Preservation of rooster post-thawed sperm epigenetic modifications, fertility potential and other quality parameters in different extenders using reduced glutathione.

Although rooster semen cryopreservation is an efficient procedure to spread qualified semen samples for reproductive goals, some post-thawed qualified semen samples resulted in poor fertility rate that could be related to epigenetic modifications during the cryopreservation process. This research was conducted to investigate the effect of reduced glutathione (GSH) in different cryopreservation extenders (Lake and Beltsville) on preservation of epigenetic modifications, fertility potential and other quality parameters of rooster sperm after thawing. Semen samples were collected and diluted in Lake and Beltsville extenders as follows: L-0: Lake without GSH, L-G: Lake with GSH, B-0: Beltsville without GSH, and B-G: Beltsville with GSH. After freeze-thawing process, sperm motility, membrane functionality, mitochondrial activity, acrosome integrity, viability, apoptosis status, lipid peroxidation, DNA fragmentation, ROS concentration, epigenetic modifications and fertility potential were evaluated. In results, the type of extender had no effect (P > 0.05) of post-thawed sperm quality. The treatments containing GSH presented higher (P ≤ 0.05) total motility, progressive motility, membrane functionality, mitochondrial activity, acrosome integrity, viability, DNA methylation, fertility as well as lower (P ≤ 0.05) lipid peroxidation, apoptosis, DNA fragmentation and ROS concentration than other treatments. Extender supplementation with GSH had no effect (P > 0.05) on histone methylation, histone acetylation and hatching rate. In conclusion, supplementation of rooster sperm cryopreservation extender with GSH could be an effective strategy to preserve post-thawed sperm DNA methylation, fertility and other quality parameters during reproductive programs.

Supplementation of sperm cooling medium with Zinc and Zinc oxide nanoparticles preserves rooster sperm quality and fertility potential.

Supplementation of sperm cooling medium with helpful additives is a reasonable method to conserve sperm fertility potential during cooling storage process. This study was aimed to determine the effect of sperm cooling medium supplementation with Zinc and Zinc oxide nanoparticles (NZn and NZnO) on rooster semen quality and fertility efficiency during storage periods. Semen samples were diluted in the Lake medium and assigned into five equal aliquots. The first was Control group and the other groups received 50 µg/ml NZn, 50 µg/ml NZnO, 100 µg/ml NZn and 100 µg/ml NZnO. Then, the samples were cooled at 5 °C and conserved up to 45 h. Total motility, progressive motility, mitochondrial activity, viability, membrane integrity and lipid peroxidation of samples were analyzed during 0, 22 and 45 h post-cooling. Artificial insemination was also performed using 22- hrs cooled semen. No difference was found among groups during quality evaluations at 0 h storage. Extender supplementation with 100 µg/ml NZn and NZnO presented higher (P ≤ 0.05) total motility, progressive motility, mitochondrial activity, viability, membrane integrity and lower lipid peroxidation compared to other groups during 22 and 45 h cooling storage. Fertility rate of 22-h cooled-stored semen samples was higher (P ≤ 0.05) in groups contained 100 µg/ml NZn and NZnO compared to the Control group. In conclusion, addition of 100 µg/ml NZn and NZnO to the sperm storage medium could be introduced as an effective method to preserve rooster semen quality during cooling storage period.

Cysteamine enhances quality and fertility potential of rooster semen in cooled storage.

This study investigated the effects of supplementing Lake extender with cysteamine (CYS) on rooster semen quality in cold storage and it's fertility performance. Semen samples were diluted with Lake extender supplemented with different concentrations of CYS (0, 1, 2, 4 and 8 mM) and were cooled and stored at 5 °C for a period of 46 h. Motility, membrane functionality, viability, lipid peroxidation, and mitochondria membrane potential were evaluated at 0, 23 and 46 h of storage. Fertility was assessed at 23 h of storage. Although at the beginning time (0 h), parameters were not affected, 1 mM of CYS improved (P ≤ 0.05) total motility, progressive motility and mitochondria membrane potential during 23 and 46 h storage. Moreover, 1 and 2 mM CYS improved (P ≤ 0.05) membrane functionality and viability compared to other groups. Lipid peroxidation was lower (P ≤ 0.05) in samples diluted with 1 and 2 mM CYS compared to the others. Artificial insemination with 23-hrs cooled-stored semen produced the higher (P ≤ 0.05) fertility rate in groups received 1 and 2 mM CYS compared to the control group. In conclusion, addition of 1 and 2 mM CYS to the extender could be helpful to protect rooster semen against structural and functional damages of cooling storage process.

A Non-cutaneous Form of Melanoma in a Goat during Meat Inspection: a Case Report.

Malignant melanoma is a neoplasm that originates from melanocytes. This tumor is observed in cutaneous and non-cutaneous forms, and it is considered one of the most life-threatening types of cancers. Non-cutaneous melanoma is a complex of unique and malignant complications that are easily separable from cutaneous type. Since the ultraviolet radiation from the sun damages DNA and is an oxidative stress factor in melanoma and there are more melanocytes in the basal layer of skin than other parts of the body, the cutaneous form has more prevalence. Most of the time, non-cutaneous form is the result of cutaneous metastasis but both forms can occur primarily. Furthermore, non-cutaneous form usually happens in mucosal layers, intestines, and eyes; moreover, the main reasons are ectopic melanocytes or their unwanted regressive growing. Malignant melanoma can occur in all domestic animals; however, they seem to be rare in sheep and goats. Herein, we describe a rare case of the primary non-cutaneous form of malignant melanoma in a three-year-old indigenous female goat. During meat inspection procedures in a slaughterhouse in Tabriz, Iran, we encountered numerous round firm black masses on visceral surfaces and serous membranes of the abdominal and thoracic cavities. The liver and lungs were prominently affected. Samples were taken from involved parts, and malignant melanoma was confirmed in the histopathological examination due to pleomorphism and polymorphism and melanin pigments in cells with eosinophilic cytoplasm. According to what was stated in the "manual on meat inspection for developing countries", the carcass was not convenient for human use and condemned by the inspector.

Modified polyethyleneimine with histidine-lysine short peptides as gene carrier.

There are several strategies that can be utilized to improve transfection efficiency while reducing the cytotoxicity of polyethyleneimine (PEI) as a promising non-viral gene delivery vector. In this study, we evaluated the potential use of lysine-histidine (KH) peptides in modifying the PEI 10 kDa structure and enhancing its efficiency while maintaining low toxicity of PEI. PEI 10 kDa was modified with 6-bromohexanoic acid (alkyl) to increase its lipophilicity. Then, ethylenediamine (EDA) was attached to the carboxylic groups of PEI-hexanoate to restore the primary amines of PEI. Subsequently, six different KH short peptides were conjugated to PEIs and evaluated for the effect of the KH sequence on vector transfection efficiency and cytotoxicity. The transfection efficiency of PEI-peptides complexed with a luciferase reporter gene (pRLCMV) in Neuro-2A murine neuroblastoma cells showed that the PEI conjugated to KHHHKKHHHK peptide had a significantly higher rate of gene transfection efficiency in comparison with other KH peptides. This peptide was conjugated to PEI-alkyl and PEI-alkyl-EDA and significant improvement in efficiency with minimal cytotoxicity was observed. The results obtained suggest that the sequence and content of KH peptides will have a significant impact on the transfection efficiency of modified PEI 10 kDa.

Adenoviral gene delivery to solid tumors by recombinant silk-elastinlike protein polymers.

PURPOSE: The purpose of this study was to investigate the potential of silk-elastinlike protein polymers (SELPs) in controlling the release rate of adenoviruses in vitro and in vivo while preserving their bioactivity. MATERIALS AND METHODS: A hydrogel system composed of SELP/adenovirus mixture was prepared. The release of the adenovirus particles from the hydrogels was quantified by Real Time-PCR and the bioactivity of the released viruses was evaluated using confocal microscopy and beta-galactosidase assay. To demonstrate the ability of SELP in entrapping virus cargo and releasing it over a prolonged period of time in vivo, a SELP/adenovirus mixture was prepared and injected directly into xenograft tumor models of breast and head and neck cancer in mice. At various time points mice were sacrificed, tumors dissected, and tissue sections studied under confocal microscope. RESULTS: In vitro studies demonstrated that SELP hydrogels release viruses over a period of 4 weeks while preserving their bioactivity. After intratumoral injection, a prolonged and localized expression of adenoviruses was observed. CONCLUSIONS: These results suggest the potential of SELPs in local adenoviral delivery to solid tumors as an alternative approach to intratumoral virus infusion.

Recombinant polymer-protein fusion: a promising approach towards efficient and targeted gene delivery.

BACKGROUND: Synthetic vectors such as polymers have the potential to reduce the safety problems associated with viral vectors; however, their low transfection efficiency limits their clinical utility. To study the critical steps involved in an efficient transgene expression, there is a need for creative approaches that allow a systematic correlation between gene carrier structure and properties necessary for successful gene transfer. Using recombinant techniques a prototype vector comprised of tandem repeating units fused to a targeting moiety was biosynthesized to mediate gene transfer in mammalian cell lines. The carrier was designed to have the structure of (KHKHKHKHKK)6-FGF2 where lysine (K) residues would allow complexation with plasmid DNA, basic fibroblast growth factor (FGF2) to target cells over-expressing FGF2 receptors (FGFR), and histidine (H) residues to facilitate escape from the endosomal compartments. METHODS: The gene carrier was biosynthesized in E. coli, purified using a Ni-NTA column, characterized, complexed with pDNA, and the complexes were used to transfect NIH 3T3, T-47D and COS-1 mammalian cell types known to express FGFR. RESULTS: Results demonstrate the successful cloning and expression of the gene carrier with over 95% purity. The molecular weight of the gene carrier was determined by MALDI-TOF to be 27 402. Amino acid content analysis and Western blot confirmed the expression of the gene carrier in E. coli. The vector was able to condense pDNA, induce cell proliferation in NIH 3T3 fibroblasts, and mediate transgene expression in NIH 3T3, T-47D and COS-1 mammalian cell types. CONCLUSION: Genetic engineering techniques show promise for systematic investigation of structure-activity relationships of non-viral gene delivery vectors.

Recombinant polymers for cancer gene therapy: a minireview.

A major challenge for successful cancer gene therapy is the development of safe and effective gene delivery vectors. Gene delivery vectors can be viral or nonviral. Among nonviral vectors various polymeric vectors have shown potential in gene delivery. However, much work needs to be done in order to correlate polymer structure with gene release at the target site and transfection efficiency. This article is a brief introduction into cancer gene therapy, barriers and methods for gene transfer with emphasis on the applications of recombinant polymers for cancer gene therapy.

A biodegradable injectable thermoplastic for localized camptothecin delivery.

Camptothecin is an example of a potent drug with a short half-life that would benefit from a localized drug depot system that maintains its stability prior to being released. For this reason, a thermoplastic, biodegradable polymer drug depot was prepared and characterized, and the in vitro release of camptothecin examined. epsilon-Caprolactone oligomers were prepared by ring-opening polymerization initiated by various alcohols. The polymers were characterized via differential scanning calorimeter (DSC) for thermal transitions, and via a parallel plate rheometer for melt viscosity. Camptothecin was loaded into the oligomers and released into PBS buffer. The viscosity of the oligomers was alterable by the initiator used. The oligomers were semi-crystalline with melting points between 37 and 45 degrees C. Camptothecin was released from the oligomers in a diffusion-controlled manner, with the release rate increasing as the melt viscosity of the oligomer decreased. The unreleased camptothecin remained in its active lactone form for a period of up to 16 weeks.

Development of biodegradable injectable thermoplastic oligomers.

Injectable thermoplastic oligomers represent a promising biomaterial for drug delivery provided they possess a melting point at or very near physiologic temperature, as well as a low melt viscosity. One approach would be to prepare an oligolactone. In this paper, we examine the role of different alcohol initiators used in the ring-opening polymerization of epsilon-caprolactone oligomers on the melting point and melt viscosity of the resultant thermoplastics. We found that the initiator used plays a significant role in the final properties of the final oligomer. For primary alcohols, the longer the chain length of the oligomer the lower its melt viscosity, until a chain length of 8 carbons, after which there was no noticeable effect. There was no significant effect observed of primary initiators on the melting point. The use of secondary alcohols produced oligomers with higher viscosities but with reduced overall crystallinity. The use of an unsaturated alcohol, oleyl alcohol, not only reduced the melting point and overall crystallinity but also reduced the melt viscosity of the oligomer. The oleyl alcohol initiated oligomer appears to be a promising vehicle for localized, sustained drug delivery applications.

Camptothecin delivery methods.

Camptothecin has shown significant antitumor activity to lung, ovarian, breast, pancreas, and stomach cancers. Camptothecin, however, like a number of other potent anticancer agents such as paclitaxel, is extremely water insoluble. Furthermore, pharmacology studies have determined that prolonged schedules of administration given continuously are required. Thus, this insolubility has restricted its clinical application. For these reasons, a number of water-soluble analogs have been synthesized and a number of different formulation approaches have been investigated. In this review, we examine each of these approaches and discuss their advantages and limitations.

Biodegradable injectable in situ forming drug delivery systems.

The ability to inject a drug incorporated into a polymer to a localized site and have the polymer form a semi-solid drug depot has a number of advantages. Among these advantages is ease of application and localized, prolonged drug delivery. For these reasons a large number of in situ setting polymeric delivery systems have been developed and investigated for use in delivering a wide variety of drugs. In this article we introduce the various strategies that have been used to prepare in situ setting systems, and outline their advantages and disadvantages as localized drug delivery systems.