Percentage Of Cleavage Research Articles

Effect of Polycyclic Aromatic Hydrocarbons Extracted from Indoor Air of Coffee Shops on Oral Squamous Carcinoma Cell (KB/C152): An in Vitro Approach

Introduction: The two main causes of indoor air pollution (IAP) are cooking and smoking. Toxic substances, including polycyclic aromatic hydrocarbons (PAHs), recognized as human carcinogens, are present in cooking and tobacco smoke. This study aims to determine the cytotoxicity and genotoxicity of PAHs collected from the indoor air of coffee shops on human cell line (KB/C152) in vitro approach.
 Materials and methods: Sampling pumps at a 2 l/min flow rate for 5 hours were applied to collect indoor air samples. Next, KB cells in cell culture medium were exposed to different concentrations of extracted PAHs using Methyl Thiazolyl Tetrazolium (MTT) test. Finally, terminal dUTP nick-end labeling (TUNEL) test and cell cycle assessments were both examined using flow cytometry.
 Results: The MTT test revealed a significant cytotoxic effect on KB cells depending on the PAHs concentrationas compared to the control cell line. The lethal concentration 50 (LC50) value against KB cells was 100 ± 3.09 µg/ml. Accordingly, exposure to extracted PAHs resulted in an arrest in the cell cycle at the sub-G1 checkpoint. The extracted PAHs suppressed the cell cycle in the sub-G1 phase, damaged DNA, and arrested KB cells from proliferating. Additionally, a statistically significant increase in DNA cleavage percentages (p ≤ 0.05) was seen in the TUNEL test, which also showed a dose-dependent increase in DNA damage.
 Conclusion: Extracted PAHs caused DNA damage and arrested in the cell cycle in epidermoid carcinoma of the mouth cells (KB/C152) in vitro mechanisms. This evaluation highlights mechanisms of exposure to extracted PAHs and their detrimental health effects.

Circulating Levels of Fibroblast Growth Factor 23 Selective for C-Terminal (FGF23-CT) in Hemodialysis Patients

Background: In hemodialysis patients, fibroblast growth factor 23 (FGF23) has reportedly been associated with the development of cardiovascular complications and a high risk of mortality. Our objective here was to study the cleavage characteristics of FGF23 in hemodialysis patients. Methods: This study design is a cross-sectional observational investigation of three facilities without intervention. To assess FGF23 concentrations, we obtained plasma samples from 97 hemodialysis patients before the hemodialysis session and from 16 healthy volunteers. We measured the FGF23 C-terminal fragment and intact FGF23 concentrations by using a commercial enzyme-linked immunosorbent assay. Results: Serum levels of the FGF23 C-terminal fragment were 189 ± 121 ng/mL in healthy volunteers and 306 ± 206 ng/mL in hemodialysis patients. The ratios of intact FGF23 to total FGF23 were 0.03 ± 0.03 in healthy volunteers and 0.44 ± 0.28 in hemodialysis patients. The ratios were positively correlated with levels of inorganic phosphate in hemodialysis patients (p < 0.001, r = 0.52). Conclusion: We measured actual levels of the serum FGF23 C-terminal fragment in hemodialysis patients by using a new commercial kit for the first time. The ratio of intact FGF23 to total FGF23 was lower in healthy controls than the ratio in hemodialysis patients. The cleavage percentage of FGF23 was considerably higher in both groups of subjects than previously thought. We suggest that hyperphosphatemia in hemodialysis patients was associated with impaired cleavage of FGF23.

Feline Wharton's jelly-derived mesenchymal stem cells as a feeder layer for oocytes maturation and embryos culture in vitro.

Due to their capacity to release growth factors and cytokines, co-culture using mesenchymal stem cells has been considered a good alternative to promoting the maturation of the oocytes and the embryo's development quality in vitro in different mammalian species. In this regard, we investigated the effect of feline Wharton's jelly MSCs as feeders layer in oocyte maturation-consequently, the development of resulting embryos in co-culture. Oocytes with dark cytoplasm and a few layers of cumulus cells were collected and subjected to in vitro maturation and embryo culture using commercial media with and without MSCs addition. The oocytes' nuclear maturation and the degree of cumulus expansion in different groups were assessed after 24 h; the development of the embryo was evaluated every 12 h until day eight. Although MSCs increased the proportion of cumulus cells oocytes exhibiting cumulus expansion, there were no significant differences in the percentage of matured oocytes (metaphase II) among the groups (p > 0.05). However, the embryo development differs significantly, with a higher cleavage, morula, and blastocyst percentage in oocytes matured with MSC co-culture conditions than in commercial media alone (p < 0.05). Also, we observed higher morula and blastocyst rates in the embryos co-cultured with MSCs during the in vitro culture (p > 0.05). Based on our results, the co-culture with MSCs during the oocyte maturation resulted in better embryo development, as well as the MSCs addition during embryo culture returned an increased number of morula and blastocysts. Further research is needed to fully understand and optimize the use of MSCs in oocyte maturation and embryo development.

Effect of use and dosage of p-follicle-stimulating hormone for ovarian superstimulation before ovum pick-up and in vitro embryo production in pregnant Holstein heifers

The benefit of ovarian superstimulation using exogenous FSH before ovum pick-up (OPU) and in vitro embryo production (IVEP) has been the subject of conflicting results. The objective of the present study, therefore, was to evaluate the effect of use and dose of porcine FSH (p-FSH) before OPU/IVEP on ovarian response and embryo production in pregnant heifers. Pregnant Holstein heifers (n = 48) were assigned randomly to receive 0, 160, or 300 mg NIH-FSH-P1 in a crossover design. Ovum pick-up was performed at 49, 63, and 77 d of gestation with a 14 d "washout" between OPU sessions. Follicle ablation was performed on D 0 (p.m.) and p-FSH treatments, consisting of 4 decreasing dose injections administered 12 h apart, were initiated 36 h after follicle ablation (D2 a.m.). Heifers underwent OPU on D 5 (a.m.), 40 h after the last p-FSH treatment, and cumulus oocyte complexes (COC) were subjected to IVEP procedures. Differences between treatment groups were evaluated using generalized linear mixed models. There were quadratic effects of treatment on both number and percentage of small (<6 mm), medium (6-10 mm) and large (>10 mm) follicles. Number and percentage of medium follicles increased with increasing p-FSH dosages, although the magnitude of the change was greater between 0 and 160 mg, than between 160 and 300 mg of p-FSH. Total number of follicles, number of COC recovered and number of viable COC increased linearly with increasing p-FSH dose. Conversely, there was no evidence for an effect of p-FSH dose on COC recovery percentage nor the percentage of viable COC. Cleavage percentage, number of cleaved oocytes, blastocyst percentage, and number of blastocysts increased linearly with increasing p-FSH dose. In conclusion, use of p-FSH before OPU resulted in greater superstimulatory response and oocyte competence which in turn increased IVEP. Furthermore, these effects were dose dependent such that use of a greater dose of p-FSH up to 300 mg progressively increased embryo yield.

Comprehensive Biological, Photodegradation, Density Functional Theory, and Docking Exploration of Zn(II) and Mn(II) complexes of Piroxicam

AbstractThe biological evaluation of coordination complexes is an active research area ascribed to structural modification and synergistic action due to metal ions. The mononuclear complexes of Zn(II) and Mn(II) piroxicam were evaluated for their encyclopedic biological activities, DFT, and docking exploration. Different antioxidant methods were used to assess their antioxidant potential. DNA cleavage and protein binding properties of the complexes were monitored through gel electrophoresis and UV‐visible spectrophotometry. The low IC50 value of both complexes implies more potency than the piroxicam. The percent cleavage and binding constant values indicated that the complexes bind with DNA and BSA more efficiently than the piroxicam. In SD rats, complexes were screened for analgesic, anti‐anxiety, anti‐inflammatory, and anti‐hyperglycemic effects. The complexes demonstrated statistically significant results for in‐vivo analysis. Photodegradation results of the metal complexes under UV lamp exhibited abrupt degradation than the piroxicam. Docking results also validate a better propensity of both complexes to bind with DNA and BSA. Hydrogen bonding and Van der Waals forces play a key role in stabilizing complexes in DNA and BSA binding pockets. These metal complexes have evident therapeutic potential more than piroxicam and could be promising drugs for relieving pain, anxiety, inflammation, and associated comorbidities with good pharmacodynamic properties.

The Role of Ionomycin in the Improvement of in vitro Fertilization and Increasing the Rate of Bovine Embryo Development

Background: The utilization of in vitro fertilization (IVF) and in vitro embryo production (IVEP) has increased significantly as an effective tool of assisted reproductive technology. Sperm capacitation is a process of membrane maturation allowing the acrosome reaction to take place. Ionomycin is a calcium ionophore that induces the acrosome reaction by increasing calcium cycling across phospholipid membranes. The aim of this study was to enhance the cleavage rate and development of bovine embryos. Methods: Bovine ovaries were collected from the central slaughter house. Oocytes were obtained from follicles and were let to mature for 24 h. Frozen bull semen thawed in 37°C water and transferred to a discontinuous percoll gradient (Sigma GE17-0891-01). The spermatozoon pellet was separated into four groups after resuspension in bovine in vitro fertilization media (BO-IVF): control without additives; with DMSO and with ionomycin (25 and 50 nM) and kept in an incubator for three hours. The oocytes matured under commonly used media (TCM-199) for 24 h. The mature oocytes were then fertilized for 18-24h. The embryos were cultured in SOF medium until the seventh day to study cleavage rate and blastocyst rate. Result: The results indicated that 50 nM ionomycin supplementation considerably reduced cleavage while DMSO and 25 nM ionomycin supplementation had no effect on cleavage percentage. Moreover, when compared to the other groups, ionomycin at 25 nM significantly increased blastocyst development. In conclusion, the highest rate of blastocyst development was observed in the capacitation medium supplemented with 25 nM ionomycin, apparently due to the importance of calcium and metabolism in spermatozoa for fertilization and embryo development.

Therapeutic effects of L-arginine, L-carnitine, and mesenchymal stem cell-conditioned medium on endometriosis-induced oocyte poor quality in an experimental mouse model.

The present study aimed to explore the potential ameliorative effects of L-arginine (LA), L-carnitine (LC), and bone marrow mesenchymal stem cell-conditioned medium (BMSC-CM) on endometriosis (EMS) model in vivo and in vitro. The animals were divided into two main groups, normal and EMS-induced mice. Normal and EMS-induced groups were injected with or without LA (250 mg/kg), LC (250 mg/kg), and BMSC-CM (a final volume of 100 μL of CM/mouse). At the end of the study, the level of total antioxidant capacity (TAC), nitric oxide (NO), and total oxidative status (TOS) were measured in plasma. Furthermore, immature oocytes were collected from two groups and cultured in a maturation medium. Subsequently, the rates of in vitro maturation, in vitro fertilization (IVF), and in vitro embryonic development were evaluated. The results revealed that administration of LA, LC, and BMSC-CM ameliorated the oxidative status through maintaining TAC and alleviating TOS and NO levels. More importantly, the maturation and fertilization rates, blastocyst development, and total blastocyst cell numbers significantly increased in LA, LC, and BMSC-CM-administrated groups compared to the control group. In both the normal and EMS groups, the highest IVF, cleavage, and blastocyst percentages were associated with BMSC-CM treatment (p < 0.05). Altogether, LA, LC, and BMSC-CM have therapeutic effects on impaired oocyte quality and promote subsequent development in vitro, probably through normalization of nitro-oxidative stress, thus offering potential alternatives to conventional therapies during assisted reproductive technologies for patients with EMS-associated sub/infertility.

Galectin-1 Used in Assisted Reproduction-Embryo Safety and Toxicology Studies.

Galectin-1 has been cited as a mediator involved in preventing early embryonic death in mammals and is implicated in maternal-fetal tolerance. Galectin-1 is also a reasonable tool to improve fertility in assisted reproduction procedures. As recommended in the ICH guidelines (S5-R2 and S6-R1) and based on bioethical concerns, we chose bovine embryos (BE) to assess in vitro embryo development as part of a larger reproductive safety and toxicology study in progress. The design considered in vitro embryo development using rHGAL-1 supplementations (in three different concentrations) of the in vitro embryo culture (IVP) media. Based on procedures for the commercial in vitro production of BE using oocytes aspirated from slaughterhouse ovaries, rHGAL-1 supplementation was performed in two experiments: In Experiment 1 on oocyte maturation, involving IVM medium supplementation, and in Experiment 2 on culture step IVC, involving supplementation with an SOF medium. IVP commercial procedures were used, with three IVP replicates per experiment, and the oocytes we distributed into four groups of treatment (one control group and three different dosages of rHGAL-1 to supplement both IVM and SOF media using 2, 20, and 40 µg·mL-1, respectively. A total of 967 (Experiment 1) and 1213 (Experiment 2) oocytes were aspirated and submitted to the IVP procedure. There was no damage to the in vitro bovine embryo growth when considering cleavage percentage (%CLE), blastocyst development (Bl, Bx, Bh, and B) at Days 7 and 8, or an amount of rHGAL-1 supplementation ≤20 µg·mL-1. The immunohistochemistry assay with D8 embryos cultivated using rHGAL-1 supplementation on the culture medium (SOF medium) demonstrated the presence of exogenous GAL-1 distributed in mass cell and trophoblastic cells, and the profile observed was dependent on exogenous supplementation, which was most evident in hatched embryos. The findings confirmed the use of a reasonable amount of rHGAL-1 for in vitro embryonic development and would make the use of rHGAL-1 in assisted reproduction in humans more reliable and safer. Even though it was not the objective of the study, we verified that supplementation with 2 µg·mL-1 significantly improved some of the evaluated parameters of embryonic development (%BlD7, %BD7, %BlD8, %BhD8, and %BD8).

Effect of Silybum marianum (L.) Geartn extract on in vitro fertilization in mice

Composition of Silybum marianum (L.) Geartn extract rich with flavonoid sylimarin which have antioxidant properties. Aim of this project study the effects of sylimarin on In vitro fertilization (IVF) and cleavage stages of embryos in mice. Fifteen adult males and females mice were divided into three groups , each group containing 5 mice , first and second groups were treated with (25 and 50 mg/kg body weight) of Silybum marianum (L.) Geartn extract (IP) while the third group treated with normal saline as control group . After 35 days of treatment the animals (male and female) were sacrificed and sperms isolated from tail of epidydymis were used for the following tests: Sperm motility, dead / live sperm, sperm abnormalities and serum was collected to assay testosterone. Isolation of oocyte from oviduct and serum blood to hormonal assay (FSH and LH) . Sperm and matured ova were incubated in TCM-199 media for insemination. The results showed increase in body weight and testes weight in groups treated with Silybum marianum (L.) Geartn. Significant increase in sperm activity and decrease in percentage of dead sperm and abnormalities in groups treated with Silybum marianum (L.) Geartn. The male testosterone and female FSH, LH increased significantly in groups treated with Silybum marianum (L.) Geartn. IVF study showed significant increase in percentage of maturation, fertilization and cleavage (2-4 cells). In conclusion sylimarin have benefits effect on IVF and cleavage stages of embryo development when injected before IVF by improve fertilization of males and females.

Effect of Trigonella Foenum-Graecum Foenugreek oil extract on some parameters of sperms and in vitro fertilization in mic

This project, aims to study the effect of trigonella seeds oil extract on body weight , testes weight and some parameter of reproductive system in male mice , and on In vitro fertilization (IVF) , cleavage stages of embryos in females. Fifteen adult (8-9 weeks) males and females mice were divided into three groups , each group containing 5 mice , first and second groups were treated with (2 and 4 mg/kg body weight) of Trigonella oil extract (intraperitonally) while the third group treated with normal saline as control group . After 35 days of treatment with Trigonella the animals (male and female) were sacrificed by dislocation of cervical vertebrate. Isolated sperms from tail of epidydymis were used for the following tests. Sperm motility, dead / live sperm, sperm abnormalities and serum ws collected to assay testosterone. Isolatation of ova from oviduct after micing in 500μl of TCM-199 media serum blood to assay FSH and LH . Sperm and matured ova were incubated in TCM-199 media for insemination. The results showed increase in body weight and testes weight in groups treated with trigonella. significant increase in sperm activity and decrease in percentage of dead sperm and abnormalities in groups treated with trigonella oil. The male testosterone and female FSH, LH increased significantly in groups treated with trigonella. IVF study showed significant increase in percentage of maturation, fertilization and cleavage (2-4 cells).

Effect of endometrial cell-conditioned medium and platelet-rich plasma on the developmental competence of mouse preantral follicles: An in vitro study.

ObjectiveThe aim of this study was to evaluate the impacts of platelet-rich plasma (PRP) and conditioned medium (CM) derived from endometrial stromal cells on mouse preantral follicle culture in a two-dimensional system to produce competent mature oocytes for fertilization.MethodsIn total, 240 preantral follicles were isolated from female mouse ovarian tissue and divided into four groups. The preantral follicles were isolated three times for each group and then cultured, respectively, in the presence of alpha minimum essential medium (control), PRP, CM, and PRP+CM. The in vitro growth, in vitro maturation, and cleavage percentage of the preantral follicles were investigated. Immunocytochemistry (IHC) was also conducted to monitor the meiotic progression of the oocytes. Additionally, the mRNA expression levels of the two folliculogenesis-related genes (Gdf9 and Bmp15) and two apoptosis-related genes (Bcl2 and Bax) were investigated using real-time polymerase chain reaction.ResultsIn the PRP, CM, and PRP+CM groups, the preantral follicle maturation (evaluated by identifying polar bodies) were greater than the control group. The cleavage rate in the CM, and PRP+CM groups were also greater than the control group. IHC analysis demonstrated that in each treatment group, meiotic spindle was normal. In the PRP+CM group, the gene expression levels of Bmp15, Gdf9, and Bcl2 were greater than in the other groups. The Bax gene was more strongly expressed in the PRP and control groups than in the other groups.ConclusionOverall, the present study suggests that the combination of CM and PRP can effectively increase the growth and cleavage rate of mouse preantral follicles in vitro.

Effect of Moringa oleifera seed extract on antimicrobial activity and in vitro fertilization ability of cryopreserved ram semen.

Cryopreservation has adverse effects on the post-thaw sperm quality due to oxidative stress and the presence of bacteria. To minimize such effects, plant extracts have been included in the composition of the semen diluents. The objective of the present study was to evaluate the antimicrobial and antioxidant effects of Moringa oleifera seed extract (MOSE) on cryopreserved ram semen, as well as its impact on in vitro fertilization. Semen from six hair rams was treated with five treatments before cryopreservation: Control (without any antibiotic), Standard (conventional antibiotic), 1.0, 10.0, and 50.0mg/ml of MOSE. Post-thawing sperm characteristics were evaluated by the computer-assisted semen analysis. Antimicrobial activity was assessed by counting colony-forming units (CFU) and the antioxidant capacity by the ferric reducing antioxidant power method. A heterologous in vitro fertilization technique was implemented to measure the fertilization rate. Progressive and rapid motility, membrane and acrosome integrity, and active mitochondria were higher (p < .05) in the 10.0mg/ml treatment compared with Standard after thawing. All M. oleifera treatments showed inhibition of CFU. The antioxidant capacity of M. oleifera seed extract was higher in the 10.0 and 50.0mg/ml treatments. Fertilization rate (cleavage percentage) was higher (p < .05) in the 10.0mg/ml (82.9 ± 10.0) and Control (82.5 ± 9.9) treatments compared with Standard (73.7 ± 9.1). The addition of 10.0mg/ml of MOSE to ram semen inhibits the development of microorganisms and improves sperm characteristics and the in vitro fertility of the semen.

Synchronization of follicle wave emergence before ovarian superstimulation with FSH and ovum pick-up improves in vitro embryo production in pregnant heifers

The objective of the present study was to evaluate effects of synchronization of timing of follicle wave emergence, before ovarian superstimulation and ovum pick-up (OPU), on ovarian response and embryo production in pregnant heifers. Pregnant (47–69 days of gestation) Holstein heifers (n = 64), 19.0 ± 0.3 months of age, were assigned in a completely randomized design to one of two groups: synchronization of follicular wave emergence using follicle ablation (Synchronized) or untreated control (Non-synchronized). Superstimulatory treatments consisting of 160 mg (280 IU) of porcine follicle stimulating hormone (p-FSH), administered in four decreasing dose treatments 12 h apart, were initiated 36 h after follicle ablation or at random stages of the follicular wave in heifers of the Synchronized and Non-synchronized group, respectively. Ovum pick-up was performed in all heifers 40 h after the last p-FSH administration and retrieved cumulus oocyte complexes (COCs) were subjected to in vitro embryo production (IVEP) procedures. Ultrasonography was performed immediately before OPU to determine number and size of ovarian follicles. Differences in treatment responses between groups were evaluated using generalized linear mixed models. Total number of follicles at the time of OPU was not different between treatment groups (P = 0.61), however, the number of small follicles (<6 mm) was greater (P = 0.05) in heifers of the Non-synchronized group, whereas number of medium size follicles (6–10 mm) tended (P = 0.09) to be greater in heifers of the Synchronized group. Heifers in the Synchronized group had a greater (P = 0.01) percentage of medium sized follicles and a lesser (P = 0.01) percentage of smaller sized follicles than heifers in the Non-synchronized group. There were no differences (P > 0.15) in total number of recovered COCs, or number of viable COCs between groups. Cleavage percentage (84.5% and 72.8%) and blastocyst percentage (48.2% and 33.4%) were greater (P < 0.01) in heifers of the Synchronized than Non-synchronized group, respectively. As a result, mean number of blastocysts per OPU/heifer was greater (P = 0.006) in the heifers of the Synchronized (8.9 ± 1.0) than the Non-synchronized (5.5 ± 0.9) group. In conclusion, synchronizing the time of follicle wave emergence in pregnant heifers, prior to ovarian superstimulation with FSH and OPU results in a greater superstimulatory response and oocyte competence leading to greater embryo production.

Effect of carbon content on interfacial microstructure and mechanical properties of a vacuum hot-compressed bonding titanium-steel composite

Vacuum hot-rolled bonding (VHRB) titanium (Ti)-steel clad composites have been increasingly serving in both severe corrosion and heavy load conditions, and their overall properties severely depend on the microstructure of interfacial reaction layer. Notably, introducing an appropriate interdiffusion barrier to minimize the brittle Fe–Ti intermetallics has still been a popular scheme for optimizing their interfacial properties. In this work, with two low-alloyed steels of 0.06 and 0.16 wt% carbon (C) contents employed, the pure Ti -steel composite samples were successfully prepared via vacuum hot-compressed bonding (VHCB) in Gleeble-3500 system under the identical temperature/reduction/rate condition of 850 °C/70 %/0.01s−1. The interfacial reaction, microstructure and tensile property in two Ti-steel composites were investigated to estimate the C content effect via various characterizations, and the mechanisms were clarified. Results indicated that the diffusion of Ti, Fe and C mainly occurred in the interfacial reaction layers of two composites, and accordingly the reaction products of TiFe, TiFe2 and TiC formed, together with the C-depleted α-Fe zone. The nanoscale TiC particles were mainly located at the steel side, and increased with the increasing C content. Correspondingly, the brittle TiFe layer and TiFe2 layer decreased due to the increased TiC layer acting as a diffusion barrier. This led to an evident decrease of percent cleavage fracture area (CA, %) from 77% to 52% and a significant increase of interfacial tensile strength from 182 to 344 MPa.

Conjugated linoleic acid improves meiotic spindle morphology and developmental competence of heat-stressed bovine oocyte

This study was conducted to elucidate the effects of introducing conjugated linoleic acid (CLA) on meiotic spindle organization of heat stressed (HS) matured oocytes and the resulting blastocysts DNA methylation as well as the expression of the genes involved in DNA methylation (DNMT3a, DNMT3b and DNMT1). Immature bovine oocytes were cultured at 41 °C for the first 12 h and 38.5 °C for the second 12 h of maturation time in the presence of 0 and 50 μM of CLA (HS and HS + CLA groups, respectively). A group of oocytes cultured in medium with no CLA supplementation at normal temperature (38.5 °C for 24 h) was considered as negative control (C). Percentage of normal spindle, and cleavage and blastocyst rates were significantly decreased in the HS group compared to the C group (P < 0.05). The global DNA methylation and expression level of DNMT3a gene were increased in HS group compared to the C groups (P < 0.05), while the expression level of DNMT3b was decreased. The CLA supplementation improved the percentage of normal microtubules shape in MII oocytes as well as the developmental competence in the HS + CLA group compared to the HS group (P < 0.05). However, global DNA methylation and expression level of DNMT3a/b were not ameliorated by CLA supplementation (P > 0.05). Based on the obtained results, CLA proved to be capable of improving the oocyte developmental competence as well as decreased the aberrant spindle organization of heat-stressed oocytes and it would not cause epigenetic alteration in the obtained blastocysts.

Effect of melatonin supplementation during IVM of dromedary camel oocytes (Camelus dromedarius) on their maturation, fertilization, and developmental rates in vitro

The positive impact of melatonin on in vitro embryo production (IVEP) has been reported in many domestic species; however, no studies have been carried out in camelids. We aimed to evaluate the effects of melatonin supplementation in maturation media on in vitro maturation, fertilization, and preimplantation embryo development of dromedary camel oocytes (experiment 1). We also evaluated the concentrations of total antioxidant capacity (TAC), and malondialdehyde (MDA) in the IVM spent medium in relation to melatonin supplementation. Cumulus oocyte complexes (COCs) were cultured in in vitro maturation media (IVM) supplemented with either 0.0, 25.0, 50.0 or 75.0 μM of melatonin for 30 h. Matured oocytes were then fertilized in vitro with epididymal camel spermatozoa. Following IVF, the resulting embryos were cultured in vitro for seven days. The percentage of maturation, fertilization, cleavage, and embryo developmental rates (morula and blastocyst) was recorded (experiment 1). TAC and MDA levels in the IVM spent maturation media were also evaluated at 30 h post-IVM (experiment 2). The results showed that supplementation of IVM media with 25 μM melatonin significantly improved oocyte nuclear maturation, fertilization (18 h post-insemination; pi), cleavage (day 3 pi), morula (day 5 pi) and blastocyst (day 7 pi) rates as compared with the controls and other melatonin-supplemented groups. Furthermore, the TAC in the IVM spent media was significantly increased (P < 0.05) in 25 μM melatonin supplemented groups than those supplemented with 0.0, 50.0, 75.0 μM melatonin. However, the concentration of MDA was significantly lower (P < 0.05) in IVM media supplemented with 25.0 μM of melatonin when compared with the control and other treatment groups. In conclusion, supplementation of IVM medium with 25 μM of melatonin could enhance the in vitro developmental capacity of dromedary camel oocytes.

Release of Anticancer Agents in the Tumor Microenvironment Using Cathepsin B and Cathepsin L Cleavable Drug‐Linker Constructs

Cysteine cathepsins are a family of cysteine proteases that are normally localized to the lysosome for maintaining homeostasis. In a number of highly invasive cancer cell lines and malignant tumors, cathepsins L and B are highly upregulated and are secreted into the extracellular matrix as pro-enzymes. Upon activation, they promote remodeling of the extracellular matrix and cancer progression and metastasis. A number of studies have demonstrated that cathepsins L and B are overexpressed in breast, ovarian, lung, and colon cancer. The goal is to use the extracellular proteolytic activity of cathepsin L and/or cathepsin B to cleave drug-linker constructs in the tumor microenvironment to release potent anticancer agents. Drug-linker constructs in this study consist of a maleimide functional group for attachment of a protein tumor-targeting agent (such as an antibody), a protease cleavable linker, and a cytotoxic effector (payload). Several pre-validated benzosuberene- and dihydronaphthalene-based effectors bind to tubulin at the colchicine binding site and are effective inhibitors of tubulin polymerization into microtubules. They demonstrate profound cytotoxicity (low nM to pM) against a wide-spectrum of human cancer cell lines. In addition to their antimitotic activity, they exhibit dual-mechanism of action and function as powerful vascular disrupting agents (VDAs), imparting widespread damage to tumor-associated vasculature that results in blood flow shutdown and massive tumor necrosis. The hypothesis is that cathepsin B and/or cathepsin L will effectively and selectively cleave these drug-linker constructs in the tumor microenvironment, thus facilitating release of the potent dual-mechanism effector agents leading to inhibition of tumor growth and metastasis. In this study, substate specificity and cleavage rates were evaluated for a series of benzosuberene- and dihydronaphthalene-based drug-linker constructs incorporating L-Val-L-Cit and other dipeptides. Assay conditions were optimized for each enzyme. Standard curves were obtained for drug-linker constructs and effectors using reversed-phase HPLC. For drug-linker constructs, the maleimide head group was first de-activated by the thiol agent, N-acetyl-L cysteine (NAC), and the products were incubated for variable times with either cathepsin L or B. After dilution of the reaction mixtures with acetonitrile, the release of effector was evaluated by HPLC. Using standard curves, the percent cleavage and the recovery were calculated for each reaction in these rate studies. The L-Val-L-Cit dipeptide linker was cleaved by both cathepsin B and cathepsin L. Rates of cleavage of drug-linker constructs with a series of different dipeptides replacing L-Val-L-Cit were determined.

68 Comparison of two Percoll gradients for selection of frozen semen for invitro production of ovine embryos

Sperm selection methods are routinely applied to prepare semen for IVF in animal species. These procedures are used to improve sperm quality characteristics as well as to remove other background material and debris. Percoll gradient is widely used in animal IVF laboratories. Different Percoll gradient concentrations and volumes can be used to improve the sperm sample motility percentage. The objective of this research was to evaluate the effect of 2 different Percoll concentrations for ovine IVF sperm selection and effects, if any, on invitro embryo production (IVP). Specifically, Percoll gradients consisted of Group 1 (G1) 40–80% and Group 2 (G2) 45–90%, 400µL each. The research was carried out in the reproduction laboratory at Palominos Ranch (Jalisco, México). The IVP was performed with a continuous invitro culture system. Ovaries (n=157) were collected from a slaughterhouse (León, México) and transported to the laboratory within 2h in physiological saline solution (0.9% NaCl) supplemented with penicillin G (100IU mL−1) and streptomycin sulphate (100µg mL−1). For IVP, IVF Bioscience™ media were used for IVM, IVF, and invitro culture (IVC). For IVM, the cumulus–oocyte complexes (COCs) were selected (only grades 1 and 2) and matured for 24h at 38.5°C in 5% CO2 in air and 100% humidity. Matured oocytes (n=800, divided equally into 4 replicates) were divided into 2 groups, G1 and G2. The IVF process was conducted with semen selected through a mini-Percoll technique with gradients at a concentration of 45% (top layer) and 90% (bottom layer) or 40% (top layer) and 80% (bottom layer) for G1 and G2, respectively, using frozen–thawed semen from the same ram, at a concentration of 2×106 sperm mL−1, for 18h in 38.5°C, 5% CO2 in air, and 100% humidity. The presumptive zygotes were denuded by pipetting and set in IVC until Day 7 at 38.5°C, 5% CO2, 5% O2, and 90% N2 at 100% humidity. The percentages of cleavage, embryos with more than 6 cells, and blastocysts on Day 7 of culture were evaluated, based on the initial number of oocytes entering into IVM. The statistical analyses were carried out with the GLM procedure of SAS software (version 9.3; SAS Institute Inc.) to evaluate the results of G1 versus G2 (α level=0.05). Cleavage rate was 47.8%±2.5 and 55.9%±2.5, respectively, in G1 and G2. The percentages of embryos with more than 6 cells were 38.1%±2.2 and 43.5%±2.2, respectively, in G1 and G2. The percentage of blastocysts on Day 7 was 27.4%±1.1 and 37.3%±1.1, respectively, in G1 and G2. There were no significant differences between groups (P&amp;gt;0.05) for percentage of cleavage and embryos with more than 6 cells, although the percentage of cleavage tended to be greater for G2 (P=0.06). Additionally, G2 had a larger percentage of blastocysts on Day 7 compared with G1 (P&amp;lt;0.05). In conclusion, under the conditions of this research, the use of a Percoll gradient at a concentration of 40–80% increased the percentage of blastocysts for ovine IVP.

69 Invitro production of ovine embryos using either fresh or frozen-thawed semen

Invitro embryo production (IVP) is an important tool for genetic improvement in small ruminants. Semen quality is one of the most important aspects to consider for the success of this assisted reproductive technique. With ovine IVP, it is a common practice to use fresh semen for IVF, but this could be a problem because the differences between ejaculates from the same animal are well documented and a source of variation in IVP results. The objective of this research was to compare the effect of fresh and frozen–thawed domestic sheep (Ovis aries) semen on IVF for ovine IVP. The research was carried out in the reproduction laboratory at the Palominos Ranch (Jalisco, México). The IVP was performed with a continuous invitro culture system. Ovaries (n=186) were collected from a slaughterhouse (León, México) and transported to the laboratory within 2h in physiological saline solution (0.9% NaCl) supplemented with penicillin G (100IU mL−1) and streptomycin sulphate (100µg mL−1). For IVP, IVF-Bioscience™ media were used for IVM, IVF, and invitro culture (IVC). For IVM, the cumulus–oocyte complexes (COCs) were selected (only grades 1 and 2) and matured for 24h at 38.5°C in 5% CO2 in air and 100% humidity. Matured oocytes (n=1000) were invitro fertilized using either fresh or frozen–thawed semen (Triladyl™; Minitube) from the same sheep, at a concentration of 2×106 sperm mL−1, for 18h in 38.5°C, 5% CO2 in air, and 100% humidity. The presumptive zygotes were denuded by pipetting and set in IVC until Day 7 at 38.5°C, 5% CO2, 5% O2, and 90% N2 at 100% humidity. The percentages of cleavage, embryos with more than 6 cells, and blastocysts on Day 7 of culture were evaluated, based on the initial number of oocytes entering into IVM. Statistical analyses were carried out with the GLM procedure of SAS software (version 9.3; SAS Institute Inc.) to evaluate the results of fresh versus frozen–thawed (α level=0.05). Rates of cleavage, embryos with more than 6 cells, and blastocysts on Day 7 were similar (P&amp;gt;0.05): fresh 52.3±3.0%, 43.6±2.6%, and 34.3±2.9%, respectively; frozen–thawed: 53.3%±3.0, 41.1±2.6%, and 33.3±2.9%, respectively. In conclusion, under the conditions of this research, the use of fresh and frozen–thawed semen had similar results for ovine IVP.

29 The effect of vitrification at the immature stage on DNA methylation in porcine oocytes and its relevance to subsequent embryo development

Oocyte vitrification is an important approach for invitro gene banking of female germplasm; however, in pigs, it hampers embryo development. In cattle, vitrification at the MII stage was reported to alter epigenetic status in oocytes and even in subsequently developing embryos (Chen et al. 2016 Theriogenology 86, 868-878). The present study investigated the effect of vitrification at the immature stage of porcine oocytes on DNA methylation status and its relevance to subsequent embryo development. Immature cumulus–oocyte complexes were vitrified in microdrops and warmed (vitrified group) or treated with cryoprotectant agents (17.5% ethylene glycol + 17.5% propylene glycol, CPA group) by our method (Appeltant et al. 2018 Cryobiology 85, 87-94). Then they were subjected to IVM, parthenogenetic activation (PA), and embryo culture. From each batch, a group of oocytes was processed without treatment (control group). Oocyte survival and polar body extrusion were recorded after IVM. Cleavage and blastocyst developmental rates were recorded on Day 2 and 6 of culture, respectively (Day 0=PA). In each replication, DNA methylation was assayed in representative oocytes at the MII stage after IVM and in embryos at the 2- to 4-cell stage on Day 2 by immunostaining with 5-methylcytosine (5mC). Relative fluorescent intensity of 5mC in the chromatin was compared among groups. The experiment was replicated 3 times. Data were analysed by ANOVA. After IVM, there was no significant difference among the control, CPA, and vitrified groups in terms of the percentage of live oocytes (99.3, 96.4, and 94.0%, respectively) or polar body extrusion (88.6, 86.9, and 79.6%, respectively). After PA of oocytes with a polar body, there was no difference between the control and CPA groups in the percentage of cleavage (84.1 and 80.7%, respectively) or blastocyst development of cleaved embryos (63.3 and 79.3%, respectively). However, in the vitrified group, cleavage and blastocyst development rates (46.6 and 33.5%, respectively) were reduced (P&amp;lt;0.05) compared with the other groups. The 5mC fluorescence in the DNA of oocytes at the MII stage in the CPA and vitrified groups were similar and significantly lower than that in the control group (0.88±0.02, 0.87±0.001, and 1.0±0.02, respectively) but higher than that in the negative control processed without primary antibody (0.33±0.02). In the embryos at the 2- to 4-cell stage, 5mC fluorescence was not significantly different among the control, CPA, and vitrified groups (1.0±0.1, 0.99±0.1, and 0.96±0.1, respectively) but was significantly higher than that of the negative control (0.36±0.04). In conclusion, CPA treatment reduced DNA methylation levels in oocytes. However, it was restored during early embryo development and did not affect blastocyst development. The results suggest that reduced DNA methylation in vitrified oocytes is caused by CPA but it may not be responsible for their reduced ability to develop to blastocysts.