Effects of different extenders on epigenetic patterns and functional parameters of bull sperm during cryopreservation process.

The cryopreservation process induces alterations in cellular parameters and epigenetic patterns in bull sperm, which can be prevented by adding cryoprotectants in the freezing extenders. The purpose of this study was to compare the protective effects of two extenders based on soybean lecithin (SLE) and egg yolk (EYE) on epigenetic patterns and quality parameters of sperm such as motility parameters, mitochondrial membrane integrity, DNA fragmentation, viability, and apoptotic-like changes of bull sperm after cryopreservation. Results demonstrated that cryopreservation significantly (p < .05) reduced the level of DNA global methylation, H3K9 histone acetylation, and H3K4 histone methylation in both frozen groups compared to the fresh sperm. Also, the level of H3K9 acetylation was lower in the frozen SLE group (21.2 ± 1.86) compared to EYE group (15.2 ± 1.86). In addition, the SLE frozen group had a higher percentage of viability, progressive motility, and linearity (LIN) in SLE frozen group compared to EYE frozen group. However, no difference was observed in mitochondrial membrane integrity and DNA fragmentation between SLE and EYE frozen groups. While soybean-lecithin-based extender showed some initial positive impacts of epigenetics and semen parameters, further investigations can provide useful information for better freezing.

Effects of hydroxytyrosol on post-thaw quality of rooster sperm.

Semen cryopreservation is one of the most important reproduction techniques in the livestock and poultry industry. Cryopreservation induces cold stress, generating reactive oxygen species (ROS) and oxidative stress causing structural and biochemical damages in sperm. In this study, we evaluated the effects of the hydroxytyrosol (HT), as an antioxidant, at the concentrations of 0, 25, 50, and 100 µg/mL on post-thaw semen quality metrics in rooster. Semen samples were collected twice a week from 10 roosters (29 weeks), processed and frozen according to experimental groups. Different quality parameters, including total motility, progressive motility, viability, morphology, membrane integrity, and malondialdehyde were measured after thawing. Results showed that 25 and 50 µg/mL of HT produced the highest percentage of total motility (51.01 ± 2.19 and 50.15 ± 2.19, respectively) and progressive motility (35.74 ± 1.34 and 35.15 ± 1.34, respectively), membrane integrity (48.00 ± 2.18 and 46.75 ± 2.18, respectively) as well as viability (53.00 ± 2.17 and 52.50 ± 2.17, respectively) compared with the other groups (p < .05). The group with 25 µg/mL of HT showed the lowest significant (p < .05) MDA concentration (1.81 ± 0.25). Our results showed that the effect of HT was not dose-dependent and optimum concentration of HT could improve functional parameters of rooster sperm after freezing-thawing. These findings suggest that HT may have protective effects on the rooster sperm during the freezing-thawing process.

Zinc oxide nanoparticles preserve the quality and fertility potential of rooster sperm during the cryopreservation process.

Sperm cryopreservation is one of the main methods for preserving rooster sperm for artificial insemination (AI) in commercial flocks. Yet, rooster sperm is extremely susceptible to reactive oxygen species (ROS) produced during the freezing process. Oxidative stress could be prevented by using nanoparticles containing antioxidants. The present study was conducted to investigate the effect of zinc oxide nanoparticles (ZnONP) in rooster semen freezing extender on quality parameters and fertility potential. For this aim, semen samples were collected and diluted in Lake extenders as follows: control: Lake without ZnONP, ZnO100: Lake with 100-µg zinc oxide (ZnO), ZnONP50: Lake with 50-µg ZnONP, ZnONP100: Lake with 100-µg ZnONP and ZnONP200: Lake with 200-µg ZnONP. After freezing and thawing, sperm motility, viability, membrane integrity, morphology, mitochondrial activity, acrosome integrity, DNA fragmentation, lipid peroxidation and ROS, as well as fertility and hatchability were assessed. According to the current results, higher rates of motility, membrane integrity, mitochondrial activity, acrosome integrity and live cells were detected in the ZnO100, ZnONP50 and ZnONP100 groups compared to other groups (p ≤ .05). Yet, the percentage of dead cells, DNA fragmentation, lipid peroxidation and ROS levels were lower in the mentioned groups (p ≤ .05). Furthermore, a higher percentage of fertility was observed in the ZnO100 and ZnONP100 groups than in the control group (p ≤ .05). In conclusion, the use of 100-µg ZnO and 50- to 100-µg ZnONP represents a valuable and safe additive material that could be used to improve the quality and fertility potential of rooster sperm under cryopreservation conditions.

Gene expression profile of placentomes and clinical parameters in the cows with retained placenta.

BACKGROUND: Retained placenta (RP) is a prevalent disorder in cattle with many health-related and economic costs for the farm owners. Its etiology has not been clarified yet and there is no definite therapy for this disorder. In this study we conducted RNA-seq, hematologic and histologic experiments to survey the causes of RP development. METHODS: Blood samples were collected from 4 RP and 3 healthy cows during periparturtion period for hematological assessments followed by placentome sampling within 30 min after parturition. Cows were grouped as RP and control in case the placenta was retained or otherwise expelled, respectively. Total RNA was extracted from placentome samples followed by RNA-sequencing. RESULTS: We showed 240 differentially expressed genes (DEGs) between the RP and control groups. Enrichment analyzes indicated immune system and lipid metabolism as prominent over- and under-represented pathways in RP cows, respectively. Hormonal assessments showed that estradiol-17ß (E2) was lower and cortisol tended to be higher in RP cows compared to controls at the day of parturition. Furthermore, histologic experiment showed that villi-crypt junctions remain tighter in RP cows compared to controls and the crypts layer seemed thicker in the placentome of RP cows. Complete blood cell (CBC) parameters were not significantly different between the two groups. CONCLUSION: Overall, DEGs derived from expression profiling and these genes contributed to enrichment of immune and lipid metabolism pathways. We suggested that E2 could be involved in development of RP and the concentrations of P4 and CBC counts periparturition might not be a determining factor.

Proteomics study reveals the molecular mechanisms underlying cryotolerance induced by mild sublethal stress in human sperm.

The preconditioning of human sperm with sublethal nitrosative stress before cryopreservation can potentially improve the thawed sperm quality. However, the underlying mechanisms behind this protective strategy are not entirely understood. We compared the cryosurvival of human sperm exposed to 0.01 µM nitric oxide (NO) throughout the cryopreservation and used multiplexed quantitative proteomics approach to identify changes in the proteome profile of preconditioned sperm cells. Semen samples were obtained from 30 normospermia donors and then each sample was divided into three equal parts: fresh (F), frozen-control (C), and frozen exposed to nitric oxide (NO). The sperm undergoing mild sublethal stress showed higher values for motility and viability compared to the frozen control sperm. Moreover, out of 2912 identified proteins, 248 proteins were detected as differentially abundant proteins (DAPs) between cryopreserved groups and fresh group (F) (p < 0.05). Gene ontology (GO) analysis of differentially abundant proteins indicated that the abundance of proteins associated with glycolysis, gluconeogenesis, and fertilization processes was reduced while oxidative phosphorylation pathway was increased in abundance in cryopreserved sperm compared to the fresh sperm. Moreover, redox protein such as thioredoxin 17 was increased in abundance in the NO group compared to the control freezing group. Therefore, the pre-conditioning of sperm prior to cryopreservation may play an important role in maintaining the redox balance in mitochondria of sperm after freezing. Overall, our results indicate that arylsulfatase A (ARSA), serine protease 37 (PRSS37), and sperm surface protein (SP17) may potentially serve as protein biomarkers associated with screening the fertilization potential of the thawed sperm.

Preconditioning of bull semen with sub-lethal oxidative stress before cryopreservation: Possible mechanism of mitochondrial uncoupling protein 2.

Preconditioning of sperm using sub-lethal oxidative stress before cryopreservation is an innovative approach that can improve sperm cryo-survival. Mitochondrial uncoupling proteins (UCPs) are critical in reducing ROS level during stress conditions. The aim of the current study was to investigate whether mild sub-lethal stress induced by low concentrations of nitric oxide and hydrogen peroxide has a protective effect on quality parameters of post-thaw bull semen through modulations of mitochondrial uncoupling protein 2 (UCP2) expression. Semen samples were collected from 6 mature Holstein bulls, then mixed and divided into 8 aliquots: fresh, frozen control and frozen groups treated with NO: 0.1 (NO-0.1), 1(NO-1), 10 µM (NO-10), and H2O2: 0.1(H2O2-0.1), 1(H2O2-1) and 10µM (H2O2-10). A significantly higher percentage of total motility, progressive motility and viability was observed in NO-1 and H2O2-10 compared to the other frozen groups (P < 0.05). Sperm exposed to 1 µM NO and 10µM H2O2 showed significantly increased percentages of mitochondria activity and membrane integrity (P < 0.05). Moreover, the lowest percentage of apoptotic percentage was observed in the NO-1 and H2O2-10 in comparison to the other frozen groups. In addition, the expression level of UCP2 was higher in the NO-1 and H2O2-10 compared to the other groups (P < 0.05). It can be concluded that stress preconditioning of bull sperm before cryopreservation can increase UCP2 expression of sperm, that can play a protective role against cryoinjury after thawing.

Epigenetic modifications at DMRs of imprinting genes in sperm of type 2 diabetic men.

High rates of infertility in type 2 diabetic (T2DM) men have led to attempts to understand the mechanisms involved in this process. This condition can be investigated from at least two aspects, namely sperm quality indices and epigenetic alterations. Epigenetics science encompasses the phenomena that can lead to inherited changes independently of the genetics. This study has been performed to test the hypothesis of the relationship between T2DM and the epigenetic profile of the sperm, as well as sperm quality indices. This research included 42 individuals referred to the infertility clinic of Royan Institute, Iran in 2019-2021. The study subjects were assigned to three groups: normozoospermic non-diabetic (control), normozoospermic diabetic (DN) and non-normozoospermic diabetic (D.Non-N). Sperm DNA fragmentation was evaluated using the sperm chromatin structure assay technique. The global methylation level was examined using 5-methyl cytosine antibody and the methylation status in differentially methylated regions of H19, MEST, and SNRPN was assessed using the methylation-sensitive high-resolution melting technique. The results showed that the sperm global methylation in spermatozoa of D.Non-N group was significantly reduced compared with the other two groups (P < 0.05). The MEST and H19 genes were hypomethylated in the spermatozoa of D.Non-N individuals, but the difference level was not significant for MEST. The SNRPN gene was significantly hypermethylated in these individuals (P < 0.05). The results of this study suggest that T2DM alters the methylation profile and epigenetic programming in spermatozoa of humans and that these methylation changes may ultimately influence the fertility status of men with diabetes.

Supplementation of freezing medium with encapsulated or free glutathione during cryopreservation of bull sperm.

The objective was to compare effects of encapsulated or free glutathione (GSH) on the quality of frozen-thawed bull sperm. Ejaculates were collected via artificial vagina from six mature Holstein bulls once weekly for 6 weeks. All ejaculates had motility ≥70%, sperm concentration ≥1.0 × 109 /ml and ≤15% morphologically abnormal sperm. Each week, semen was pooled and diluted with lecithin-based extenders containing various concentrations of encapsulated (E0, E1, E2.5 and E5 mM) or free (F0, F1, F2.5 and F5 mM) GSH, with total glutathione content determined before and after cryopreservation. Total GSH in fresh semen was (mean+SEM) 4.8 ± 0.2 nmol/108 sperm, whereas in frozen-thawed semen of group F0 (control), it decreased to 1.4 ± 0.2 nmol/108 sperm, a 70.8% reduction (p < .05). In addition, total GSH in frozen-thawed semen from groups E2.5, E5 and F5 were 2.4 ± 0.2, 2.8 ± 0.2 and 1.8 ± 0.2 nmol/108 sperm, respectively (E5 versus. F0, p < .05). Compared to group F0, frozen-thawed sperm from group E2.5 had greater (p < .05) percentages of sperm that were viable (Annexin-V) (61.1 ± 1.8 versus. 71.1 ± 1.8) and that had cell membrane integrity (eosin-nigrosin) (64.5 ± 3.1 versus. 80.0 ± 3.1). Furthermore, frozen-thawed sperm from group E2.5 had the numerically highest total and progressive motility (CASA) and cell membrane functionality (HOS) and the lowest percentage of early apoptotic sperm (Annexin-V). However, acrosome membrane integrity (PSA) of E5 had the lowest mean (p < .05), whereas E2.5 caused a small nonsignificant decrease (69.1 ± 1.4%) compared to E0 and F0. In conclusion, 2.5 mM encapsulated GSH in semen extender significantly improved the quality of frozen-thawed bull sperm.

Membrane lipid replacement with nano-micelles in human sperm cryopreservation improves post-thaw function and acrosome protein integrity.

RESEARCH QUESTION: Membrane lipid replacement (MLR) of oxidized membrane lipids can restore sperm cellular membrane functionality and help improve surface protein stability during cryopreservation. What are the effects of MLR with nano-micelles made from a glycerophospholipid (GPL) mixture and cholesterol-loaded cyclodextrin (CLC), on the cryosurvival and expression of acrosome-related proteins in thawed human spermatozoa? DESIGN: Twenty samples were used to determine the optimum level of nano-micelles by incubation of semen with different concentrations of GPL (0.1 and 1%) and CLC (1 and 2 mg/ml) (including GPL-0.1, GPL-1, CLC-1, CLC-2, CLC-1/GPL-0.1, CLC-2/GPL-0.1, CLC-1/GPL-1 and CLC-2/GPL-1) before cryopreservation. Then, 30 semen samples were collected, and each sample was divided into the following three aliquots: fresh, frozen control and frozen incubated with optimum level of nano-micelles (0.1% GPL and 1 mg/ml CLC). RESULTS: CLC-1/GPL-0.1 and GPL-0.1 significantly increased motility parameters. CLC-1, GPL-0.1 and CLC-1/GPL-0.1 significantly improved viability rate compared with frozen control group. Significantly higher mitochondrial activity and acrosome integrity, and a lower rate of apoptosis, were observed in the CLC-1/GPL-0.1 compared with the frozen control group. The expression ratios of arylsulfatase A (ARSA), serine protease 37 (PRSS37), serine protease inhibitor Kazal-type 2 (SPINK2) and equatorin (EQTN) significantly increased compared with the frozen control group. CONCLUSIONS: Modification of membrane cholesterol and GPL mixtures in spermatozoa enhances their acrosome protein integrity by inhibiting early apoptotic changes and spontaneous acrosome reactions.

Evaluation of used Purslane extracts in Tris extenders on cryopreserved goat sperm.

This study aimed to evaluate the comparative effects of Purslane aqueous extract (PAE), Purslane methanolic extract (PME) and Purslane ethanolic extract (PEE on the quality of frozen-thawed goat spermatozoa. Collected semen with motility >75% and sperm concentration >1.0 × 109 sperm/ml was pooled and divided into 10 equal aliquots and supplemented by basic extender containing 25, 50 or 100 µg/ml of Purslane aqueous extract (PAE25µg/ml, PAE50µg/ml, PAE100µg/ml, respectively), basic extender containing 25, 50 or 100 µg/ml of Purslane methanolic extract (PME25µg/ml, PME50µg/ml, PME100µg/ml, respectively), basic extender containing 25, 50 or 100 µg/ml of Purslane ethanolic extract (PEE25µg/ml, PEE50µg/ml, PEE100µg/ml, respectively). Control diluent contained no additives. For the determination of sperm quality, frozen straws were thawed and then the sperm characteristics were assessed. Results indicated that higher (P < 0.05) percentages of total motility, viability, mitochondrial activity and lower percentages of malondialdehyde (MDA) for PAE50µg/ml, PME50µg/ml and PEE50µg/ml than those of the control. In addition, PME50µg/ml resulted in the highest) P < 0.05) total motility and the lowest (P < 0.05) MDA levels compared to other treatments. Compared to the control group, PME50µg/ml resulted in higher integrity (P < 0.05) of plasma membranes and in lower amounts of apoptotic and dead spermatozoa. PME50µg/ml and PAE50µg/ml showed higher (P < 0.05) percentages of progressive motility, DNA integrity and live post-thawed spermatozoa than those of the control. No significant differences in the motility, viability, mitochondrial activity and number of live sperms were observed between PME50µg/ml and PAE50µg/ml treatments. In conclusion, the results of this study indicated that 50 µg/ml purslane extracts could be used for the cryopreservation. However, the results of methanolic extract was more beneficial compared to other extracts.

Dietary L-carnitine affects the expression of genes involved in apoptosis and fatty acid metabolism in rooster testes.

Thirty-six 12-week-old breeder roosters (Ross 308) were randomly allocated into three groups to receive L-carnitine (LC): LC-0, LC-250 or LC-500 mg/kg of diet to evaluate the effects of dietary LC on the expression of apoptotic-related genes and desaturases and elongase mRNA transcript levels, in the cockerel testicles. Alteration of Bak (Bcl2 antagonist/killer), Bcl2, Cas3, Cas8, Cas9, Elovl2, Elovl4, Elovl5, Fads1, Fads2 and Scd expression at 24 and 34 weeks of age was compared by real-time quantitative PCR. The expression of Bcl2 and Elovl5 was significantly up-regulated (p < .05), while Cas8 expression (p < .05) and Bak/Bcl2 ratio were reduced (p < .02) in the cockerel testicles at 24 weeks of age. Although Bak mRNA abundance decreased by dietary LC, Bak/Bcl2 ratio was not affected by the treatments at 34 weeks of age. The expression of Cas3 was down-regulated, while Fads2 was up-regulated in the cockerel testicles by dietary LC at 34 weeks of age (p < .05). The results demonstrate the beneficial effects of LC supplementation in suppression of the Bak/Bcl2 ratio by altering Bak and Bcl2 mRNA abundance and, ultimately, prevention of apoptosis. Furthermore, LC increased the expression of Elovl5 and Fads2 genes which are involved in the metabolism of long chain fatty acids.

Inclusion of ovine enriched serum with vitamin E and polyunsaturated fatty acids in the freezing medium: a new strategy to improve human frozen-thawed sperm parameters.

The objective was to evaluate the effect of inclusion of 2.5% and 5% ovine serum, enriched with vitamin E (Vit E) and fish oil (FO), in human sperm freezing medium. Serum samples were prepared from sixteen rams (n = 4) feeding on a without supplemented diet, and diets supplemented with Vit E, FO and Vit E + FO. Semen samples, from 60 normozoospermic men, were frozen in: (I) a commercial freezing medium (SpermFreeze™; control medium), (II) the commercial freezing medium containing foetal bovine serum, (III) the commercial freezing medium + nonenriched serum (serum group), (IV) the commercial freezing medium + Vit E enriched serum (Vit E group), (V) the commercial freezing medium + FO enriched serum (FO group) and (VI) the commercial freezing medium + Vit E + FO enriched serum (Vit E + FO group). Sperm total and progressive motility, morphology, viability and plasma membrane integrity were significantly higher (p ≤ .05) in Vit E and Vit E + FO groups compared with the control group. Mitochondrial membrane potential did not differ between treatments (p > .05). It was concluded that ovine serum enriched with vitamin E and vitamin E + FO improved the quality of human spermatozoa but enriched serum containing FO could not improve the sperm cryo-injuries.

Preconditioning of sperm with sublethal nitrosative stress: a novel approach to improve frozen-thawed sperm function.

RESEARCH QUESTION: Can sublethal stress induced by nitric oxide on fresh human spermatozoa protect the functional properties of post-thaw human spermatozoa? DESIGN: Semen samples were obtained from 46 donors. Twenty semen samples were used to determine toxicity level of nitric oxide by incubation of semen with different concentrations of nitric oxide (0.01 to 400 µM). Then, 26 semen samples were cryopreserved with optimized ranges of nitric oxide: control (NO-0.00), 0.01 µM nitric oxide (NO-0.01), 0.1 µM nitric oxide (NO-0.1), 1 µM nitric oxide (NO-1), 10 µM nitric oxide (NO-10), 100 µM nitric oxide (NO-100). Frozen-thawed spermatozoa were assessed for motion characteristics, viability, morphology, apoptosis-like changes, caspase 3 activity, DNA fragmentation and intracellular reactive oxygen species levels. Fertilization potential was investigated by heterologous piezo-intracytoplasmic sperm injection (piezo-ICSI) of human spermatozoa into mouse oocytes. RESULTS: In fresh spermatozoa, nitric oxide did not induce a negative effect, except a significant reduction in motility and viability at 200 µM and 400 µM (P < 0.05). Cryopreservation significantly reduced sperm motility and increased reactive oxygen species, apoptosis-like changes, caspase 3 activity, and DNA damage (P < 0.05). NO-0.01 significantly increased total and progressive motility versus the other groups (P < 0.05). The lowest percentage of caspase 3 activity was in the NO-0.01 and NO-0.1 compared with the other freezing groups. In the fertilization trial, the rate of two-cell embryo formation after heterologous piezo-ICSI was higher (P < 0.05) in NO-0.01 (69%) versus controls (42%). CONCLUSIONS: Sublethal oxidative stress induced by nitric oxide might improve human sperm function after cryopreservation.

Pre-conditioning with Xanthine oxidase to improve post thawed quality of bull sperm.

The purpose of this study was to examine the effects of sub-lethal concentration of Xanthine oxidase (XO) on the post-thawed bull sperm quality. Semen samples were collected from four Holstein bulls, twice a week and during three consecutive weeks (n = 24 total ejaculates). After collection in each replicate, semen samples were pooled and then frozen by semen extender containing different concentrations [0 (XO-0), 0.05 (XO-0.05), 0.5 (XO-0.5), 5 (XO-5), 50 (XO-50) and 500 (XO-500) µM] of XO. After thawing, motion parameters (SCA), plasma membrane functionality (HOST), apoptosis status (Phosphatidylserine translocation assay), mitochondrial activity (Rhodamine 123), and acrosome integrity (PSA), were evaluated. The results showed that total motility, VAP, VSL, VCL, STR, and LIN were lower in XO-50 and XO-500 compared to other groups (P < 0.05). Progressive motility were higher in XO-0.05 and XO-0.5 compared to XO-0, XO-50, and XO-500 (P < 0.05). Mitochondrial activity was highest in XO-0.05 and XO-0.5 groups. Sperm plasma membrane functionality was significantly greater in XO-0, XO-0.05, XO-0.5, and XO-5 than that of XO-50 and XO-500. Xanthine oxidase had not significant effects on acrosome integrity and dead spermatozoa. Higher percentage of live spermatozoa was recorded for XO-0, XO-0.05, XO-0.5, and XO-5; however, the lower amount of apoptotic spermatozoa was detected in the aforementioned groups (P < 0.05). In conclusion, it seems that XO at lower doses may have beneficial effects on post-thawed bull sperm quality.

Dietary trans and saturated fatty acids effects on semen quality, hormonal levels and expression of genes related to steroid metabolism in mouse adipose tissue.

Our objectives were to assess sperm alteration and adipose tissue (AT) genes expression related to steroid metabolism subsequent to fatty acids consumption. Twenty-nine mature male mice were divided into: fat diet (FD; n = 15) and the control group (n = 14). FD group was fed with low level of trans and saturated fatty acids source for 60 days. Sperm parameters, levels of hormones and the mRNA abundance of the target genes in AT were assessed. The sperm concentration, total and progressive motilities were lower in FD group compared to that of control (p < 0.01). Blood estradiol levels increased in FD (p < 0.001), whereas no significant difference was observed in testosterone. The mRNA levels of StAR, CYP11A1, CYP17A1, 17ßHSD7 and 17ßHSD12 in AT of FD were higher than those of the control (p < 0.05). In contrast, mRNA level of Cyp19a1 in FD was significantly (p < 0.05) lower than that of control. 17ßHSD12 and 17ßHSD7 (as oestrogenic genes) increased, while 17ßHSD5 and 17ßHSD3 (as androgenic genes) remained unchanged, indicating that dietary trans/saturated fatty acids affect AT genes expression. Probably, sperm parameters were altered by increment of expression level of genes involved in oestrogenic metabolism rather than those engaged in androgenic metabolism after fatty acids consumption.

In vitro improvement of quail primordial germ cell expansion through activation of TGF-beta signaling pathway.

Avian primordial germ cells (PGCs) have valuable potentials to cell-based approaches for transgenic bird production. In this regard, improvement of avian PGC expansion in vitro is necessary. Among experimental avian species, quail is a good model for transgenic technology, especially due to its short generation time. In the present study, we have examined the proliferative effects of transforming growth factor ß (TGF-ß) on the quail PGCs. After isolation of quail PGCs from blood (Hamburger-Hamilton [HH stages 13-15]) and gonads (HH stages 28-30), these cells were cultured on quail embryonic fibroblasts (QEF). Our results indicated th at cultured gonadal-derived PGCs proliferated 400 times in comparison to 100 times for blood PGCs over 40-50 days. Upon in vitro exposure to TGF-ß inducers by Activin or the inducer of definitive endoderm 1 (IDE1) small molecule, the number of gonad PGCs significantly increased to 26% and 64%, respectively. In contrast, inhibition of the TGF-ß signaling pathway by SB431542 resulted in a significant reduction in the numbers of PGCs (P < 0.001). Moreover, Phosphorylation of SMAD2/3 in the IDE1 group was higher compared to the Activin-treated ones. We confirmed the PGC identification with periodic acid-Schiff (PAS) staining, anti-SSEA1, ß-catenin, ß-integrin, and Nanog immunofluorescence staining. Exogenously IDE1 treated-PGCs migrated toward the embryonic gonads after transplantation into the heart of the recipient embryo at HH stages 13-15. Our results suggested that the application of IDE1 small molecule into the culture of quail PGCs represented a step toward achieving efficient expansion of the avian PGCs.

Proteomic analysis and microtubule dynamicity of human sperm in electromagnetic cryopreservation.

The proteomic changes, microtubule dynamicity, and quality parameters of human sperm were investigated during cryopreservation in an extremely low electromagnetic field (ELEF) condition. Semen samples were obtained from 210 healthy individuals with normospermia and then were divided into three experimental groups: fresh control, frozen control, and frozen ELEF group. Shotgun proteomics was performed to assess the identification of microtubule proteins of the sperm in experimental groups. Microtubule dynamicity, secondary, and tertiary structure modifications of tubulins, characteristics of transmission electron microscopy of sperm as well as sperm quality parameters were evaluated. The expression ratios of α- and ß-tubulins were significantly increased after cryopreservation compared with fresh control while this ratio was not significantly different in frozen ELEF group. The expression ratio of tubulin polymerization-promoting protein was significantly decreased after cryopreservation compared with fresh control. The length, width, and the activity of microtubule, secondary, and tertiary structures of tubulins, motility, and the viability of the sperm were decreased in frozen control as compared with fresh control. The microtubule activity, secondary, and tertiary structures of sperm tubulin in frozen ELEF group were higher than frozen control. Transmission electron microscopy of microtubules showed that the size of the width and length of the microtubules in frozen ELEF group were greater than frozen control. Motility, viability, and reactive oxygen species levels were improved in frozen ELEF group when compared with frozen control. While the microtubule dynamicity of the sperm was affected by the cryopreservation, this trait was improved during the electromagnetic cryopreservation resulted in better motility and viability.

Sperm cryopreservation: A review on current molecular cryobiology and advanced approaches.

The cryopreservation of spermatozoa was introduced in the 1960s as a route to fertility preservation. Despite the extensive progress that has been made in this field, the biological and biochemical mechanisms involved in cryopreservation have not been thoroughly elucidated to date. Various factors during the freezing process, including sudden temperature changes, ice formation and osmotic stress, have been proposed as reasons for poor sperm quality post-thaw. Little is known regarding the new aspects of sperm cryobiology, such as epigenetic and proteomic modulation of sperm and trans-generational effects of sperm freezing. This article reviews recent reports on molecular and cellular modifications of spermatozoa during cryopreservation in order to collate the existing understanding in this field. The aim is to discuss current freezing techniques and novel strategies that have been developed for sperm protection against cryo-damage, as well as evaluating the probable effects of sperm freezing on offspring health.

Dietary fish oil supplemented with vitamin E improves quality indicators of rooster cold-stored semen through reducing lipid peroxidation.

Cockerel semen is sensitive to cooling, which limits chilled storage of semen for more than 24 h. Results of artificial insemination with cold-stored semen are not desirable. This study was conducted to evaluate the effects of dietary fish oil and vitamin E (vitE) for cold-storage of rooster semen and its effects on parameters of semen during 48 h cooling preservation. Roosters were assigned into four dietary treatments; 1) control group received a basal diet, 2) vitE group received a basal diet supplemented with 200 mg/kg vitE, 3) fish oil group (FO) received a basal diet supplemented with 2% fish oil and 4) fish oil and vitE group received a basal diet supplemented with 2% fish oil and 200 mg/kg vitE (FO + vitE). Semen samples were collected after 40 days of feeding and then diluted and cooled to 5 °C for preservation up to 2 days. Several quality indicators of sperm such as motion characteristics, membrane integrity, and viability, and abnormal morphology, activity of mitochondria, lipid peroxidation and acrosome integrity of the sperm were assessed at different times of storage (0, 24 and 48 h). None of sperm were significantly affected by the diets at the start of storage (0 h, p > 0.05). FO and FO + vitE improved the percentage of total motility, viability, and mitochondria activity at 24 h (P ≤ 0.05). After 48 h, only FO + vitE group produced the higher percentage of total motility, viability and membrane integrity (P ≤ 0.05). Lipid peroxidation was significantly reduced in sperm obtained from roosters fed diets of FO + vitE and vitE compared to FO and control (P ≤ 0.05) at times of 24 and 48 h. There was no significant difference between control and vitE groups in none of parameters (P > 0.05). Integrity of acrosome and abnormal morphology were not significantly affected by the diets (P > 0.05). Supplementation of roosters' diet with 2% fish oil and 200 mg/kg vitamin E improved the quality of cold-stored semen by supporting several indicators of sperm quality through reducing lipid peroxidation.

Fertility response of artificial insemination methods in sheep with fresh and frozen-thawed semen.

The aim of this study was to evaluate the fertility response of artificial insemination (AI) methods with fresh and frozen sperm in sheep. In experiment 1, one hundred and fifty fat tailed Zandi ewes were assigned into 3 equal groups and inseminated with three AI methods consisting of vaginal, laparoscopic and trans-cervical AI with fresh semen. In experiment 2, a factorial study (3 AI methods × 2 extenders) was used to analyze the effects of three AI methods and two freezing extenders containing soybean lecithin (SL) or Egg yolk (EY) on reproductive performance of 300 fat tailed Zandi ewes. Also, total motility, progressive motility, viability and lipid peroxidation of semen were evaluated after freeze-thawing in two extenders. In result, there was no significant difference among three AI methods when fresh semen was used. In experiment 2, the highest percentage of pregnancy rate, parturition rate and lambing rate were obtained in laparoscopic AI group (P < 0.05). Although pregnancy rate, parturition rate and lambing rate in trans-cervical group were higher (P < 0.05) than vaginal group, the results were not as high as laparoscopic group. No difference was observed between SL and EY extenders and their performance was close to each other. It can be concluded that although no difference was observed on reproductive performance for fresh semen, trans-cervical AI was more efficient than vaginal method when frozen-thawed semen was used, but its efficiency was not as high as laparoscopic method. Also, SL extender can be an efficient alternative extender to preserve ram sperm during cryopreservation procedure without adverse effects of EY.