The Effects of Ferulic Acid, Tryptophan, and L-Glutamine on the Cryopreservation of Mouse Spermatozoa.

In 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°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.

Effect of Ascorbic Acid and Proline Amino Acid Supplementations on Cryosurvival and Fertility Rates of Cryopreserved Honey Bee (Apis mellifera) Semen.

This 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.

Comparison of cryosurvival and spermatogenesis efficiency of cryopreserved neonatal mouse testicular tissue between three vitrification protocols and controlled-rate freezing.

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.

Human Fetal Testicular Tissue Xenotransplantation: A Platform to Study the Effect of Gonadotropins on Human Germ Cell Development In Utero.

PURPOSE: We examined the effects of long-term hCG stimulation on germ cell maturation, and Sertoli and Leydig cell function in a xenotransplantation model of the human fetal testis. MATERIALS AND METHODS: A total of 20 human fetal testes were ectopically xenografted on 20 castrated NCr male nude mice. Grafts were collected for analysis 24 weeks later. Mice were treated with saline as the control or with hCG beginning 4 weeks after the grafts were transplanted. RESULTS: Of the grafts 65% survived at 24 weeks. In contrast to untreated pregrafted samples, hCG stimulated xenografts showed significantly increased density of seminiferous tubule formation with Sertoli cell migration to the basement membrane. Germ cell proliferation and differentiation from gonocytes (M2A(+)) to prespermatogonia (MAGE-4A(+)) were observed in graft samples recovered from the hCG and nonhCG treated groups at 24 weeks of treatment. Leydig cells in hCG treated grafts produced significantly more testosterone than nonhCG treated grafts. Although further studies are required to investigate the potential for further differentiation and maturation of xenografted human fetal testes, normal in utero testicular development was reproduced under long-term hCG stimulation. CONCLUSIONS: This model represents a means to study long-term effects of gonadotoxins or hormonal stimulation on the maturation of human fetal testes.

Effect of cholesterol-loaded cyclodextrin on cryosurvival and fertility of cryopreserved carp (Cyprinus carpio) sperm.

Addition 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 cryoprotectant 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 3.0mg per 120×10(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°C for 15 min and frozen in liquid nitrogen vapor. Fertilization was conducted using a ratio of 1×10(5) 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.

An evaluation of soybean lecithin as an alternative to avian egg yolk in the cryopreservation of fish sperm.

Plant-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 °C for 15 min and frozen in liquid nitrogen vapor. Fertilization was conducted using a ratio of 1 × 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.

Effect of different cryoprotectant agents on spermatogenesis efficiency in cryopreserved and grafted neonatal mouse testicular tissue.

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.

Comparison of sperm quality and DNA integrity in mouse sperm exposed to various cooling velocities and osmotic stress.

The first objective was to compare sperm quality following conventional manual sperm freezing (cryovials held 1, 2, 3, and 4 cm, respectively, above liquid nitrogen (LN(2)) for 10 min, resulting in cooling velocities of approximately -14.9, -10.1, -6.6, and -5.1 °C/min, respectively), and cooling velocities of -5, -20, -40, and -100 °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 LN(2) or in an automated freezer, 2 cm above LN(2) and -100 °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.

Fresh and frozen-thawed sperm quality, nuclear DNA integrity, invitro fertility, embryo development, and live-born offspring of N-ethyl-N-nitrosourea (ENU) mice.

Efficient collection, freezing, reliable archiving of sperm, and re-derivation of mutant mice are essential components for large-scale mutagenesis programs in the mouse. Induced mutations (i.e. transgenes, targeted mutations, chemically induced mutations) in mice may cause inherited or temporary sterility, increase abnormal sperm values, or decrease fertility. One purpose of this study was to compare the effect(s) on fresh and frozen-thawed sperm quality, spermatozoa DNA integrity, unassisted in vitro fertility (IVF) rate, in vitro embryo development rate to blastocysts, and live-born offspring rates in non-ENU (control) animals and the F1-generation of N-ethyl-N-nitrosourea (ENU)-treated male mice (765mg/kg C57BL6/J or 600mg/kg 129S1/SvImJ total dose). The second purpose was to determine the effect(s) of parental oocyte donor strain on in vitro fertilization, in vitro embryo development to blastocysts, and live-born offspring rates using sperm and unassisted IVF to re-derive animals from non-ENU control and ENU mice. Sperm assessment parameters included progressive motility, concentration, plasma membrane integrity, membrane function integrity, acrosome integrity, and DNA integrity. There were no significant differences in fresh sperm assessment parameters, DNA integrity, unassisted in vitro fertility rate, in vitro embryo development rate to blastocysts, and live-born offspring rates between non-ENU and C3B6F1/J or B6129S1F1/J ENU mice. In addition, there were no significant differences in frozen-thawed sperm assessment parameters and DNA integrity rates for non-ENU control and ENU C3B6F1/J or B6129SF1/J mice. In vitro fertilization and in vitro embryo development to blastocysts were effected from strain genetic variability (P<0.05). However, the cryopreservation process caused an increase of DNA fragmentation in non-ENU control and ENU C3B6F1/J or B6129S1F1/J hybrid mice compared to fresh control sperm (P<0.01). Unlike the combinations of hybrid sperm and hybrid oocyte, increasing frozen-thawed sperm DNA fragmentation decreased the embryo development rate to blastocyst compared to fresh sperm when C57BL6, C3H, or 129S inbred mice were used as oocyte donors (P<0.05).

Effects of cryopreservation on sperm quality, nuclear DNA integrity, in vitro fertilization, and in vitro embryo development in the mouse.

Efficient freezing, archiving, and thawing of sperm are essential techniques to support large scale research programs using mouse models of human disease. The purpose of this study was to investigate the effects of variable combinations and concentrations of cryoprotectants on sperm-assessment parameters of frozen-thawed mouse sperm in order to optimize cryopreservation protocols. Sperm was frozen using combinations of 3% skim milk + 0.2 or 0.3 M nonpermeating raffinose with either permeating glucose, fructose, propylene glycol, ethylene glycol, glycerol, or sodium pyruvate in CD-1, C3FeB6F1/J, B6129SF1, C57BL/6NCrIBR, 129S/SvPaslco, and DBA/2NCrIBR mice. Sperm-assessment parameters included progressive motility, plasma membrane integrity (SYBR-14 + PI), in vitro fertilization rate, and in vitro embryo development rate to blastocyst. DNA content analysis of sperm was measured by the sperm chromatin structure assay (SCSA). 0.3 M raffinose with 0.1 M fructose significantly improved post-thaw sperm-assessment parameters for CD-1, C3B6F1, B6129SF1 mice (P < 0.05-0.01), whereas 0.2 M raffinose with 0.1 M glycerol or 0.1 M fructose enhanced sperm assessment values for C57BL/6 and 129S mice (P < 0.01), compared to 0.3 M raffinose alone. DNA fragmentation during cryopreservation was significantly increased in all strains evaluated when compared with fresh control sperm in a strain-dependent manner (P < 0.01). Supplementation with permeating glycerol or fructose to the cryoprotectant (CPA) solution showed a significant protective effect to DNA integrity when cryopreserving sperm from C57BL/6 and 129S mice. Damage to sperm DNA significantly decreased the rate of in vitro embryo development to blastocyst in C57BL/6 mice. The type of monosaccharide sugar or polyols, CPA molarity, and combination of permeating and nonpermeating cryoprotectant are significant factors for improving progressive motility, plasma membrane integrity, DNA integrity, in vitro fertilization rate, and in vitro embryo development rate to blastocyst in cryopreserved mouse sperm.

Common carotid intima-media thickness measurement is not a pertinent predictor for secondary cardiovascular events after coronary bypass surgery. A prospective study.

OBJECTIVE: We aimed to assess the utility of common carotid intima-media thickness (CCA-IMT) to predict secondary cardiovascular events after coronary artery bypass grafting (CABG). In primary prevention, carotid-IMT is known as a valuable cardiovascular risk marker, but its interest in secondary prevention has been less studied. We hypothesized that CCA-IMT could be used for peri-operative and long-term risk stratification in candidates for CABG. METHODS: A total of 609 patients (66.8+/-9.2 years) were prospectively enrolled for preoperative CCA-IMT measurement and follow-up. The primary end-point combined cardiovascular death, non-fatal acute coronary syndromes, stroke, secondary coronary revascularization and peripheral arterial surgery during follow-up. The secondary end-point was the 1-month post-operative death. Univariate and multivariate analysis were performed by usual methods. RESULTS: A subgroup of 150 patients (24.6%) was individualized with a CCA-IMT above 90th percentile (>0.90 mm) or presenting plaques in their CCA. At 1 month, there was no significant difference in the prevalence of elevated CCA-IMT between deceased patients and survivors (16.7 vs. 24.9%, P=ns). During a mean follow-up of 41.8+/-16 months, 121 patients (19.8%) met the primary end-point. High CCA-IMT was predictive (OR=1.67, 95% CI 1.14-2.46, P=0.009) in the univariate analysis. In the multivariate analysis, age (OR=1.03, 95% CI 1.00-1.05, P=0.029) concomitant valvular surgery (OR=2.17, P=0.003) arrhythmia (OR=2.20, P=0.021), and peripheral arterial disease (OR=2.41, P<0.001) were significant independent prognostic factors whereas CCA-IMT failed to remain independently significant. CONCLUSIONS: Pre-operative CCA-IMT can provide prognostic information for candidates to CABG. However, clinical data present stronger prognostic values.

Effects of a GnRH agonist on oocyte number and maturation in mice superovulated with eCG and hCG.

The objective was to investigate the effects of a gonadotropin-releasing hormone agonist (GnRH) on ovulation rate and the number and maturation of oocytes in mice superovulated with equine chorionic gonadotropin (eCG) and human chorionic gonadotropin (hCG). Thirty 3-month-old BALB/C female mice (weight: 25-30 g) were assigned to three experimental groups: control, superovulated, and superovulated with GnRH pretreatment (n=10 per group). Control mice received an i.p. injection of 0.1 ml physiological saline solution. Superovulation was induced with 5 IU eCG (i.p.) and 5 IU hCG 48 h later. Mice in the superovulated with GnRH pretreatment group were given GnRH (20 mg/kg Fertirelin, i.m.), 24 h before superovulation. Thirteen hours after hCG administration, mice were sacrificed by cervical dislocation and blood samples were collected to determine serum progesterone concentration (by radioimmunoassay). Ovaries and oviducts were also harvested to enumerate corpora lutea and cumulus-enclosed oocytes. Progesterone concentrations were not significantly different among groups. The oocyte number and the maturation, ovulation rate, and the number of corpora lutea were higher in GnRH-treated mice than both controls and superovulated mice. In conclusion, GnRH given 24 h before superovulation with eCG-hCG increased the number and maturation of oocytes and the rate of ovulation in mice.