Borassus fine fibers possess superior tensile properties and are an important renewable, natural reinforcement material for composites. This paper reports the improved tensile properties of natural ...fibers extracted from Borassus fruit. Changes occurring in Borassus fibers when treated with a 5% concentration sodium hydroxide solution for different periods (1, 4, 8 and 12h) were characterized using tensile testing, chemical analysis, Fourier transform infrared spectroscopy, X-ray diffraction, scanning electron microscopy and thermogravimetric analysis. The tensile properties (strength, modulus and % elongation) of the fibers improved by 41, 69 and 40% respectively, after 8h of alkali treatment. Based on the properties determined for the Borassus fibers, we expect that these fibers will be suitable for use as a reinforcement in green composites.
Contents
The objective of this study was to find out the effect of l‐carnitine on oocyte maturation and subsequent embryo development, with l‐carnitine‐mediated alteration if any in transcript level ...of antioxidant enzymes (GPx, Cu/Zn‐SOD (SOD1) and Mn‐SOD (SOD2) in oocytes and developing sheep embryos produced in vitro. Different concentrations of l‐carnitine (0 mm, 2.5 mm, 5 mm, 7.5 mm and 10 mm) were used in maturation medium. Oocytes matured with 10 mm l‐carnitine showed significantly (p < 0.05) higher cleavage (66.80% vs 39.66, 41.76, 44.64, 64.31%), morula (48.50% vs 20.88, 26.01, 26.99, 44.72%) and blastocyst (33.22% vs 7.66, 9.19, 10.71, 28.57%) percentage as compared to lower concentrations (0 mm, 2.5 mm, 5 mm and 7.5 mm). Cleavage percentage between 10 mm and 7.5 mm l‐carnitine were not significantly different. Maturation rate was not influenced by supplementation of any experimental concentration of l‐carnitine. There was a significant (p < 0.05) decrease in intracellular ROS and increase in intracellular GSH in 10 mm l‐carnitine‐treated oocytes and embryos than control group. Antioxidant effect of l‐carnitine was proved by culturing oocytes and embryos with H2O2 in the presence of l‐carnitine which could be able to protect oocytes and embryos from H2O2‐induced oxidative damage. l‐carnitine supplementation significantly (p < 0.05) upregulated the expression of GPx and downregulated the expression of SOD2 genes, whereas the expression pattern of SOD1 and GAPDH (housekeeping gene) genes was unaffected in oocytes and embryos. It was concluded from the study that l‐carnitine supplementation during in vitro maturation reduces oxidative stress‐induced embryo toxicity by decreasing intracellular ROS and increasing intracellular GSH that in turn improved developmental potential of oocytes and embryos and alters transcript level of antioxidant enzymes.
Background
Early embryonic mortality is one of the major intriguing factors of reproductive failure that causes considerable challenge to the mammalian cell biologists. Heat stress is the major ...factor responsible for reduced fertility in farm animals. The present study aimed to investigate the influence of heat stress on prostaglandin production and the expression of key genes, including
COX-2
,
PGES
,
PGFS
,
ITGAV
and
LGALS15
, in buffalo endometrial epithelial cells.
Methods and results
Buffalo genitalia containing ovaries with corpus luteum (CL) were collected immediately post-slaughter. The stages of the estrous cycle were determined based on macroscopic observations of the ovaries. Uterine lumens of the mid-luteal phase (days 6–10 of the estrous cycle) were washed and treated with trypsin to isolate epithelial cells, which were then cultured at control temperature (38.5 °C for 24 h) or exposed to elevated temperatures 38.5 °C for 6 h, 40.5 °C for 18 h; Heat Stressed (HS). The supernatant and endometrial epithelial cells were collected at various time points (0, 3, 6, 12, and 24 h) from both the control and treatment groups. Although heat stress (40.5 °C) significantly (
P
< 0.05) increased
COX-2
,
PGES
, and
PGFS
transcripts in epithelial cells but it did not affect the in vitro production of PGF
2α
and PGE
2
. The expression of
ITGAV
and
LGALS15
mRNAs in endometrial epithelial cells remained unaltered under elevated temperature conditions.
Conclusion
It can be concluded that elevated temperature did not directly modulate prostaglandin production but, it promoted the expression of
COX-2
,
PGES
and
PGFS
mRNA in buffalo endometrial epithelial cells.
Abstract
STUDY QUESTION
Is it feasible to disseminate testicular tissue cryopreservation with a standardized protocol through a coordinated network of centers and provide centralized ...processing/freezing for centers that do not have those capabilities?
SUMMARY ANSWER
Centralized processing and freezing of testicular tissue from multiple sites is feasible and accelerates recruitment, providing the statistical power to make inferences that may inform fertility preservation practice.
WHAT IS KNOWN ALREADY
Several centers in the USA and abroad are preserving testicular biopsies for patients who cannot preserve sperm in anticipation that cell- or tissue-based therapies can be used in the future to generate sperm and offspring.
STUDY DESIGN, SIZE, DURATION
Testicular tissue samples from 189 patients were cryopreserved between January 2011 and November 2018. Medical diagnosis, previous chemotherapy exposure, tissue weight, and presence of germ cells were recorded.
PARTICIPANTS/MATERIALS, SETTING, METHODS
Human testicular tissue samples were obtained from patients undergoing treatments likely to cause infertility. Twenty five percent of the patient’s tissue was donated to research and 75% was stored for patient’s future use. The tissue was weighed, and research tissue was fixed for histological analysis with Periodic acid-Schiff hematoxylin staining and/or immunofluorescence staining for DEAD-box helicase 4, and/or undifferentiated embryonic cell transcription factor 1.
MAIN RESULTS AND THE ROLE OF CHANCE
The average age of fertility preservation patients was 7.9 (SD = 5) years and ranged from 5 months to 34 years. The average amount of tissue collected was 411.3 (SD = 837.3) mg and ranged from 14.4 mg—6880.2 mg. Malignancies (n = 118) were the most common indication for testicular tissue freezing, followed by blood disorders (n = 45) and other conditions (n = 26). Thirty nine percent (n = 74) of patients had initiated their chemotherapy prior to undergoing testicular biopsy. Of the 189 patients recruited to date, 137 have been analyzed for the presence of germ cells and germ cells were confirmed in 132.
LIMITATIONS, REASONS FOR CAUTION
This is a descriptive study of testicular tissues obtained from patients who were at risk of infertility. The function of spermatogonia in those biopsies could not be tested by transplantation due limited sample size.
WIDER IMPLICATIONS OF THE FINDINGS
Patients and/or guardians are willing to pursue an experimental fertility preservation procedure when no alternatives are available. Our coordinated network of centers found that many patients request fertility preservation after initiating gonadotoxic therapies. This study demonstrates that undifferentiated stem and progenitor spermatogonia may be recovered from the testicular tissues of patients who are in the early stages of their treatment and have not yet received an ablative dose of therapy. The function of those spermatogonia was not tested.
STUDY FUNDING/COMPETING INTEREST(S)
Support for the research was from the Eunice Kennedy Shriver National Institute for Child Health and Human Development grants HD061289 and HD092084, the Scaife Foundation, the Richard King Mellon Foundation, the Departments of Ob/Gyn & Reproductive Sciences and Urology of the University of Pittsburgh Medical Center, United States-Israel Binational Science Foundation (BSF), and the Kahn Foundation. The authors declare that they do not have competing financial interests.
Cyclooxygenase-2 (COX-2) has important functions in the synthesis and release of endometrial prostaglandin F2α (PGF2α). Excessive production of COX-2 leads to an increase in endometrial PGF2α ...synthesis and subsequently causes luteolysis and early embryonic mortality. The aim of this study was to investigate in goats the effects of COX-2 small interference RNA (siRNA) on COX-2 mRNA abundance and the secretion of PGF2α and PGE2 in goat endometrial cells. Endometrial cells isolated from goat uteri were cultured at 38.5 °C and 5% CO2. The cells were treated with different concentrations (0, 10, 25, 50, 100, 250, 500, 750 and 1000 nM per well) of three different COX-2 siRNAs at confluency for 24 h. At 24 h post culture, COX-2 mRNA abundance was quantified using qPCR and PGF2α and PGE2 concentrations were quantified in the culture medium. There was a lesser relative abundance of COX-2 mRNA in endometrial cells at 100 to 1000 nM siRNA. The greatest extent of abundance suppression, however, was observed with 1000 nM siRNA. Transfection of COX-2 siRNA (1000 nM) to endometrial cells suppressed the COX-2 mRNA abundance by 77%, 82%, and 84% with siRNA 1, 2, 3, respectively. Furthermore, with COX-2 siRNA transfected cells, there was a lesser (P < 0.05) PGF2α concentration than in cells not transfected, whereas PGE2 secretion was not affected. The results of the study provide evidence that COX-2 siRNA used in this study suppresses COX-2 mRNA abundance and PGF2α secretion but there was no association between PGE2 concentrations and COX-2 mRNA abundance in goat endometrial epithelial cells.
Attainment of puberty in animals is dependent on their age, body weight, nutritional status, genetic and environmental conditions. Nutritionally, organic minerals are suggested to improve semen ...production, sperm motility and male fertility. In this context, role of organic zinc (Zn) and copper (Cu) in advancing male puberty and semen characters in Osmanabadi goats were studied. Forty one (n = 41) bucks (Aged 5 months) were divided into ten groups and the dietary treatments comprised of a control group (basal diet; without additional trace mineral supplementation) and nine treatment groups that received, in addition to the basal diet, various doses of trace minerals (mg) on per kg dry matter basis, organic Zn as low Zn20, medium Zn40 and high Zn60, organic Cu as low Cu12.5, medium Cu25, high Cu37.5 and combination of organic Zn + Cu as low Zn20 + Cu12.5, medium Zn40 + Cu25, high Zn60 + Cu37.5, respectively fed for a period of 8 months. Bucks fed organic trace minerals reached puberty 28–35 days earlier than control group. In addition, improvement (P < .01) in testosterone hormone (ng/ml) levels (control: 1.63 ± 0.07 VS Zn60: 2.54 ± 0.02; Cu12.5: 6.17 ± 0.05; Cu25: 3.01 ± 0.04; Cu37.5: 2.39 ± 0.06; Zn20 + Cu12.5: 1.94 ± 0.02; Zn60 + Cu37.5: 2.44 ± 0.16 at 240 days), semen production capacity (sperm concentration, volume, mass motility) and semen quality (higher progressive motility, velocity, sperm membrane integrity and acrosome integrity) were observed in supplemented groups (P < .05) than the control bucks. The present study demonstrated that, additional feeding of organic Zn and Cu to growing male goats advanced onset of puberty and improved quantitative and qualitative semen characteristics. The results also implied that the organic Cu had a significant effect on overall performances of bucks as compared to Zn alone or Zn and Cu in combination.
Abstract Background Despite revisions in 2005 and 2014, the Gleason prostate cancer (PCa) grading system still has major deficiencies. Combining of Gleason scores into a three-tiered grouping (6, 7, ...8–10) is used most frequently for prognostic and therapeutic purposes. The lowest score, assigned 6, may be misunderstood as a cancer in the middle of the grading scale, and 3 + 4 = 7 and 4 + 3 = 7 are often considered the same prognostic group. Objective To verify that a new grading system accurately produces a smaller number of grades with the most significant prognostic differences, using multi-institutional and multimodal therapy data. Design, setting, and participants Between 2005 and 2014, 20 845 consecutive men were treated by radical prostatectomy at five academic institutions; 5501 men were treated with radiotherapy at two academic institutions. Outcome measurements and statistical analysis Outcome was based on biochemical recurrence (BCR). The log-rank test assessed univariable differences in BCR by Gleason score. Separate univariable and multivariable Cox proportional hazards used four possible categorizations of Gleason scores. Results and limitations In the surgery cohort, we found large differences in recurrence rates between both Gleason 3 + 4 versus 4 + 3 and Gleason 8 versus 9. The hazard ratios relative to Gleason score 6 were 1.9, 5.1, 8.0, and 11.7 for Gleason scores 3 + 4, 4 + 3, 8, and 9–10, respectively. These differences were attenuated in the radiotherapy cohort as a whole due to increased adjuvant or neoadjuvant hormones for patients with high-grade disease but were clearly seen in patients undergoing radiotherapy only. A five–grade group system had the highest prognostic discrimination for all cohorts on both univariable and multivariable analysis. The major limitation was the unavoidable use of prostate-specific antigen BCR as an end point as opposed to cancer-related death. Conclusions The new PCa grading system has these benefits: more accurate grade stratification than current systems, simplified grading system of five grades, and lowest grade is 1, as opposed to 6, with the potential to reduce overtreatment of PCa. Patient summary We looked at outcomes for prostate cancer (PCa) treated with radical prostatectomy or radiation therapy and validated a new grading system with more accurate grade stratification than current systems, including a simplified grading system of five grades and a lowest grade is 1, as opposed to 6, with the potential to reduce overtreatment of PCa.
Contents
The objective of this study was to find out the impact of L‐carnitine (10 mM) on developmental regulation of preimplantation sheep embryos cultured in vitro when supplemented in maturation ...medium and post‐fertilization medium separately. Subsequent objective was to observe the L‐carnitine‐mediated alteration in expression of apoptotic genes (Bcl2, Bax, Casp3 and PCNA) in sheep oocytes and developing embryos produced in vitro. Oocytes matured with L‐carnitine showed significantly (p < .05) higher cleavage (67.23% vs 43.12%), morula (47.65% vs 28.58%) and blastocysts (32.12% vs 13.24%) percentage as compared to presumptive zygotes cultured with L‐carnitine during post‐fertilization period. So it is suggested to use L‐carnitine during maturation than post‐fertilization period. Antiapoptotic and proliferative effects of L‐carnitine were confirmed by inducing culture medium with actinomycin D (apoptotic agent) and TNFα (antiproliferative agent), respectively, with and without L‐carnitine. Oocytes and embryos cultured with actinomycin D and TNFα showed developmental arrest with significant (p < .05) decrease in morula and blastocysts percentage but supplementation of L‐carnitine to actinomycin D and TNFα induced culture medium showed similar result as that of control. L‐carnitine supplementation during IVM significantly (p < .05) upregulated the expression of Bcl2 and PCNA genes in majority of the developmental stages. Although L‐carnitine upregulated the expression of Bax in initial developmental stages but downregulated at latter part, whereas the expression of Casp3 was upregulated upto 16‐cell stage but after that there was no difference in expression. Expression of GAPDH gene was not affected by L‐carnitine supplementation. In conclusion, L‐carnitine acted as an antiapoptotic and proliferative compound during embryo development and supplementation of L‐carnitine during IVM altered the expression of apoptotic genes in the developmental stages of embryos.
In Vitro Embryo Production in Sheep Mondal, S; Mor, A; Reddy, I J ...
Methods in molecular biology (Clifton, N.J.),
2019, Letnik:
2006
Journal Article
The recent advances in biotechnological research have led to development of many advanced reproductive techniques and biological tools which are set to revolutionize the productive efficiency of ...livestock species. The development of technology for sequencing of whole genomes and mass screening of gene regulation has widened our approach to genetic profiling and mapping, as well as furthering our understanding of underlying physiological mechanisms. The newer biotechnologies of gene transfer, in vitro fertilization and embryo production, cloning, and stem cell technology have been developed and are being refined with efficiencies suitable for use in animal farming. Efficient in vitro systems for maturing oocytes, fertilizing, and developing embryos have resulted in commercial in vitro production of embryos. Here we describe in vitro maturation, in vitro fertilization, embryo production, embryo culture, and quantitation of gene expression in sheep embryos.
Contents
Early embryonic mortality is one of the main sources of reproductive loss in domestic ruminants including sheep. Fibroblast growth factor‐2 (FGF‐2) is a member of FGFs family that mediates ...trophoblast activities and regulates embryonic development in various species. In this study, we have cloned, characterized sheep FGF2 cDNA (KU316368) and studied the expression in sheep embryos. Ovaries of non‐pregnant sheep were collected from local abattoir and matured in culture medium at 38.5ºC, 5% CO2, 95% humidity for 22–24 hr. The matured oocytes were inseminated with capacitated spermatozoa in Brackett and Oliphant medium and resulted embryos were cultured in CO2 incubator for 6–7 days to complete the developmental stages from two cells to blastocyst stage. Total RNA was extracted from immature oocytes (n = 100), mature oocytes (n = 100) and different stages of embryos such as 2 cell (n = 50), 4 cell (n = 25), 8 cell (n = 12), 16 cell (n = 6), morula (n = 5) and blastocyst (n = 3). The total RNA isolated from the oocytes and embryos was reverse transcribed and subjected to real‐time polymerase chain reaction using sequence‐specific primers and SYBR green as the DNA dye. On sequence analysis, the nucleotide sequence of sheep FGF2 exhibited highest sequence similarity with cattle (100%) and least with rat and mouse (69.2%). At the deduced amino acid level, a highest degree of similarity was noticed with cattle, buffalo, goat, pig, camel and horse (100%) and lowest degree of identity with rat, human and mouse (98.2%). The FGF2 mRNA expression was higher in immature and mature oocytes and gradually decreases from 2‐cell stage of embryo to the blastocyst stage. More over a significant differences in FGF2 mRNA expression (p < .05) were observed between immature oocytes and all pre‐implantation stages of embryo. It can be concluded that FGF‐2 plays a significant role in pre‐implantation and early development of embryos in sheep.
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