 18% raffinose + 3% skimmed milk powder sperm extender on the freezing of mouse spermatozoa in liquid nitrogen were investigated. The combination of 18% raffinose + 3% skimmed milk powder without additives was used as the control group. Frozen spermatozoa were thawed in a 37 degrees C water bath for 30 seconds. After freeze-thawing, motility, dead spermatozoa ratio, plasma membrane integrity, abnormal acrosome ratio, motility endurance (for 4 hours), and cell apoptosis tests were performed in Human Tubal Fluid (HTF). Compared with the control group after freezing and thawing, the highest motility and plasma membrane integrity were obtained in the 10 mM L-glutamine group with 56.6% +/- 2.11% and 77.8% +/- 0.87%, respectively (p < 0.05). In addition, when compared to the control group, the lowest rate of dead spermatozoa and abnormal acrosome was found in the 10 mM L-glutamine group as 26.0% +/- 1.46% and 6.3% +/- 1.09%, respectively (p < 0.05). The highest motility values for spermatozoa endurance were determined in the 10 and 50 mM L-glutamine groups up to the 4th hour compared to the control group (p < 0.05). In the evaluation of apoptosis in semen samples, there was no significant difference between the control, 0.1 mM ferulic acid, and 10 mM L-glutamine groups (p > 0.05). As a result, it was determined that the addition of 10 mM L-glutamine to the spermatozoa extender increased the motility, viable spermatozoa, functional membrane integrity, intact acrosome ratios, or motility endurance after freeze-thawing and could be used successfully in the freezing extender of mouse spermatozoa.Öğe An evaluation of soybean lecithin as an alternative to avian egg yolk in the cryopreservation of fish sperm(Academic Press Inc Elsevier Science, 2013) Yildiz, Cengiz; Bozkurt, Yusuf; Yavas, IlkerPlant-derived lecithin has been used as a more sanitary alternative to avian egg yolk in livestock sperm cryopreservation protocols but its efficacy for cryopreserving fish sperm has not previously been tested comparatively. Here various concentrations of soybean lecithin were evaluated for the cryopreservation of carp (Cyprinus carpio) sperm. Sexually mature fish were induced to spermiation and ovulation with ovopel. The extenders were prepared by using 300 mM glucose, 10% DMSO, supplemented with different ratios of lecithin (5%, 10%, 15%, and 20%) and 10% egg yolk (control I). Negative control was made without egg yolk and soybean lecithin (control II). The pooled semen was diluted separately at ratio of 1:3 (v/v) by using egg yolk and soybean-based extenders. Diluted semen placed into 0.25 ml straws were equilibrated at 4 degrees C for 15 min and frozen in liquid nitrogen vapor. Fertilization was conducted using a ratio of I x 10(5) spermatozoa/egg. Supplementation of 10% lecithin to extender showed the best cryoprotective effect for sperm motility and duration of motility against freezing damage compared to 15%, 20% and control II groups (p < 0.05). Cryopreserved sperm with extender containing 10% lecithin provided a greater result in terms of fertilization success when compared to extenders containing 20% lecithin or control II (p < 0.05). It is concluded that the animal protein-free extender containing 10% soybean lecithin has a similar cryoprotective actions with conventional egg yolk-based extender against freezing damages and fertilization. Therefore, soybean lecithin is a suitable alternative to avian egg yolk for the cryopreservation of fish sperm. (C) 2013 Elsevier Inc. All rights reserved.Öğe Vitrification of Common Carp (Cyprinus Carpio) Spermatozoa, Post-Thaw Sperm Quality, and Fertility(Israeli Journal Of Aquaculture-Bamidgeh, 2014) Bozkurt, Yusuf; Yildiz, Cengiz; Yavas, IlkerThe aim of this investigation was to test a new technology, vitrification, or ultra-rapid freezing of the spermatozoa of common carp, and to study the ability of glucose, BSA, and other cryoprotectants to protect these cells from cryo-injuries. Spermatozoa were isolated and vitrified using 10 different cryoprotectant solutions: (1) Glucose based medium (GBM) + 1% bovine serum albumin (BSA); (2) GBM + 1% BSA + 10% DMSO; (3) GBM + 1% BSA + 20% DMSO; (4) GBM + 1% BSA + 30% DMSO; (5) GBM + 1% BSA + 10% DMA; (6) GBM + 1% BSA + 20% DMA; (7) GBM + 1% BSA + 30% DMA; (8) GBM + 1% BSA + 10% methanol; (9) GBM + 1% BSA + 20% methanol; (10) GBM + 1% BSA + 30% methanol. Fertilization rates for vitrification experiments were low and the use of low concentrations of cryoprotectants yielded lower fertilization rates than the vitrification solutions containing high cryoprotectant concentrations. In conclusion, this study reported successful vitrification of common carp spermatozoa by direct transfer into liquid nitrogen.
