284 IN VITRO FUNCTION OF FROZEN - THAWED BOTTLENOSE DOLPHIN (TURSIOPS TRUNCATUS) SPERM UNDERGOING SORTING AND RECRYOPRESERVATION

2011 ◽  
Vol 23 (1) ◽  
pp. 240 ◽  
Author(s):  
G. A. Montano ◽  
D. C. Kraemer ◽  
C. C. Love ◽  
T. R. Robeck ◽  
J. K. O'Brien
Keyword(s):  
Dna Fragmentation ◽  
Incubation Period ◽  
Bottlenose Dolphin ◽  
Membrane Integrity ◽  
Sperm Chromatin ◽  
Computer Assisted ◽  
Sperm Dna Fragmentation ◽  
Hoechst 33342

Artificial insemination (AI) using sex-selected sperm of bottlenose dolphins is currently used for the reproductive and social management of captive populations, but distance of males to the sorting facility represents a limitation of the procedure. Sorting and recryopreservation of previously frozen–thawed (FSF) sperm would facilitate the global application of this technology. Although a calf has been produced using FSF sperm (O’Brien et al. 2009 Theriogenology 71, 98–107), a comprehensive examination of the in vitro quality of such samples is needed. The objective was to compare the in vitro quality of nonsorted (CNTR) and sorted (FSF) dolphin sperm before and after recryopreservation using straw (STR) and directional freezing (DF) methods. At all assessment intervals, sperm were evaluated for 1) motility parameters with computer-assisted sperm analysis (CASA); 2) plasma membrane integrity (viability) and acrosome integrity using propidium iodide/fluorescein isothiocyanate-labeled peanut agglutinin (PI/FITC-PNA) staining and 3) DNA denaturation using the sperm chromatin structure assay (SCSA). Semen from 3 ejaculates × 3 males was cryopreserved by DF. After thawing, samples were divided into CNTR and FSF. The CNTR sperm were recryopreserved using STR and DF methods with assessments performed after the first thaw (PT1) and before recryopreservation (PF2). The FSF sperm were prepared for sorting using a density gradient centrifugation (DGC) method, stained with Hoechst 33342, sorted (SX MoFlo®, Dako, Fort Collins, CO, USA), then recryopreserved using STR and DF methods. The FSF sperm were assessed post-PT1, post-DGC, post-stain, post-sort, and at PF2. After the second thaw (PT2), CNTR and FSF samples were diluted (1:0.1, vol/vol) with Androhep Enduraguard™ (AE; Minitube of America, Verona, WI, USA), incubated at room temperature, and assessed at 0, 6, 12, 18, and 24 h PT2. The PT1 samples retained high proportions of their PF1 total motility (TM) and progressive motility (PM) (mean ± SD; 87.9 ± 7.3% and 92.2 ± 5.9%, respectively). The FSF sperm had improved (ANOVA; P < 0.05) motility (TM, PM, VAP, VCL, VSL) and viability at PF2 compared with PF1. The FSF sperm recryopreserved using DF had higher (P < 0.05) motility over the 24-h post-thaw incubation period compared with STR. The CNTR sperm DNA fragmentation remained unchanged throughout the process. The DNA fragmentation of FSF samples increased after staining (P < 0.05), then decreased during the PT2 incubation period, stabilising at lower values (P < 0.05) than CNTR from 6 to 24 h PT2. This unusual pattern indicates a possible interaction between Hoechst 33342 and acridine orange. After recryopreservation, the viability of FSF sperm was higher (P < 0.05) than that of CNTR sperm. Results indicate that bottlenose dolphin sperm undergoing cryopreservation, sorting, and recryopreservation are of adequate quality for use in AI.

P–030 A sperm chromatin damage >15% negatively impacts on the quality of embryos obtained from ovum donation ICSI cycles of unselected couples

2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
A Pachec. Castro ◽  
I Hervas ◽  
R Rivera-Egea ◽  
M Gi. Julia ◽  
A Navarro-Gomezlechón ◽  
...  
Keyword(s):  
Clinical Practice ◽  
Dna Fragmentation ◽  
Embryo Quality ◽  
Blastocyst Stage ◽  
Sperm Chromatin ◽  
Sperm Dna Fragmentation ◽  
Cleavage Stage ◽  
Ovum Donation ◽  
Embryo Fragmentation

Abstract Study question Is embryo quality downgraded in couples with elevated sperm DNA fragmentation (SDF) in the ejaculated semen of male partner using donated eggs? Summary answer The rate of good quality embryos at day 3 and blastocyst-stage is statistically inferior in males with SDF&gt;15% undergoing ICSI cycles with donated oocytes. What is known already The effect of a damaged paternal chromatin will be shown from the 8-cell stage of embryo development, a time which the genome of the embryo is transcriptionally active. Fertilization with a spermatozoon with fragmented DNA may impair the quality of the embryos obtained per cycle, and therefore reduce the chances of pregnancy. The use of donated oocytes is an ideal model to evaluate the real effect of SDF on embryo quality by standardizing the female factor. In addition, we have a large cohort of ovum donation cases in our history, which allows a more proper evaluation of the effect. Study design, size, duration Retrospective multicentric study including the clinical data of 864 couples of ovum donation program who underwent 1903 ICSI cycles between January 2000 and March 2019. The DNA fragmentation of their ejaculated spermatozoa was measured by TUNEL assay (Terminal deoxynucleotidyl transferase dUTP nick end labeling). Two study groups were created according to the SDF level: ≤15% (low) (n = 1626) or &gt; 15% (high) (n = 277). Participants/materials, setting, methods Embryos were evaluated throughout embryonic development according to classical morphological parameters at day 3 (D3), on cleavage-stage, and at day 5 (D5), on blastocyst-stage (trophectoderm (TE) and inner cell mass (ICM)), following ASEBIR guidelines, categorized from A to D. Embryos scored as A and B were considered to be good quality. The proportion of embryos was calculated according to the total number of correctly fertilized oocytes or zygotes. A p &lt; 0.05 was considered significant. Main results and the role of chance A total of 6130 embryos were evaluated. The SDF average of ≤ 15% group was 5.9% (95%CI 5.7–6.1) and 24.3% (95%CI 23.2–25.3) in the &gt;15% group. The cycle-related characteristics and the seminal parameters were comparable. The proportion of optimal cleavage-stage embryo (number of A+B embryos at D3) per cycle was 21.7% (95%CI 19.0–24.5) (8.1 average cells number, 0.8 embryo fragmentation average, symmetry 1, mononucleated cells) in ≤ 15% SDF group versus 21.1% (95%CI 13.9–28.3) (8.2 cells number average, 1.3 embryo fragmentation average, symmetry 1, mononucleated cells) (p &lt; 0.001). The blastocyst-stage arrival rate (number of embryos at D5) per cycle was higher in the &gt;15% SDF group (p &lt; 0.001), 53.4% (95%CI 48.8–58.1) (TE quality A:20.5%, B:42.5%, C:22.7%, D:14.8%, and the ICM quality A:26.1%, B:52.1%, C:13.2%, D:6.2%) versus 49.9% (95%CI 48.1–51.6) (TE quality A:21.1%, B:42.8%, C:21.85, D:14.1% and ICM A:26.6%, B:55.5%, C:11.1%, D:4.7%) in the low SDF group. The rate of good quality blastocyst (number of quality A+B embryos in D5) per cycle was significantly higher in the couples with low SDF (24.8% (95%CI 23.6–25.9)) than in those with elevated SDF (23.5% (95%CI20.9–26.2)) (p &lt; 0.001). Accordingly, the A+B blastocyst rate divided by the total number of blastocysts was 59.1% (95%CI 56.7–61.4) versus 55.9% (95%CI 49.9–62.0) (p &lt; 0.001), respectively. Limitations, reasons for caution The main limitation is that retrospective design of the study may not eliminate the potential unaccounted-for bias derived from the clinical practice of multiple centers even though both groups were statistically comparable. Also, the assessment of embryo quality is still remaining highly subjective to embryologists. Wider implications of the findings: Although the effect size is small, it may be useful in clinical practice when an ICSI cycle yields no good-quality embryos, as one of the underlying causes of that fact. Knowing the SDF level can be a helpful tool in making subsequent clinical decisions aimed at improving outcomes for couples. Trial registration number Not applicable

105 Increasing fertility of interspecific hybrid males using biotechnological approaches

2021 ◽  
Vol 33 (2) ◽  
pp. 159
Author(s):  
A. Vetokh ◽  
A. Tadzhieva ◽  
B. Iolchiev ◽  
N. Volkova ◽  
V. Bagirov
Keyword(s):  
Dna Fragmentation ◽  
Interspecific Hybrid ◽  
Semen Quality ◽  
Sperm Quality ◽  
Semen Analysis ◽  
Differential Staining ◽  
Computer Assisted ◽  
Sperm Dna Fragmentation ◽  
Russian Science

The results of AI depend on many factors, with the quality of semen being one of the most important. Not all male hybrids can meet the requirements for semen quality, because they often have reduced fertility following cryopreservation. Thus, it is necessary to improve semen processing before use in AI. The aim of the study was to evaluate the effectiveness of using the “swim-up” flotation method to improve sperm quality of hybrid males of the Ovis genus. Semen from interspecific hybrid rams (1/4 Argali×3/4 Romanov, n=15; 1/8 Argali×7/8 Romanov, n=15) was freshly obtained, frozen–thawed, and processed by the swim-up method. Evaluation of sperm motility was determined using computer-assisted semen analysis. Statistical analysis was performed using SPSS vs.15.0 (ANOVA and t-test; SPSS Inc.). Semen was collected during the breeding season (October–December) via artificial vagina. Assessment of acrosome integrity was determined using differential staining with a Diachem diff-quick kit (NPF ABRIS+). The degree of sperm DNA fragmentation was determined using the acridine-orange test. The sperm freezing/thawing cycle was accompanied by sperm damage and an increase in the proportion of immobile sperm from 10 to 58%, with non-progressive movement increasing from 9 to 19.3%. The number of spermatozoa with abnormal morphology doubled, and the DNA fragmentation index increased from 16 to 26%. Use of the swim-up procedure allowed us to sort progressively motile spermatozoa. The content of progressively motile spermatozoa in the samples obtained from the supernatant was 86%, which was 2.3 times higher than in frozen–thawed sperm (P≤0,01). The obtained results show the effective use of the swim-up procedure to determine the quality of semen in hybrid rams. These studies were carried out with financial support from the Russian Science Foundation, grant No. 18-16-00079 and the Ministry of Science and Higher Education of the Russian Federation.

scholarly journals Antibiotics Versus Natural Biomolecules: The Case of In Vitro Induced Bacteriospermia by Enterococcus Faecalis in Rabbit Semen

Molecules ◽  
2019 ◽  
Vol 24 (23) ◽  
pp. 4329 ◽  
Author(s):  
Michal Duracka ◽  
Norbert Lukac ◽  
Miroslava Kacaniova ◽  
Attila Kantor ◽  
Lukas Hleba ◽  
...  
Keyword(s):  
Dna Fragmentation ◽  
Enterococcus Faecalis ◽  
Antioxidant Properties ◽  
Membrane Integrity ◽  
Bioactive Molecules ◽  
Human Reproduction ◽  
Sperm Chromatin ◽  
Dna Integrity ◽  
Computer Assisted ◽  
Microbiological Analysis

Male subfertility is a global issue in human reproduction as well as in animal reproduction. Bacterial infection and semen contamination are still widely overlooked. As the collection of ejaculates is not a sterile process, it is necessary to add antimicrobial agents to avoid a possible depreciation of semen samples. As traditionally used antibiotics have been questioned because of an ever-increasing bacterial resistance, natural bioactive molecules could offer an alternative because of their antibacterial and antioxidant properties. As such, we decided to compare the effects of selected natural biomolecules (resveratrol-RES, quercetin-QUE and curcumin-CUR) with routinely used antibiotics in animal biotechnologies (penicillin-PEN, gentamicin-GEN and kanamycin-KAN) on the rabbit sperm vitality in the presence of Enterococcus faecalis. Changes in the sperm structural integrity and functional activity were monitored at 0, 2, 4 and 6 h. Computer-assisted sperm analysis (CASA) was used for the assessment of spermatozoa motility. Production of reactive oxygen species (ROS) was evaluated using chemiluminiscence, while the mitochondrial membrane potential (ΔΨm) was examined using the JC-1 dye. Finally, the sperm chromatin dispersion (SCD) test was used to assess DNA fragmentation, and changes to the membrane integrity were evaluated with the help of annexin V/propidium iodide. The motility assessment revealed a significant sperm motility preservation following treatment with GEN (p < 0.001), followed by PEN and CUR (p < 0.01). QUE was the most capable substance to scavenge excessive ROS (p < 0.001) and to maintain ΔΨm (p < 0.01). The SCD assay revealed that the presence of bacteria and antibiotics significantly (p < 0.05) increased the DNA fragmentation. On the other hand, all bioactive compounds readily preserved the DNA integrity (p < 0.05). In contrast to the antibiotics, the natural biomolecules significantly maintained the sperm membrane integrity (p < 0.05). The microbiological analysis showed that GEN (p < 0.001), KAN (p < 0.001), PEN (p < 0.01) and CUR (p < 0.01) exhibited the strongest antibacterial activity against E. faecalis. In conclusion, all selected biomolecules provided protection to rabbit spermatozoa against deleterious changes to their structure and function as a result of Enterococcus faecalis contamination. Therefore, administration of RES, QUE and/or CUR to rabbit semen extenders in combination with a carefully selected antibacterial substance may be desirable.

In vitro Supplementation of Linoleic Acid Improves Quality of Cryopreserved Buffalo Semen

2020 ◽  
Author(s):  
R Ejaz ◽  
S Qadeer ◽  
M S Ansari ◽  
B A Rakha ◽  
S Shamas ◽  
...  
Keyword(s):  
Linoleic Acid ◽  
Membrane Integrity ◽  
Sperm Chromatin ◽  
Standard Procedures ◽  
Buffalo Semen ◽  
Chromatin Integrity ◽  
Live Sperm ◽  
And Control

Present study was designed to evaluate the effect of linoleic acid (LA) supplementation in extender on post thaw quality of cryopreserved buffalo semen. Semen was collected from three adult Nili Ravi buffalo bulls of same age with artificial vagina (42°C) for five weeks (replicates; N=30). Qualified semen ejaculates (>1mL volume, >60% motility, >0.5 billion/mL concentration) were diluted in tris-citric acid extender containing 0.0 (control), 5.0, 10.0 and 20.0ng mL-1 of LA and were cryopreserved using standard procedures. Sperm motility and plasma membrane integrity were improved plessthan0.05) in extender containing 10.0 ng mL-1 of LA compared to other treatments and control while number of acrosome intact live sperm, chromatin integrity and number of morphologically normal sperms remained the same. In conclusion, LA supplementation in extender at 10.0 ng mL-1 was found to be beneficial to improve post thaw quality of cryopreserved buffalo semen.

Effect of sperm preparation on development of bovine blastocyst in vitro

Zygote ◽  
2016 ◽  
Vol 24 (6) ◽  
pp. 825-830 ◽  
Author(s):  
Maria Celina Abraham ◽  
Anders Johannisson ◽  
Jane M. Morrell
Keyword(s):  
Assisted Reproduction ◽  
Membrane Integrity ◽  
Single Layer ◽  
Human Sperm ◽  
Sperm Chromatin ◽  
Computer Assisted ◽  
Cleavage Rate ◽  
Preparation Methods ◽  
Sperm Preparation

SummarySperm preparation is an important step in the in vitro production of embryos. Centrifugation through colloids has been used to select normal sperm for assisted reproduction in several species. Animal models can sometimes be used as a preliminary step to investigate sperm preparation methods that are potentially of use for human fertility treatments. In this study bovine semen was prepared using three variants of the single-layer centrifugation sperm selection technique (Small, Mini, Mini-EP) with Bovicoll (Androcoll-B). Computer-assisted sperm motility analysis, the hypo-osmotic swelling test, and the sperm chromatin structure assay were performed on unselected (control) and SLC-selected sperm samples. Mini and Mini-EP gave the highest yield of motile spermatozoa, progressive motility and membrane integrity. In vitro fertilization trials were performed to investigate the fertilizing ability of the frozen–thawed bovine spermatozoa selected with Bovicoll. Mini-SLC (single-layer centrifugation) and swim-up (Control) were performed and cleavage rate and blastocyst rate did not differ significantly between groups. As there was a trend to an increased number of cells in blastocysts in the SLC group, the Mini-SLC method is at least as good as swim-up for selecting frozen–thawed bull spermatozoa for in vitro fertilization (IVF). This method could potentially be used to prepare human sperm for assisted reproduction.

219 SORTING OF EQUINE SPERM USING A MICROFLUIDIC DEVICE AS A METHOD OF SPERM SELECTION FOR IN VITRO FERTILIZATION AND INTRACYTOPLASMIC SPERM INJECTION

2016 ◽  
Vol 28 (2) ◽  
pp. 241 ◽  
Author(s):  
R. Gonzalez ◽  
E. Carnevale
Keyword(s):  
Dna Fragmentation ◽  
Microfluidic Device ◽  
Membrane Integrity ◽  
Sperm Parameters ◽  
Dna Integrity ◽  
Sperm Dna Fragmentation ◽  
Normal Morphology ◽  
Vitro Fertilization ◽  
Sperm Sorting

Microfluidic technology can be used for sperm separation. Microfluidic devices generate a fluid flow to sort sperm from a media reservoir into a collection chamber. In the human and mouse, the use of microfluidic devices resulted in the selection of sperm with improved sperm motility, normal morphology, and DNA integrity for in vitro fertilization (IVF), intrauterine insemination (IUI), and intracytoplasmic sperm injection (ICSI). With the use of microfluidic sperm separation, centrifugation can be eliminated, diminishing the risk of reactive oxygen species exposure and DNA damage. We hypothesised that equine sperm can be separated using a microfluidic sorting device (Fertile PlusTM Sperm Sorting Chip; DxNow, Worcester, MA, USA) to improve the quality of sperm for ICSI. The aim of our research was to evaluate sperm parameters, including motility, morphology, membrane integrity, and DNA integrity, in frozen-thawed samples of equine semen before and after sorting using the Fertile Plus Sperm Sorting Chip. Two experiments were performed. In Experiment 1, the microfluidic device was used to separate frozen-thawed semen samples (n = 10) from research stallions (n = 3) with good quality frozen semen; all semen was frozen by one method in our laboratory. In Experiment 2, clinical samples of frozen-thawed semen (n = 11) from 7 stallions were evaluated. The semen was of variable quality and frozen at different facilities. Sperm analyses included (1) motility, (2) morphology (Hancock stain, Animal Reproduction Systems, Chino, CA, USA), (3) live-dead sperm (Hancock stain), (4) membrane integrity (HOS, hypo-osmotic swelling test), and (5) DNA fragmentation (SCD, sperm chromatin dispersion). Two sample t-tests were used to compare sperm parameters. In Experiment 1, use of the Fertile Plus Sperm Sorting Chip improved sperm parameters between the original and sorted samples, respectively: sperm motility (37.2 ± 13.0% and 62.2 ± 15.6%; P = 0.002), normal morphology (60.1 ± 12.2% and 75.5 ± 9.7%; P = 0.006), percentage live sperm (55.8 ± 16.0% and 73.6 ± 12.9%; P = 0.03), HOS (33.7 ± 7.2% and 48 ± 9.7%; P = 0.001) and sperm DNA fragmentation (12.3 ± 4.4% and 5.6 ± 4.4%; P = 0.004). When the Fertile Plus Sperm Sorting Chip was used in Experiment 2 to separate frozen-thawed semen from various sources, improvements were noted between the original and sorted samples, respectively, with increased motility (22.0 ± 13.0% and 57.0 ± 11.6%; P = 0.0009), normal morphology (58.4 ± 9.6% and 74.0 ± 10.3%; P = 0.005), a higher percentage of live sperm (55.5 ± 11.2% and 68.3 ± 14.2%; P = 0.04), and decreased sperm DNA fragmentation (22.3 ± 14.7% and 8.2 ± 8.3%; P = 0.004); no effect was observed on HOS (21.2 ± 6.0% and 24.9 ± 11.5%; P = 0.19). Our results demonstrate that use of the Fertile Plus Sperm Sorting Chip resulted in a subpopulation of sperm with improved quality parameters. Separation of sperm using a microfluidic device has the potential to select sperm with desirable characteristics for equine assisted reproductive techniques.

Influence of bovine sperm DNA fragmentation and oxidative stress on early embryo in vitro development outcome

Reproduction ◽  
2013 ◽  
Vol 146 (5) ◽  
pp. 433-441 ◽  
Author(s):  
Renata Simões ◽  
Weber Beringui Feitosa ◽  
Adriano Felipe Perez Siqueira ◽  
Marcilio Nichi ◽  
Fabíola Freitas Paula-Lopes ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Dna Fragmentation ◽  
Sperm Chromatin ◽  
Dna Integrity ◽  
Sperm Dna Fragmentation ◽  
Cleavage Rate ◽  
Sperm Dna Integrity ◽  
Sperm Dna ◽  
Significant Difference

Sperm chromatin fragmentation may be caused by a number of factors, the most significant of which is reactive oxygen species. However, little is known about the effect of sperm oxidative stress (OS) on DNA integrity, fertilization, and embryonic development in cattle. Therefore, the goal of this study was to evaluate the influence of sperm OS susceptibility on the DNA fragmentation rate and in vitro embryo production (IVP) in a population of bulls. Groups of cryopreserved sperm samples were divided into four groups, based on their susceptibility to OS (G1, low OS; G2, average OS; G3, high OS; and G4, highest OS). Our results demonstrated that the sperm DNA integrity was compromised in response to increased OS susceptibility. Furthermore, semen samples with lower susceptibility to OS were also less susceptible to DNA damage (G1, 4.06%; G2, 6.09%; G3, 6.19%; and G4, 6.20%). In addition, embryo IVP provided evidence that the embryo cleavage rate decreased as the OS increased (G1, 70.18%; G2, 62.24%; G3, 55.85%; and G4, 50.93%), but no significant difference in the blastocyst rate or the number of blastomeres was observed among the groups. The groups with greater sensitivity to OS were also associated with a greater percentage of apoptotic cells (G1, 2.6%; G2, 2.76%; G3, 5.59%; and G4, 4.49%). In conclusion, we demonstrated that an increased susceptibility to OS compromises sperm DNA integrity and consequently reduces embryo quality.

Sperm DNA fragmentation measured by sperm chromatin dispersion impacts morphokinetic parameters, fertilization rate and blastocyst quality in ICSI treatments

Zygote ◽  
2021 ◽  
pp. 1-8
Author(s):  
Shikai Wang ◽  
Weihong Tan ◽  
Yueyue Huang ◽  
Xianbao Mao ◽  
Zhengda Li ◽  
...  
Keyword(s):  
Dna Fragmentation ◽  
Embryo Quality ◽  
Fertilization Rate ◽  
Sperm Chromatin ◽  
Sperm Dna Fragmentation ◽  
Blastocyst Formation ◽  
High Quality ◽  
Morphokinetic Parameters ◽  
Sperm Dna ◽  
Blastocyst Quality

Summary To determine the effects of sperm DNA fragmentation (SDF) on embryo morphokinetic parameters, cleavage patterns and embryo quality, this retrospective study analyzed 151 intracytoplasmic sperm injection (ICSI) cycles (1152 embryos collected) between November 2016 and June 2019. SDF was assessed using sperm chromatin dispersion. The cycles were divided into two groups based on the SDF rate: SDF < 15% (n = 114) and SDF ≥ 15% (n = 37). The embryo morphokinetic parameters, cleavage patterns, and embryo quality were compared between the two groups. The morphokinetic parameters tPNf, t2, t3, t4, t5, t6, and t8 were achieved significantly earlier in the SDF < 15% group compared with in the SDF ≥ 15% group. The fertilization and 2PN rates seemed to be significantly higher in the SDF < 15% group compared with in the SDF ≥ 15% group, while the abnormal cleavage rates were similar. However, a significantly higher rate of chaotic cleavage (CC) was observed in the SDF ≥ 15% group. The D3 high-quality embryo and available embryo rates were similar between the two groups. The blastocyst formation, high-quality blastocyst, and available blastocyst rates in the SDF < 15% group were significantly higher than those in the SDF ≥ 15% group. With an increase in SDF level, the chemical pregnancy, clinical pregnancy and implantation rates tended to decrease, while the miscarriage rate increased. This study demonstrated that SDF ≥ 15% reduces the fertilization rate of ICSI cycles and affects certain morphokinetic parameters. A higher SDF level can also induce a higher rate of CC, with subsequent decreases in the blastocyst formation rate and blastocyst quality.

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