MAKNA SUNGAI DALAM RUANG HIDUP YANG BERUBAH: STUDI KASUS DAYAK NGAJU, KALIMANTAN TENGAH

The rivers flow on the island of Borneo have had a significant function for human life since a long time ago. The river has had a role in the dynamics of civilization and environmental evolution in human life until now. With the construction of road infrastructure, the significance of rivers for riverside communities has also changed. This study sees how Ngaju people interpret the river in a living space that changes with development. The research was conducted in two villages which show the characteristics of different locations, society, and development rates. The villages are Talingke village on the bank of the Katingan River and Pangi Village on the bank of the Kahayan River. Data collection were through observation, interviews, and literature study. The study shows that the characteristics of the river area, the community, and the development rates affect the community in interpreting the river. On the one hand, Pangi people start to leave activities in the river, but they still hold the values of the river in their lives. On the other hand, Talingke’s people still focus their activities on the river but no longer have river values in their lives.

DNA damage in cumulus cells generated after the vitrification of in vitro matured porcine oocytes and its impact on fertilization and embryo development

Background The evaluation of the DNA damage generated in cumulus cells after mature cumulus-oocyte complexes vitrification can be considered as an indicator of oocyte quality since these cells play important roles in oocyte developmental competence. Therefore, the aim of this study was to determine if matured cumulus-oocyte complexes exposure to cryoprotectants (CPAs) or vitrification affects oocytes and cumulus cells viability, but also if DNA damage is generated in cumulus cells, affecting fertilization and embryo development. Results The DNA damage in cumulus cells was measured using the alkaline comet assay and expressed as Comet Tail Length (CTL) and Olive Tail Moment (OTM). Results demonstrate that oocyte exposure to CPAs or vitrification reduced oocyte (75.5 ± 3.69%, Toxicity; 66.7 ± 4.57%, Vitrification) and cumulus cells viability (32.7 ± 5.85%, Toxicity; 7.7 ± 2.21%, Vitrification) compared to control (95.5 ± 4.04%, oocytes; 89 ± 4.24%, cumulus cells). Also, significantly higher DNA damage expressed as OTM was generated in the cumulus cells after exposure to CPAs and vitrification (39 ± 17.41, 33.6 ± 16.69, respectively) compared to control (7.4 ± 4.22). In addition, fertilization and embryo development rates also decreased after exposure to CPAs (35.3 ± 16.65%, 22.6 ± 3.05%, respectively) and vitrification (32.3 ± 9.29%, 20 ± 1%, respectively). It was also found that fertilization and embryo development rates in granulose-intact oocytes were significantly higher compared to denuded oocytes in the control groups. However, a decline in embryo development to the blastocyst stage was observed after CPAs exposure (1.66 ± 0.57%) or vitrification (2 ± 1%) compared to control (22.3 ± 2.51%). This could be attributed to the reduction in both cell types viability, and the generation of DNA damage in the cumulus cells. Conclusion This study demonstrates that oocyte exposure to CPAs or vitrification reduced viability in oocytes and cumulus cells, and generated DNA damage in the cumulus cells, affecting fertilization and embryo development rates. These findings will allow to understand some of the mechanisms of oocyte damage after vitrification that compromise their developmental capacity, as well as the search for new vitrification strategies to increase fertilization and embryo development rates by preserving the integrity of the cumulus cells.

Melatonin Promotes In Vitro Maturation of Vitrified-Warmed Mouse Germinal Vesicle Oocytes, Potentially by Reducing Oxidative Stress through the Nrf2 Pathway

Previously it was reported that melatonin could mitigate oxidative stress caused by oocyte cryopreservation; however, the underlying molecular mechanisms which cause this remain unclear. The objective was to explore whether melatonin could reduce oxidative stress during in vitro maturation of vitrified-warmed mouse germinal vesicle (GV) oocytes through the Nrf2 signaling pathway or its receptors. During in vitro maturation of vitrified-warmed mouse GV oocytes, there were decreases (p < 0.05) in the development rates of metaphase I (MI) oocytes and metaphase II (MII) and spindle morphology grades; increases (p < 0.05) in the reactive oxygen species (ROS) levels; and decreases (p < 0.05) in expressions of Nrf2 signaling pathway-related genes (Nrf2, SOD1) and proteins (Nrf2, HO-1). However, adding 10−7 mol/L melatonin to both the warming solution and maturation solutions improved (p < 0.05) these indicators. When the Nrf2 protein was specifically inhibited by Brusatol, melatonin did not increase development rates, spindle morphology grades, genes, or protein expressions, nor did it reduce vitrification-induced intracellular oxidative stress in GV oocytes during in vitro maturation. In addition, when melatonin receptors were inhibited by luzindole, the ability of melatonin to scavenge intracellular ROS was decreased, and the expressions of genes (Nrf2, SOD1) and proteins (Nrf2, HO-1) were not restored to control levels. Therefore, we concluded that 10−7 mol/L melatonin acted on the Nrf2 signaling pathway through its receptors to regulate the expression of genes (Nrf2, SOD1) and proteins (Nrf2, HO-1), and mitigate intracellular oxidative stress, thereby enhancing in vitro development of vitrified-warmed mouse GV oocytes.

P–032 Assessment of embryonic developmental outcome of direct unequal cleavage in patients with non-obstructive azoospermia and/or obstructive azoospermia

Study question Does direct unequal cleavage (DC) affect embryonic development after ICSI with testicular sperm (TESE-ICSI) in patients with non-obstructive azoospermia (NOA) and/or obstructive azoospermia (OA)? Summary answer The incidence of DC at the first cleavage (DC1) was extremely high and DC1 negatively affected embryonic development in NOA patients. What is known already It has been reported that the blastocyst development of embryos with direct cleavage (DC) was significantly lower than that without DC, but the clinical pregnancy rate after blastocyst transfer was not different with or without DC. The incidence of DC has been reported to be significantly higher after ICSI with testicular sperm (TESE-ICSI) than ICSI with ejaculated sperm (Ej), but to our knowledge, there are few reports investigating that the embryos with DC after TESE-ICSI affect embryonic development. Study design, size, duration We conducted a retrospective cohort study using time-lapse incubators (Geri, Genea Biomedx, Australia) from September 2018 to November 2020. Of 1033 two-pronuclear (2PN) embryos from TESE-ICSI, 486 and 547 embryos were from OA (35.9±5.5 years) and NOA (33.7±5.2 years), respectively. As an age matched control, we chose 581 embryos from ICSI using Ej (36.5±4.4 years). Participants/materials, setting, methods DC embryos were classified as DC1 (DC at first cleavage), DC2 (DC at second cleavage), and non-DC (without DC). The incidences of DC1 or DC2 and blastocyst development rates were compared among OA, NOA and Ej groups. In TESE-ICSI group, we compared blastocyst development rates with or without DC between good and poor quality embryos on day 3. Good quality embryos were defined as 8 cells with G3 or more by the Veeck’s classification. Main results and the role of chance DC1 incidence was significantly higher in NOA (37.3%) than OA (27.8%) and Ej (22.7%) (P &lt; 0.01), whereas DC2 incidence was not statistically different among three groups; NOA (15.7%), OA (15.0%) and Ej (13.4%). Blastocyst development rates in DC1 were 17.8%, 19.5% and 25.8% for NOA, OA and Ej, respectively, which were significantly lower compared to non-DC in corresponding three groups (65.1%, 67.7%, and 68.5%, respectively, P &lt; 0.01). In TESE-ICSI group, good-quality embryo rate on day 3 was significantly lower in DC1 (34.5%, P &lt; 0.01) than DC2 (60.9%) or non-DC (54.2%). Additionally, blastocyst development rates in DC1 and DC2 were significantly lower than non-DC regardless of embryonic grades on day 3 (35.1%, 51.0%, and 81.6% for good-quality embryos on day 3, 10.1%, 27.0%, and 49.1% for poor-quality embryos on day 3, respectively, P &lt; 0.05). When immotile sperm was used for TESE-ICSI, DC1 incidence was 40.0% (6/15), which did not show statistically differences. When performing single frozen-thawed blastocyst transfers, no pregnancies resulted from either DC1 (n = 13) or DC2 (n = 3) embryos in TESE-ICSI group. Limitations, reasons for caution We had a few data about the pregnancy rates after blastocyst transfers with DC, because embryos with DC were seldom transferred due to those lower priority. Although DC might be influenced by the sperm, we did not analyze the incidence of DC by taking the semen factors into account. Wider implications of the findings: The incidence of DC1 was extremely high and DC1 negatively affected embryonic development in NOA patients. Therefore, it is important to observe embryos using time-lapse incubator in order to recognize embryos with/without pregnancy potential, especially for embryos with DC1 in NOA patients. Trial registration number Not applicable

DNA damage in cumulus cells generated after the vitrification of in vitro matured porcine oocytes and its impact on fertilization and embryo development

Oocyte vitrification has become an important tool for the improvement of assisted reproduction in humans and other mammalian species. The toxicity and use of high cryoprotectants concentrations have been a limiting factor for cryopreservation success. The evaluation of the DNA damage generated in cumulus cells after mature cumulus-oocyte complexes vitrification can be considered as an indicator of oocyte quality since these cells play important roles in oocyte developmental competence. Alterations produced in these cells could compromise oocyte maturation, fertilization, and embryo development. Therefore, the aim of this study was to determine if matured cumulus-oocyte complexes exposure to cryoprotectants or vitrification affects both oocytes and cumulus cells viability, but also if DNA damage is generated in cumulus cells, affecting fertilization and embryo development. The DNA damage in cumulus cells was measured using the alkaline comet assay and expressed as Comet Tail Length and Olive Tail Moment. Results demonstrate that oocyte exposure to cryoprotectants or vitrification reduced oocyte and cumulus cells viability compared to control. Also, significantly higher DNA damage was generated in the cumulus cells after exposure to cryoprotectants and vitrification compared to control. In addition, fertilization and embryo development rates also decreased after exposure to cryoprotectants and vitrification. It was also found that fertilization and embryo development rates in granulose-intact oocytes were significantly higher compared to denuded oocytes in the control groups. However, a decline in oocyte fertilization and embryo development to the blastocyst stage was observed after cryoprotectants exposure or vitrification. This could be attributed to the reduction in both cell types viability, and the generation of DNA damage in the cumulus cells. These findings will allow to understand some of the mechanisms of oocyte damage after vitrification, and the search for new vitrification strategies to increase fertilization and embryo development rates.

Effects of Porcine Immature Oocyte Vitrification on Actin Microfilament Distribution and Chromatin Integrity During Early Embryo Development in vitro

Vitrification is mainly used to cryopreserve female gametes. This technique allows maintaining cell viability, functionality, and developmental potential at low temperatures into liquid nitrogen at −196°C. For this, the addition of cryoprotectant agents, which are substances that provide cell protection during cooling and warming, is required. However, they have been reported to be toxic, reducing oocyte viability, maturation, fertilization, and embryo development, possibly by altering cell cytoskeleton structure and chromatin. Previous studies have evaluated the effects of vitrification in the germinal vesicle, metaphase II oocytes, zygotes, and blastocysts, but the knowledge of its impact on their further embryo development is limited. Other studies have evaluated the role of actin microfilaments and chromatin, based on the fertilization and embryo development rates obtained, but not the direct evaluation of these structures in embryos produced from vitrified immature oocytes. Therefore, this study was designed to evaluate how the vitrification of porcine immature oocytes affects early embryo development by the evaluation of actin microfilament distribution and chromatin integrity. Results demonstrate that the damage generated by the vitrification of immature oocytes affects viability, maturation, and the distribution of actin microfilaments and chromatin integrity, observed in early embryos. Therefore, it is suggested that vitrification could affect oocyte repair mechanisms in those structures, being one of the mechanisms that explain the low embryo development rates after vitrification.

Optimized culture systems for the preimplantation ICR mouse embryos with wide range of EDTA concentrations

In this study, in vitro preimplantation embryo culture media especially for outbred stock mice (Institute of Cancer Research (ICR)) were optimized with different concentrations of ethylenediaminetetraacetic acid (EDTA). A plot with embryo development rates against EDTA concentrations ranging from 0 to 500 µM showed a unique pattern with two characteristic peaks. Two hundred micromolar was adopted as an optimal concentration of EDTA. The optimized media were also evaluated with two culture systems: conventional large volume culture system (1 ml) and micro-droplet culture system. In the conventional large volume culture system, the blastocyst development rates were compared among three different media (F-10, KSOM and KSOM with the optimized 200 µM EDTA). The rates were 0.4%, 16.7% and 57.6%, respectively. The development rates for the micro-droplet (10 µl) culture system were 73.9%. In conclusion, 200 µM EDTA concentration in 10 µl droplets in the KSOM medium was found as the most suitable culture conditions for ICR mouse embryos, as the blastocyst development rate was higher in the micro-droplet culture system than in the traditional conventional large volume culture system.

Sperm selection by rheotaxis improves sperm quality and early embryo development

The objective of this work was to elucidate whether a sperm selection method that combines rheotaxis and microfluidics can improve the selection of spermatozoa over density gradient and swim-up. For this purpose human sperm selected by rheotaxis were compared against density gradient, swim-up and a control group of non-selected spermatozoa in split frozen-thawed (FT) and fresh (F) semen samples. Sperm quality was assessed in terms of motility, morphology, DNA fragmentation index (DFI), viability, acrosome integrity and membrane fluidity. Using a mouse model, we compared fertilisation and embryo development rates after performing ICSI with spermatozoa, sorted using rheotaxis or swim-up. Selection by rheotaxis yielded a sperm population with reduced DFI than the control (P < 0.05), improved normal morphology (P < 0.001) and higher total motility (TM; P < 0.001) than the other techniques studied in F and FT samples. Swim-up increased TM compared to density gradient and control in FT or F samples (P < 0.001), and yielded lower DFI than the control with F samples (P < 0.05). In FT samples, selection by rheotaxis yielded sperm with higher viability than control, density gradient and swim-up (P < 0.01) while acrosomal integrity and membrane fluidity were maintained. When mouse spermatozoa were selected for ICSI using rheotaxis compared to swim-up, there was an increase in fertilisation (P < 0.01), implantation (P < 0.001) and foetal development rates (P < 0.05). These results suggest that, in the absence of non-destructive DNA testing, the positive rheotaxis can be used to select a population of low DNA fragmentation spermatozoa with high motility, morphology and viability, leading to improved embryo developmental rates.