Can roscovitine and trichostatin A be alternatives to standard protocols for cell cycle synchronization of ovine adult and foetal fibroblast cells?

Synchronization of donor cells is an important step for the success of somatic cell nuclear transfer application and facilitates the development of embryos. Contact inhibition, serum starvation and different chemical agents are used in synchronizing different types of somatic cells. In this study, to synchronize the primary ovine adult (POF) and foetal (POFF) fibroblast cells to G0/G1 phases, the contact inhibition, the serum starvation, roscovitine and trichostatin A (TSA) methods were used. In the first part of the study, roscovitine (10, 15, 20 and 30 µM) and TSA (25, 50, 75 and 100 nM) were applied for 24 h to determine the optimal concentration for POF and POFF cells. In the second part, optimal concentrations of roscovitine and TSA for these cells were compared with contact inhibition and serum starvation methods. Cell cycle distribution and apoptotic activity analysis were performed by flow cytometry to compare this synchronization methods. Serum starvation method resulted in higher cell synchronization rate in both cells compared to other groups. Although contact inhibition and TSA also achieved high success rates of synchronized cell value, it was observed that the difference between serum starvation and these groups was significant (p < .05). When the apoptosis rates of the two cell types were examined, it was observed that the early apoptotic cells in contact inhibition and late apoptotic cells in the serum starvation were higher than the other groups (p < .05). Although the 10 and 15 µM concentrations of roscovitine gave the lowest apoptosis rates, it was observed that it failed to synchronize both the ovine fibroblast cells to G0/G1 phase. As a result, it was concluded that while roscovitine was not successful to synchronize both the POFF and POF cell lines, TSA (50 nM for POF cells and 100 nM for POFF cells) can be used efficiently as an alternative to the contact inhibition and the serum starvation methods.

Effects of triton X-100 pretreatment of lyophilized and frozen-thawed ram sperm on preimplantation embryo developmental competence.

In this study, it was aimed to determine the effect of destruction of lyophilized and frozen-thawed ram sperm plasma and acrosomal membrane on development of embryos produced by intracytoplasmic sperm injection (ICSI). Semen samples were divided into two groups for lyophilization (L) and freezing (F). For the removal of the plasma membrane, L and F groups were incubated with Triton X-100 (LTX-100 and FTX-100, respectively). Integrities of the plasma membrane, acrosome and chromatin structure were evaluated. Oocytes were injected with these sperm groups. Although no plasma membrane and acrosome integrities of the L (0.0%) group were detected, the plasma membrane integrity of the F group (69.4%) was significantly higher than the FTX-100 group (23.6%) (p < 0.05). The acrosome integrity of the FTX-100 group (3.80%) was significantly lower than the F group (55.6%) (p < 0.05). The chromatin integrities of L and F groups were higher than the Triton X-100 treated groups (p < 0.05). ICSIs with L, LTX-100, F and FTX-100 sperm were produced similar cleavage and blastocyst rates. In conclusion, data presented here confirm that ram spermatozoa can effectively be lyophilized and injected into oocytes for initiation of embryonic development and Triton X-100 pretreatment is not necessary while using lyophilized and frozen semen.

Relationship between phosphorylamine-modification and molecular weight on transfection efficiency of chitosan.

The aim of this study is both to design the chitosan (Chi) nanoparticles with different Mw containing the phosphoester bonds and increase their amino function for the transfection. The phosphorylamine-modification of Chi and depolymerized Chi (DChi) was realized using o-phosphorylethanolamine (o-PEA) and characterized, for the first time. The nanoparticles (nMChi and nMDChi) were prepared by ionic gelation and their particle size, polydispersity index (PDI), zeta potential, stability, gene binding capacity and cytotoxicity were examined. The effects of the Mw of Chi on the cytotoxicity, gene binding capacity, and in vitro transfection efficiency of the nanoparticles on Human Embryonic Kidney 293 (HEK293) cells were also examined. Green Fluorescent Protein Circular Plasmid DNA (pEGFN1) loaded nanoparticles (gnMChi and gnMDChi) were used in the transfection. This study showed that the Mw of phosphorylamine-modified Chi significantly affected the characteristics, cytotoxicity, gene binding capacity and transfection efficiency of the nanoparticles.

Evaluation of synergic effects of iodixanol and trehalose on cryosurvival of electroejaculated ram semen.

The primary aim of the study was to investigate whether iodixanol and trehalose would have a synergic effect on the cryosurvival of electroejaculated ram semen. Tris-based diluter was used to prepare 9 different extenders by the addition of iodixanol or trehalose alone or varying combinations of these substances. Diluters were prepared as follows: Tris (control), Io5 (5% iodixanol), Tr25 (25 mmol/L trehalose), Tr50 (50 mmol/L trehalose), Tr50 + Io1.25 (50 mmol/L trehalose and 1.25% iodixanol), Tr50 + Io2.5 (50 mmol/L trehalose and 2.5% iodixanol), Tr50 + Io5 (50 mmol/L trehalose and 5% iodixanol), Tr25 + Io5 (25 mmol/L trehalose and 5% iodixanol) and Tr12.5 + Io5 (12.5 mmol/L trehalose and 5% iodixanol). Supplementation of the freezing extender with trehalose or iodixanol alone supported the protection of both morphological and functional integrity of ram spermatozoa and total motility at 1 and 4 hr post-thawing respectively. However, beyond these positive effects, the combination of trehalose (25 mmol/L) and iodixanol (5%) significantly increased post-thaw sperm longevity and motion properties at the end of 4-hr incubation. The results of the study clearly showed that there was positive synergic effect of iodixanol and trehalose on cryosurvival of ram semen.

Effect of the linear aliphatic amine functionalization on in vitro transfection efficiency of chitosan nanoparticles.

The aim of this study is to prepare the long linear aliphatic amine pendant group-functionalized chitosan based nanoparticulate gene carrier system with improved properties for the efficient transfection. The amine functionalized chitosan (MChi) was synthesized by using N-(2-hydroxyethyl)ethylenediamine (HE-EDA) and characterized for the first time. The nanoparticles of MChi (nMChi) were prepared by ionic gelation method, and their particle size, polydispersity (PDI), zeta potential (mV), gene binding capacity and cytotoxicity were determined. Green Fluorescent Protein circular plasmid DNA (pEGFN1) loaded nanoparticles (gnMChi) were used in the transfection studies. The results showed that nMChi with a particle size of 102.9 nm and zeta potential of 41.9 ± 5.63 mV was non-toxic, had high transfection efficiency in Human Embryonic Kidney 293 and Primary Ovine Fibroblast cell lines and would be used as an efficient gene carrier system.

Administration time of misoprostol affects fertility rate in artificially inseminated Kivircik ewes with frozen-thawed ram semen.

The aim of this study was to determine the effects of the administration time of misoprostol (11 h (Miso11) and 6 h (Miso6) before artificial insemination) on fertility rates in Kivircik ewes (control: n = 41, Miso11: n = 32 and Miso6: n = 33) during breeding season. Artificial insemination (AI) was performed 48 h after sponge removal using frozen-thawed semen (150 million sperm per dose in 0.25 ml straws). Estrus synchronization parameters (onset and duration) and lambing rate were evaluated. No significant difference was observed among groups for the estrus onset and duration hours (P > 0.05). The lambing rates in the control, Miso11 and Miso6 groups were 39.0, 62.5 and 54.5%, respectively. There were significant differences among the control, Miso11 and Miso6 groups according to lambing rates (P < 0.05). In conclusion, misoprostol treatment significantly improved fertility in ewes when using frozen-thawed semen in AI. Administration of misoprostol 11 h before AI resulted in a higher lambing rate than that at 6 h before AI; therefore, treatment of misoprostol 11 h before AI can effectively be used.

The effects of the thiolation with thioglycolic acid and l-cysteine on the mucoadhesion properties of the starch-graft-poly(acrylic acid).

The aim of this study is to investigate the effects of the thiolation on the mucoadhesion characteristics of the gelatinized and crosslinked wheat starch-graft-poly(acrylic acid) [(WS-g-PAA)gc] for potential use in drug delivery via vaginal route. Thiolation of (WS-g-PAA)gc was first time realized using l-cysteine hydrochloride monohydrate (CyS) and thioglycolic acid (TGA). These conjugates [(WS-g-PAA)gcth] were characterized using FTIR. The free SH group, mucoadhesion, cytotoxicity characteristics and the mechanism of the thiolation were also evaluated. To obtain fundamental data for possible application such as drug carrier, in vitro and in vivo progesterone release profiles from the mucoadhesive tablet formulations were also determined. The results showed that, vaginal tablet containing (WS-g-PAA)gc-TGA, which has not contain free SH groups in its structure, displays higher mucoadhesion than (WS-g-PAA)gc and (WS-g-PAA)gc-CyS. This tablet formulation can also be used as a drug carrier in vaginal applications.

Development of starch based mucoadhesive vaginal drug delivery systems for application in veterinary medicine.

The aim of this study was to prepare and evaluate the mucoadhesive, biocompatible and biodegradable progesterone containing vaginal tablets based on modified starch copolymers for the estrus synchronization of ewes. Starch-graft-poly(acrylic acid) copolymers (S-g-PAA) were synthesized and characterized. The vaginal tablets were fabricated with S-g-PAA and their equilibrium swelling degree (Qe) and matrix erosion (ME%) were determined in lactate buffer solution. In vitro, mucoadhesive properties of the tablets were investigated by using ewe vaginal mucosa and in vivo residence time were also investigated. In vitro and in vivo progesterone release profiles from the tablets were compared with two commercial products. Tablet formulation containing wheat starch based grafted copolymer (WS-g-PAA)gc indicated promising results and might be convenient as an alternative product to the commercial products in veterinary medicine.

Comparison of cryoprotective effects of iodixanol, trehalose and cysteamine on ram semen.

This study was conducted to improve cryosurvival of electroejaculated (EE) ram semen in the presence of iodixanol (OptiPrep™), trehalose or cysteamine. A tris-based extender was used to prepare 12 extenders containing OptiPrep™ (Op), trehalose (Tr) or cysteamine (Cy) alone, or different combinations of these compounds. Extenders were designated as follows: Tris (control), Op1.25 (1.25% Op, v/v), Op2.5 (2.5% Op, v/v), Op5 (5% Op, v/v), Tr50 (50mM Tr), Tr100 (100mM Tr), Cy (5mM Cy), OpTr (2.5% Op and 100mM Tr), OpCy (2.5% Op and 5mM Cy), TrCy (100mM Tr and 5mM Cy), OpTrCy1 (2.5% Op, 100mM Tr and 5mM Cy) and OpTrCy2 (1.25% Op, 50mM Tr and 2.5mM Cy). A two-step dilution was used and glycerol was added at 5°C in the second step. Diluted samples were equilibrated for 1h, loaded in 0.25mL straws and frozen in a programmable freezing machine. Supplementation of 5% OptiPrep™ significantly protected post-thaw progressive motility, membrane integrity, acrosomal integrity and morphological damages. Trehalose supplementation protected membrane integrity of ram sperm; however, it did not help post-thaw motility and morphology. Supplementation of 5mM cysteamine had detrimental effect on cryosurvival of EE ram semen. These results demonstrate that the supplementation of iodixanol increases the cryosurvival of EE ram semen in a dose-dependent manner.

Using cell banks as a tool in conservation programmes of native domestic breeds: the production of the first cloned Anatolian Grey cattle.

The aim of this study was to clone native Anatolian Grey cattle by using different donor cell types, such as fibroblast, cartilage and granulosa cells cryopreserved in a gene bank and oocytes aspirated from ovaries of Holstein cows as the recipient cytoplasm source. One male calf from fibroblast, three female calves from granulosa cells and one female calf from cartilage cells were born healthy and at normal birthweights. No calves were lost after birth. The results demonstrated that the cloned calves had the same microsatellite alleles at 11 loci as their nuclear donors. However, the mtDNAs of the five Anatolian Grey cloned calves had different haplotypes from their donor cells and mtDNA heteroplasmy could not be detected in any of the clones. The birth of healthy clones suggests that the haplotype difference between the cell and oocyte donor did not affect the pre- or post-implantation development of the bovine nuclear transfer derived embryos in our study. The results showed that well established nuclear transfer protocols could be useful in conserving endangered species. In conclusion, somatic cell banking can be suggested as a tool in conservation programmes of animal genetic resources.

Effects of estrous cycle stage and transport temperature of ovaries on in vitro maturation of canine oocytes.

Unlike other domestic animals, in vitro maturation (IVM) of canine oocytes still has limited success. The present study investigated the effects of estrous cycle stage and transport temperature of ovaries on in vitro maturation of canine oocytes. The donor bitches were categorized into three groups based on stage of estrus cycle: follicular (proestrus or estrous), luteal (diestrus) and anestrus. One ovary of each pair collected from 39 mature bitches was transported in Phosphate Buffer Saline (PBS) at 4 degrees C while the other was transported at 37 degrees C. A total of 1138 Grade I COCs obtained from all ovaries were grouped and matured in modified synthetic oviduct fluid (mSOF) supplemented with follicle stimulating hormone (FSH), luteinizing hormone (LH), essential and non-essential amino acids at 38.5 degrees C in a humidified 5% CO(2), 5% O(2), and 90% N(2) atmosphere for 72 h. The nuclear maturation rates were evaluated by aceto-orcein staining. Oocytes harvested from follicular and luteal ovaries have a significantly higher maturation rates (MI+MII) than the oocytes from anestrual ovaries in the 37 degrees C group (p<0.05). However, oocytes harvested from anestrual ovaries transported at 4 degrees C had the highest maturation (MI+MII) rate, and the difference between anestrual and luteal ovary groups was significant (p<0.05). The oocytes from anestrual ovaries transported at 4 degrees C have significantly higher maturation rates than those transported at 37 degrees C (p<0.0001). However, the transport temperature (37 or 4 degrees C) did not significantly affect the maturation (MI+MII) rates of oocytes harvested from the luteal (p=0.61) and follicular (p=0.48) stage ovaries. It can be concluded from this study that (1) both transport temperature and transport temperaturexestrus cycle stage interaction effected the maturation rates, while estrus cycle stage alone did not, and (2) transporting canine ovaries at 4 degrees C can improve in vitro maturation rates in oocytes harvested from anestrous ovaries.

Developmental competence of domestic cat oocytes from ovaries stored at various durations at 4 degrees C temperature.

Temporal storage of ovaries can provide opportunity to rescue oocytes from ovaries of endangered felids. The objective of the study was to examine the effect of different storage periods (2, 24 and 48h) of ovaries at 4 degrees C for maturation of cat oocytes in vitro. Ovaries were collected from 25 domestic cats at various stages of the estrous cycle by routine ovariohysterectomy following anesthesia at different local veterinary clinics, and maintained in physiological saline at 4 degrees C for 2, 24 or 48h until oocytes recovery. Selected COCs were maturated at 38 degrees C for 48h in four-well petri dishes, which included 500microL modified synthetic oviduct fluid (mSOF) medium under mineral oil in a humidified 5% CO(2), 5% O(2), and 90% N(2) atmosphere incubator. After the in vitro maturation period, there were no differences between the rate of oocytes matured at MII stages in 2 and 24h storage groups (50.7% and 48.2% respectively, p>0.05). However, the same result for the 48h group was significantly lower than the 2 and 24h groups (28.0%, p<0.001). Our results suggest that while 2 or 24h storage of ovaries at 4 degrees C does not affect the meiotic competence of oocytes in vitro, 48h storage of ovaries decrease the results dramatically.

Evaluation of short estrus synchronization methods in dairy cows.

In the present study, two new short estrus synchronization methods have been developed for lactating dairy cows. The study was completed in three consecutive phases. In experiment (Exp) 1, 32 cows, that were not detected in estrus since calving between the 50th and 84th post-partum days, were treated with PGF2alpha (PGF, d-cloprostenol, 0.150 mg), estradiol propionate (EP, 2mg) and GnRH (lecirelina, 50 microg) at 24h intervals, respectively, and timed artificial insemination (TAI) was performed 48 h after PGF. Different from Exp 1, EP and GnRH were given at 48 and 60 h, respectively after PGF in Exp 2 (n=20), instead of 24 and 48 h. Ovulations were investigated by ultrasound for 7 days starting from the day of PGF treatment, and ovulation rates were compared with the ones obtained in Exp 1. In Exp 3, cows were given the same treatments as Exp 2, but treatments started at certain estrus stages. Cows detected in estrus and with a confirmed ovulation (n=27) after the second PGF given 11 days apart were assigned to three treatment groups. Treatment was initiated at Day 3 (group metestrus, n=9), Day 12 (group diestrus, n=9) and Day 18 (group proestrus, n=9) after ovulation. All cows included in Exp 3 were TAI between 16 and 20 h after GnRH treatment. In Exp 2 and 3, blood samples were obtained once every 2 days, starting from Day 0 to the 10th day after GnRH injection, and once every 4 days between the 10th and the 22nd days after GnRH to examine post-treatment luteal development. During the study, animals exhibiting natural estrus were inseminated and served as controls (n=85). The rate of estrus was found to be significantly higher in cows with an active corpus luteum (CL) at the start of Exp 1 (72.7% vs. 30.0%, P<0.05) and the pregnancy rate tended to be higher than cows without an active CL (40.9% vs. 10.0%, P=0.08). Compared to those in Exp 1, cows in Exp 2 had higher rates of synchronized ovulation (94.1% vs. 59.1%, P=0.013). In Exp 3, estrus (P<0.001) and pregnancy rates (P=0.01) were found to be significantly higher in cows in the proestrus group than in those in the metestrus group. Comparable pregnancy rates were obtained from the first and second inseminations in Exp 1 and 3 with results from those inseminated at natural estrus (P>0.05). It was concluded from the study that the treatment in Exp 1 and 3 could result in comparable pregnancy rates after timed AI of lactating dairy cows at random stages of the estrus cycle relating to those inseminated at natural estrus, but the stage of the estrus cycle can have significant effects on pregnancy rates.

Effect of transport and storage temperature of ovaries on in vitro maturation of bitch oocytes.

In this study, the effects of ovary transport and storage temperature on in vitro maturation of bitch oocytes were investigated. Ovaries were collected from 23 mature bitches and one randomly selected ovary of each pair (n=23 pairs) was transported in physiologic saline at 4 degrees C, while the other one at 35-38 degrees C for 2-4h. A total of 316 cumulus oocyte complexes (COCs) were obtained from the 4 degrees C group and 301 COCs from the 35-38 degrees C group. All COCs were matured in modified synthetic oviduct fluid (mSOF) supplemented with follicle stimulating hormone (FSH), essential and non-essential amino acids at 38 degrees C in a humidified 5% CO2, 5% O2, and 90% N2 atmosphere for 72 h. At the end of the in vitro maturation period, nuclear maturation of oocytes was classified as germinal vesicle (GV), germinal vesicle breakdown (GVBD), metaphase I (MI), metaphase II (MII), undetermined nuclear maturation (UDNM), and MI+MII. The nuclear maturation rates to MI, MII, and MI+MII stages were 60.44%, 10.75%, and 71.20% in the 4 degrees C group and 37.20%, 7.64%, and 45.85% in the 35-38 degrees C group, respectively. The data demonstrated that oocytes obtained from ovaries transported at 4 degrees C had higher maturation rates than from the ones transported at 35-38 degrees C (p<0.001).