15 EFFECT OF SEMEN THAW METHOD ON CONCEPTION RATE IN DAIRY HEIFER HERDS

Semen processed with procedures permitting a flexible thaw method is used to breed millions of cows yearly. “Pocket thawing” is widely used as an alternative to warm-water thawing with such semen. To pocket thaw, a straw is retrieved from cryostorage, immediately wrapped in a folded paper towel, and moved to a thermally protected pocket for 2 to 3 min of thawing within the pocket before AI gun loading. Published field data are lacking for comparisons of such a thaw method with those for semen prepared to permit flexible-thawing. We measured the effect of warm-water or pocket thaw on conception rate in four dairy heifer herds using semen prepared with methods previously optimized for flexible-thawing success. Semen processing (Anderson S et al. 1994 J. Dairy Sci. 77, 2302–2307) includes two-step whole-milk extension, static vapor tank freezing (0.5-mL straws), and IMV Digitcool mechanical freezing (0.25-mL straws). It is unclear which specific processing steps permit flexible thawing. These procedures have been developed using breeding results from decades of field trials by professional inseminators using both pocket and warm-water thaw. Semen prepared from each of 12 sires produced equal straw units at 10 and 15 million total sperm per straw, in both 0.5- and 0.25-mL straw packages. Professional inseminators used each combination evenly over 16 months. Additional commercial semen (55% of total) from the same source was used. The thaw methods alternated weekly. Thaw effect on conception status, from 70 day non-return data for 11,215 services (67.6% conception rate), was estimated by a generalized linear mixed model. Neither thaw method nor total sperm per straw significantly affected conception rate (P = 0.658, 0.769, respectively). Bull, herd, inseminator within herd, year, season, and straw size did significantly affect conception rate (P < 0.05). No thaw method interactions with herd, sperm number, season, and straw package size were significant (P = 0.297, 0.526, 0.365, 0.723, respectively). This suggests that if semen has been prepared with procedures specific to flexible-thawing, it can be either pocket thawed or warm-water thawed within a range of herdsman or inseminator practices, season, or straw packaging choices. Even at 10 million, the lowest total sperm per straw, pocket thaw was equally as successful as warm-water thaw. We generally observe that in vitro sperm quality, as expected, is maximal for rapidly thawed straws, with slower thawing resulting in lower values. However, while it appears that conventional measures of in vitro semen quality are improved with fast thaw rates, these measures do not appear to correspond to higher in vivo fertility for semen prepared intentionally to be flexibly thawed. We conclude that, for semen prepared with procedures that permit flexible thawing, the thaw method, whether pocket or warm-water thaw, does not affect conception under commercial conditions and with routine semen handling methods. We thank the herd owners and their staff, the inseminators, and Hap Allen, Ron Hunt, Gordon Nickerson, and Bryan Krick of Genex for their help and cooperation.

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Optimization of Sperm Cryopreservation Protocol for Mediterranean Brown Trout: A Comparative Study of Non-Permeating Cryoprotectants and Thawing Rates In Vitro and In Vivo

Animals ◽

10.3390/ani9060304 ◽

2019 ◽

Vol 9 (6) ◽

pp. 304 ◽

Cited By ~ 1

Author(s):

Giusy Rusco ◽

Michele Di Iorio ◽

Pier Paolo Gibertoni ◽

Stefano Esposito ◽

Maurizio Penserini ◽

...

Keyword(s):

Brown Trout ◽

Semen Quality ◽

Sperm Quality ◽

Egg Yolk ◽

Dna Integrity ◽

Experimental Conditions ◽

C 30 ◽

Thawing Rate

The aim of our study was to test the effects of different non-permeating cryoprotectants (NP-CPAs), namely low-density lipoproteins (LDLs), sucrose, and egg yolk, and thawing rates on the post-thaw semen quality and fertilizing ability of the native Mediterranean brown trout. Pooled semen samples were diluted 1:3 (v:v) with 2.5%, 5%, 10%, or 15% LDL; 0.05, 0.1, or 0.3 M sucrose; or 10% egg yolk. At the moment of analysis, semen was thawed at 30 °C/10 s or 10 °C/30 s. The post-thaw semen quality was evaluated, considering motility, the duration of motility, viability, and DNA integrity. Significantly higher values of motility and viability were obtained using egg yolk/10 °C for 30 s, across all treatments. However, LDL and sucrose concentrations affected sperm cryosurvival, showing the highest post-thaw sperm quality at 5% LDL and 0.1 M sucrose. Based on the in vitro data, egg yolk, 5% LDL, and 0.1 M sucrose thawed at 10 °C or 30 °C were tested for the in vivo trial. The highest fertilization and hatching rates were recorded using egg yolk/10 °C (p < 0.05). According to these in vitro and in vivo results, egg yolk emerged as the most suitable NP-CPA and 10 °C/30 s as the best thawing rate for the cryopreservation of this trout sperm, under our experimental conditions.

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Making the most of sperm activation responses: experiments with boar spermatozoa and bicarbonate

Reproduction Fertility and Development ◽

10.1071/rd17476 ◽

2018 ◽

Vol 30 (6) ◽

pp. 842 ◽

Cited By ~ 4

Author(s):

William V. Holt ◽

Nana Satake

Keyword(s):

Semen Quality ◽

Current Knowledge ◽

Sperm Quality ◽

Environmental Chemicals ◽

Information Value ◽

In Vitro Tests ◽

Sperm Activation ◽

Activation Mechanisms

Attempting to extract useful and reliable information about semen quality and its fertility potential remains a difficult exercise, partly because the sperm heterogeneity within samples often renders simple statistical analyses rather meaningless. In fact, a mean and standard deviation may reflect neither the very fast swimming activities of the most active cells nor the slow and sluggish activities of others. Herein we propose that the information value within semen samples can be maximised if current knowledge about sperm activation mechanisms is exploited before undertaking the measurements. We explain, using boar semen as an example, that estimating and defining relative sperm subpopulation sizes, after activation by bicarbonate, provides a means of quantifying sperm quality. Although such estimates may indeed be related to in vivo fertility, the general approach also suggests potential new avenues that could be exploited for the elaboration of novel in vitro tests for the characterisation of toxic environmental chemicals and, indeed, to reduce the number of animals used in such testing programs.

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Multiparametric Study of Antioxidant Effect on Ram Sperm Cryopreservation—From Field Trials to Research Bench

Animals ◽

10.3390/ani11020283 ◽

2021 ◽

Vol 11 (2) ◽

pp. 283

Author(s):

Marta F. Riesco ◽

Mercedes Alvarez ◽

Luis Anel-Lopez ◽

Marta Neila-Montero ◽

Cristina Palacin-Martinez ◽

...

Keyword(s):

Sperm Quality ◽

Redox Balance ◽

Field Trials ◽

Low Fertility ◽

High Fertility ◽

Sperm Cryopreservation ◽

Ram Sperm ◽

Positive Effect

The optimization of sperm cryopreservation protocols in ram is a feasible tool to reinforce artificial insemination technologies considering the desirable application of sperm by vaginal/cervical or transcervical deposition. Cryopreservation provokes different types of damage on spermatozoa and many of these detrimental effects are triggered by redox deregulation. For this reason, the antioxidant supplementation in sperm cryopreservation protocols to decrease reactive oxygen species (ROS) levels and to equilibrate redox status has been widely employed in different species. Despite this, more fertility trials are necessary to provide the definitive tool to ensure the antioxidant effectiveness on sperm quality. For this reason, in this work, we performed a multiparametric analysis of some previously tested antioxidants (crocin, GSH and Trolox) on ram sperm cryopreservation from field trials to sperm quality analyses focused on new strategies to measure redox balance. Attending to fertility trial, Trolox supplementation registered an improvement concerning to fertility (when we considered high fertility males) and multiple lambing frequency and other complementary and descriptive data related to lambing performance such as prolificacy and fecundity. This positive effect was more evident in multiple lambing frequency when we considered low fertility males than in global male analysis. In vitro analyses of sperm quality confirmed in vivo trials registering a positive effect on sperm viability and redox balance. In this study, we provided the definitive evidence that the role of trolox on redox balance maintenance has a direct effect on fertility parameters, such as prolificacy. The effectiveness of antioxidant treatments was tested, for the first time in ovine species, using an integrative and multiparametric approach combining in vivo and in vitro analyses and novel approaches, such as RedoxSYS. These types of strategies should be applied to improve sperm conservation methods and optimize AI technologies upgrading the correlation between in vitro and in vivo analyses.

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191 SexedULTRA™, A NEW METHOD OF PROCESSING SEX SORTED BOVINE SPERM IMPROVES POST-THAW SPERM QUALITY AND IN VITRO FERTILITY

Reproduction Fertility and Development ◽

10.1071/rdv29n1ab191 ◽

2017 ◽

Vol 29 (1) ◽

pp. 204 ◽

Cited By ~ 9

Author(s):

C. Gonzalez-Marin ◽

R. W. Lenz ◽

T. B. Gilligan ◽

K. M. Evans ◽

C. E. Gongora ◽

...

Keyword(s):

Mixed Model ◽

Semen Quality ◽

Sperm Quality ◽

Random Effect ◽

Fixed Effect ◽

Computer Assisted ◽

Motile Sperm ◽

Bovine Sperm ◽

Mixed Model Anova

Since the first publications 30 years ago showing that flow cytometry was a reliable method to separate X and Y chromosome bearing sperm, the process has been subject to continual refinement. Numerous experiments have been performed in the last few years with the objective of developing an improved sex-sorted product, branded SexedULTRA™ (Sexing Technologies, Navasota, TX, USA) that retains sperm integrity to improve post-thaw sperm quality, in vitro embryo production, and field fertility compared with the previous XY method. Laboratory evaluations were performed on semen from 12 bulls at the Sexing Technologies laboratory in Navasota (TX, USA). Each ejaculate was divided in 2 aliquots and then processed in 1 of 2 methods (XY method or SexedULTRA™). Post-thaw sperm motilities were classified into percent total and progressively motile after thawing (0 h) and after a 3-h incubation at 37°C using a computer-assisted sperm motility analyzer (Hamilton Thorne IVOS II system, Hamilton Thorne Biosciences, Beverly, MA, USA). Percent intact acrosomes was also estimated after a 3-h incubation. Results were analysed by a mixed model ANOVA with the fixed effect of treatment and random effect of bull. Percent total motile SexedULTRA™ sperm was greater (P < 0.001) than sperm processed following the XY method at 0 (78.8 v. 67.2%) and 3 h (51.0% v. 39.0%) post-thaw. Likewise, there was a higher percent of progressively motile sperm both at 0 (50.7 v. 44.9%) and 3 h (31.5 v. 4.4%) post-thaw in the SexedULTRA™ sperm. Percent intact acrosomes was also greater in SexedULTRA™ sperm compared with the sperm processed following previous method (78.0 v. 64.0%). In vitro fertilizations were performed as a measure of sperm competence using 8 ejaculates from 2 bulls in Sexing Technologies IVF laboratory in Laceyville (PA, USA). Five to 10 oocytes and 5,000 motile sperm/oocyte were placed per IVF drop for the analysis. A total of 3 straws and a minimum of 800 oocytes per treatment group (ejaculate × treatment) were included in the comparison for development to 8-cell stage (cleavage rate) and to Day 7 blastocyst stage, measured as total (grades 1 to 4) and freezable (grades 1 and 2) embryos. Results were analysed using a mixed model ANOVA with treatment as a fixed effect and bull, ejaculate within bull, and IVF cycle as random effects. Results from IVF trials are shown in Table 1. Total and freezable embryo numbers were significantly higher (P < 0.05) when using SexedULTRA™ compared with XY sperm. Maintaining a suitable environment for sperm to progress through the various steps of the sex-sorting process results in better semen quality post-thaw as well as improved in vitro fertility. The SexedULTRA™ method confers a significant benefit in maintaining sperm integrity that, if translated into field fertility, could reduce the conception rate gap between conventional and sex-sorted bovine sperm. Table 1. Results from IVF and embryo culture using frozen-thawed, sex-sorted semen processed using the XY or the SexedULTRA™ method

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249 Increasing Structural Fiber Improves Growth Performance of Nursery Pigs

Journal of Animal Science ◽

10.1093/jas/skab054.149 ◽

2021 ◽

Vol 99 (Supplement_1) ◽

pp. 93-93

Author(s):

Sara Ebarb ◽

Sabrina May ◽

Mark D Newcomb

Keyword(s):

Growth Performance ◽

Mixed Model ◽

Linear Mixed Model ◽

Two Phase ◽

Nursery Pigs ◽

High Fiber ◽

Rice Hulls ◽

Fiber Treatment ◽

Wheat Middlings

Abstract Fiber ingredients in swine diets have various components that affect the intestinal tract distinctively. The objective of this study was to determine the effect of structural fiber sources on growth performance of nursery pigs. A total of 1,760 weanling pigs (initial BW = 6.12 ± 0.3 kg, 16 reps/trt, 22 pigs/pen) were used in a study with 5 dietary treatments: 1) Complex nursery diet with no additional fiber (CON); 2) CON + 2% rice hulls; 3) CON + 4% rice hulls; 4) CON + 6% wheat middlings; 5) CON + 12% wheat middlings. Fiber ingredients were added at the expense of corn and some processed soybean meal to maintain similar dietary protein levels. Using an in vitro fermentation estimation of fermentation for rice hulls and wheat middlings, diets were constructed to add similar levels of structural (non-fermentable NDF) fiber between low and high additions of rice hulls and wheat middlings. Pens across two barns were randomly allotted independent of one another and within a location block to one of the five treatments on d 0 of a two-phase study (d 0–11 and d 11–20.5 post-weaning). Data were analyzed by general linear model in R. Mortality and removal data were analyzed as a generalized linear mixed model with a binomial distribution. Contrasts tested the effect of additional fiber (CON vs treatments 2–5), effect of medium vs high fiber (treatment 2/4 vs 3/5), effect of source of fiber (treatment 2/3 vs 4/5), and the interaction of level and source of fiber. Overall (Table 1), additional fiber resulted in increased ADFI (P < 0.05) and tended to increase ADG (P < 0.10). Probability of mortality and removal was reduced (P < 0.05) when additional fiber was included. In summary, increasing the level of structural fiber improved performance and livability of nursery pigs.

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Validation of the Turkey Semen Cryopreservation by Evaluating the Effect of Two Diluents and the Inseminating Doses

Animals ◽

10.3390/ani10081329 ◽

2020 ◽

Vol 10 (8) ◽

pp. 1329

Author(s):

Michele Di Iorio ◽

Giusy Rusco ◽

Roberta Iampietro ◽

Lucia Maiuro ◽

Achille Schiavone ◽

...

Keyword(s):

Sperm Quality ◽

Sperm Concentration ◽

Membrane Integrity ◽

In Vivo Test ◽

Sperm Analysis ◽

Fertilizing Ability ◽

Vitro Analysis ◽

Hatching Rates

This study was designed to test the fertilizing ability of cryopreserved turkey semen, and here, two experiments were performed: an in vitro analysis to assess the effects of Tselutin and Lake diluents and an in vivo test to determine the fertility and hatching rates by also studying the feat of three insemination doses (250, 400 and 600 × 106 sperm/hen). Pooled semen samples were diluted with Tselutin or Lake extender which contained 20% of dimethylsulfoxide and 1 mM of Ficoll at final sperm concentration of 3 × 109 sperm/mL. Thereafter, semen was packaged into straws and frozen on liquid nitrogen. The post-thaw sperm quality was evaluated considering motility (computer-aided sperm analysis—CASA system) and membrane integrity (flow cytometry). Significantly higher values of progressive motility and some kinetic parameters in semen frozen with Lake were found. When we compared the extenders in vivo, no significant effects were detected, whilst sperm concentration significantly affected both fertility and hatching rates, with the best results obtained with the sperm concentration of 400 × 106 sperm/hen. From the results obtained, it emerged that the extender type only affected sperm motility characteristics, not the fertilizing ability of frozen-thawed semen, while inseminating dose markedly affected fertility and hatching rates.

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FTIR Spectroscopy to Reveal Lipid and Protein Changes Induced on Sperm by Capacitation: Bases for an Improvement of Sample Selection in ART

International Journal of Molecular Sciences ◽

10.3390/ijms21228659 ◽

2020 ◽

Vol 21 (22) ◽

pp. 8659

Author(s):

Maria Pachetti ◽

Luisa Zupin ◽

Irene Venturin ◽

Elisa Mitri ◽

Rita Boscolo ◽

...

Keyword(s):

Lipid Composition ◽

Semen Quality ◽

Sperm Quality ◽

Sample Selection ◽

Poor Quality ◽

Interesting Possibility ◽

The Many ◽

Α Helix ◽

Analytical Approaches

Although being a crucial step for Assisted Reproduction Technologies (ART) success, to date sperm selection is based only on morphology, motility and concentration characteristics. Considering the many possible alterations, there is a great need for analytical approaches allowing more effective sperm selections. The use of Fourier Transform Infrared (FTIR) may represent an interesting possibility, being able to reveal many macromolecular changes in a single measurement in a nondestructive way. As a proof of concept, in this observational study, we used a FTIR approach to reveal features related to sperm quality and chemical changes promoted by in vitro capacitation. We found indication that α-helix content is increased in capacitated sperm, while high percentages of the β-structures seem to correlate to poor-quality spermatozoa. The most interesting observation was related to the lipid composition, when measured as CH2/CH3 vibrations (ratio 2853/2870), which resulted in being strongly influenced by capacitation and well correlated with sperm motility. Interestingly, this ratio is higher than 1 in infertile samples, suggesting that motility is related to sperm membranes stiffness and lipid composition. Although further analyses are requested, our results support the concept that FTIR can be proposed as a new smart diagnostic tool for semen quality assessment in ART.

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Lippia citriodora (verbascoside) extract supplementation: Effect on rabbit semen quality in vivo and in vitro

Czech Journal of Animal Science ◽

10.17221/35/2018-cjas ◽

2019 ◽

Vol 64 (No. 1) ◽

pp. 1-10 ◽

Cited By ~ 3

Author(s):

F. Vizzarri ◽

M. Palazzo ◽

D. Casamassima ◽

L. Ondruska ◽

M. Massanyi ◽

...

Keyword(s):

New Zealand ◽

Semen Quality ◽

Antioxidant Activities ◽

Zealand White Rabbit ◽

Free Radical Scavengers ◽

Feed Additive ◽

Feed Mixture ◽

Lippia Citriodora

Verbascoside, the main component of Lippia citriodora extract, is one of the most powerful free radical scavengers exhibiting a wide biological activity. In in vivo study 20 adult New Zealand white rabbit bucks were divided into two homogeneous groups, one control (CON) and one verbascoside-supplemented (0.1%) in feed mixture (EXP) and later in vitro effects of verbascoside on the motility aspects of rabbit spermatozoa were analysed. The spermatozoa concentration, ejaculate volume, spermatozoa motility, progressive motility, distance parameters, velocity parameters and type of spermatozoa movement were negatively affected by Lippia citriodora leaves extract after the first 4 weeks of dietary treatment, till the end of experiment (8 weeks). Four weeks after the suspension of feed additive supplementation, all spermatozoa traits values returned to the normality, and in line with CON group. For in vitro findings, ejaculates from 10 male New Zealand white bucks were collected using an artificial vagina. Then it was diluted in physiological saline solution containing different concentrations of verbascoside at the concentration of 0, 0.0024, 0.0219, 0.157, 120.0 mg/ml (Ctrl, VB1, VB2, VB3, VB4 groups, respectively), using a dilution ratio of 1 : 4. The obtained data proved that verbascoside at the concentration of 0.0024 and 0.0219 mg/ml had no adverse effect on spermatozoa. Additionally, we found that verbascoside at higher concentrations (0.157 and 120.0 mg/ml) significantly altered all the motility parameters analysed in the experiment. In conclusion a possible negative effect of verbascoside supplementation into feed mixture (0.1%) on semen quality parameters in rabbit bucks as well as in vitro can be stated, obviously considering that target organs of antioxidant activities of phenylpropanoid glycosides are various. In addition it has to be emphasized that the extract showed a reversible action, since the semen traits of treated animals returned to the normality after the dietary administration period.

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Verticillium Wilt in Oilseed Rape—the Microbiome is Crucial for Disease Outbreaks as Well as for Efficient Suppression

Plants ◽

10.3390/plants9070866 ◽

2020 ◽

Vol 9 (7) ◽

pp. 866 ◽

Cited By ~ 1

Author(s):

Daria Rybakova ◽

Mariann Wikström ◽

Fia Birch-Jensen ◽

Joeke Postma ◽

Ralf Udo Ehlers ◽

...

Keyword(s):

Verticillium Wilt ◽

Severe Disease ◽

Disease Outbreaks ◽

Field Trials ◽

Growth Conditions ◽

Assessment Method ◽

Serratia Plymuthica ◽

Hierarchical Selection

Microbiome management is a promising way to suppress verticillium wilt, a severe disease in Brassica caused by Verticillium longisporum. In order to improve current biocontrol strategies, we compared bacterial Verticillium antagonists in different assays using a hierarchical selection and evaluation scheme, and we integrated outcomes of our previous studies. The result was strongly dependent on the assessment method chosen (in vitro, in vivo, in situ), on the growth conditions of the plants and their genotype. The most promising biocontrol candidate identified was a Brassica endophyte Serratia plymuthica F20. Positive results were confirmed in field trials and by microscopically visualizing the three-way interaction. Applying antagonists in seed treatment contributes to an exceptionally low ecological footprint, supporting efficient economic and ecological solutions to controlling verticillium wilt. Indigenous microbiome, especially soil and seed microbiome, has been identified as key to understanding disease outbreaks and suppression. We suggest that verticillium wilt is a microbiome-driven disease caused by a reduction in microbial diversity within seeds and in the soil surrounding them. We strongly recommend integrating microbiome data in the development of new biocontrol and breeding strategies and combining both strategies with the aim of designing healthy microbiomes, thus making plants more resilient toward soil-borne pathogens.

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Development of sperm sexing and associated assisted reproductive technology for sex preselection of captive bottlenose dolphins (Tursiops truncatus)

Reproduction Fertility and Development ◽

10.1071/rd05108 ◽

2006 ◽

Vol 18 (3) ◽

pp. 319 ◽

Cited By ~ 63

Author(s):

J. K. O'Brien ◽

T. R. Robeck

Keyword(s):

Tursiops Truncatus ◽

Sperm Quality ◽

Bottlenose Dolphins ◽

Sperm Concentration ◽

Motility Index ◽

Liquid Storage ◽

Directional Freezing

Research was conducted to develop sperm sorting and novel sperm preservation methodologies for sex predetermination in the bottlenose dolphin (Tursiops truncatus) using artificial insemination. In Study 1, the effect of seminal plasma (SP), sperm concentration and freezing rate (FR) on in vitro sperm quality of liquid-stored, non-sorted spermatozoa was examined. There was no effect (P > 0.05) of prefreeze SP addition on post-thaw quality (progressive motility, kinetic rating, sperm motility index (SMI), viability and acrosome integrity). Post-thaw motility parameters and viability were higher (P < 0.05) for slow FR than fast FR samples. In Study 2 investigating the effects of liquid storage and sorting on sperm quality, motility and SMI after sorting and centrifugation were lower (P < 0.05) than those of the initial ejaculate. The sort rate for enrichment (91 ± 4% purity) of X- and Y-bearing spermatozoa was 3400 ± 850 spermatozoa sex−1 s−1. In Study 3, compared with a modified straw method, directional freezing resulted in enhanced in vitro quality of sorted and non-sorted spermatozoa derived from liquid-stored semen (P < 0.05). In Study 4, endoscopic insemination of three dolphins with sorted, frozen–thawed X-bearing spermatozoa resulted in one conception and the birth of a female calf. High-purity sorting of dolphin spermatozoa, derived from liquid-stored semen, can be achieved with minimal loss of in vitro sperm quality and samples are functional in vivo.

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