61 Swim-up and microfluidic techniques improve the kinetic parameters of selected bovine spermatozoa for invitro fertilization: Preliminary results

2021 ◽  
Vol 33 (2) ◽  
pp. 137
Author(s):  
J. Vega ◽  
M. Rodriguez ◽  
D. Dipaz-Berrocal ◽  
J. Rivas ◽  
C. Huayhua ◽  
...  
Keyword(s):  
Kinetic Parameters ◽  
Dna Fragmentation ◽  
Density Gradient ◽  
Gradient Method ◽  
Sperm Chromatin ◽  
Computer Assisted ◽  
Sperm Selection ◽  
Embryo Production ◽  
Microfluidic Sorting ◽  
Microfluidic Technique

For invitro embryo production, spermatozoa with fertilizing capacity must possess optimal kinetic, morphometric, vitality, and DNA integrity characteristics. The objective of this study was to evaluate the effect of 3 sperm selection methods on sperm quality and invitro embryonic development in bovine. Frozen commercial semen (0.5 mL/straws) from one bull with known fertility was thawed at 37°C for 20s and was divided for 3 sperm selection techniques: density gradient, swim-up, and microfluidic sperm sorting. The sperm kinetic parameters (VCL=curvilinear velocity, VSL=straight line velocity, VAP=average path velocity, ALH=lateral displacement of sperm head, BCF=beat frequency cross, STR=path straightness) were assessed using computer-assisted sperm analysis (CASA). Sperm morphometric parameters were evaluated using Diff-Quick staining followed by automated analysis. To assess vitality, the sperm were stained with propidium iodide and acridine orange, then analysed under a fluorescence microscope. In addition, DNA fragmentation was assessed using sperm chromatin dispersion method. Last, the fertilizing capacity of the selected sperm was tested by fertilizing cumulus–oocyte complexes (2×106 sperm mL−1) obtained from slaughterhouse ovaries and matured invitro for 24h. A standardized invitro embryo production protocol was used with commercial medium from Vitrogen. The cleavage rate and blastocyst yield were measured on Day 2 and 7, respectively (fertilization=Day 0). The results were calculated with analysis of variance and Tukey’s test (P<0.05). The values of sperm kinetic parameters obtained with swim-up (VCL 132.5µm/s; VSL 73.5µm/s) and microfluidic technique (VCL 129.5µm/s; VSL 64.4µm/s) were significantly higher (P<0.05) than those obtained by density gradient (VCL 98.3µm/s; VSL 45.01µm/s). However, the total and progressive motility by density gradient method was slightly higher (89% and 57%) compared with that assessed by swim-up (64% and 43%) or microfluidic technique (74% and 54%) respectively. Microfluidic sorting (11.3%) showed lower (P<0.05) DNA fragmentation levels compared with density gradient method (16.6%), whereas the swim-up technique (12.5%) was similar between both groups. No significant difference was detected between the 3 groups for sperm morphometric and vitality parameters. Moreover, cleavage rates were similar (P>0.05) between the 3 sperm selection techniques: density gradient (84.0%), swim-up (75.2%), and microfluidic sorting (67.3%). However, blastocyst yield was significantly higher (P<0.05) using sperm selected by density gradient (28.1%) and swim-up (21.9%) compared with microfluidic sorting (15.3%). In conclusion, sperm selection using microfluidic and swim-up techniques improved kinetic parameters with lower levels of DNA fragmentation, without affecting sperm morphometry. However, both the density gradient and swim-up techniques are efficient systems for producing invitro bovine embryo.

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.

247 COMPARISON OF THE DNA FRAGMENTATION INDEX BETWEEN CRYOPRESERVED EJACULATED SPERM AND EPIDIDYMAL SPERM IN STALLIONS

2013 ◽  
Vol 25 (1) ◽  
pp. 271
Author(s):  
G. A. Monteiro ◽  
C. P. Freitas-DellAqua ◽  
P. N. Guasti ◽  
Y. F. R. Sancler-Silva ◽  
C. Ramires-Neto ◽  
...  
Keyword(s):  
Dna Fragmentation ◽  
Sperm Motility ◽  
Epifluorescence Microscopy ◽  
Sperm Chromatin ◽  
Computer Assisted ◽  
Epididymal Sperm ◽  
Fragmentation Index ◽  
Motion Parameters ◽  
Motility Parameters ◽  
Dna Fragmentation Index

The development of a reliable technique for freezing epididymal semen would provide a unique opportunity to preserve valuable genetic material from unexpectedly lost stallions. The semen analysis method with the best ability to predict fertility is an examination of the sperm chromatin structure. This test evaluates the susceptibility of spermatozoa DNA to denaturation. The ability of spermatozoal DNA to maintain an intact double-stranded configuration is determined by exposure to an acid environment. The aim of this study was to compare the DNA fragmentation index of sperm obtained from ejaculate (G1) and sperm from the cauda epididymis (G2). For G1, two ejaculates from each of seven stallions were collected and then subjected to cryopreservation using BotuCrioTM extender. One week after the last semen collection, the stallions underwent bilateral orchiectomy. Sperm from the cauda epididymis was harvested immediately after castration (G2) by retrograde flushing of the caudal portion of the epididymis using a skim milk-based extender (BotuSemenTM). The recovered sperm was then cryopreserved using BotuCrioTM extender. The sperm motility parameters were analysed by computer-assisted sperm analysis (HTM IVOS 12, Hamilton Thorne Inc., Beverly, MA, USA), and the DNA fragmentation index was estimated using acridine orange test epifluorescence microscopy. The samples were evaluated immediately (0 h) and 8 h after thawing. The total motility, progressive motility, and percentage of rapid cells of the G1 v. G2 samples at 0 h were, respectively, 62.3 ± 12.9a v. 72.6 ± 8.4a, 31.6 ± 9.2a v. 35.3 ± 10.32a, and 49.3 ± 14.33a v. 59.7 ± 13.59a. At 8 h, the results were 26.0 ± 21.6b v. 54.7 ± 12.2a, 6.1 ± 6.4b v. 17.4 ± 8.54a, and 13.7 ± 14.85b v. 37.6 ± 14.15a. Evaluation of the DNA fragmentation in the G1 and G2 samples yielded 6.7 ± 1.41a v. 5.7 ± 1.60a at 0 h and 8.3 ± 1.78b v. 7.2 ± 1.19b at 8 h for percentage of DNA fragmentation after thawing. At 0 h, no differences in the sperm parameters were observed between groups, but statistical differences were observed in the sperm motility parameters between the treatment groups after 8 h. For the DNA fragmentation index, no difference was found at 0 and 8 h between the groups. However, after thawing, a higher percentage of DNA fragmentation was observed in the ejaculated sperm (8 h) as compared with the epididymal sperm (0 h). On the basis of these results, we can conclude that frozen–thawed cauda epididymal sperm had similar or higher motion parameters than ejaculated sperm after thawing. In addition, incubating the sperm at 20°C for 8 h after thawing resulted in higher motion parameters and less DNA fragmentation of the epididymal sperm. This finding suggests that epididymal sperm are more resistant to the cold shock caused by cryopreservation. FAPESP for financial support and Botupharma for donation of BotuSemenTM and BotuCrioTM extender.

scholarly journals Variation of DNA Fragmentation Levels During Density Gradient Sperm Selection for Assisted Reproduction Techniques

Medicine ◽  
2016 ◽  
Vol 95 (20) ◽  
pp. e3624 ◽  
Author(s):  
Monica Muratori ◽  
Nicoletta Tarozzi ◽  
Marta Cambi ◽  
Luca Boni ◽  
Anna Lisa Iorio ◽  
...  
Keyword(s):  
Dna Fragmentation ◽  
Density Gradient ◽  
Assisted Reproduction ◽  
Sperm Selection ◽  
Assisted Reproduction Techniques ◽  
Selection For

scholarly journals Sperm selection by rheotaxis improves sperm quality and early embryo development

Reproduction ◽  
2021 ◽  
Vol 161 (3) ◽  
pp. 343-352 ◽  
Author(s):  
Jon Romero-Aguirregomezcorta ◽  
Ricardo Laguna-Barraza ◽  
Raúl Fernández-González ◽  
Miriama Štiavnická ◽  
Fabian Ward ◽  
...  
Keyword(s):  
Membrane Fluidity ◽  
Dna Fragmentation ◽  
Density Gradient ◽  
Embryo Development ◽  
Sperm Quality ◽  
Early Embryo ◽  
Control Group ◽  
Sperm Selection ◽  
Foetal Development ◽  
Development Rates

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.

Probiotic administration improves sperm quality in asthenozoospermic human donors

2017 ◽  
Vol 8 (2) ◽  
pp. 193-206 ◽  
Author(s):  
D.G. Valcarce ◽  
S. Genovés ◽  
M.F. Riesco ◽  
P. Martorell ◽  
M.P. Herráez ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Cell Viability ◽  
Dna Fragmentation ◽  
Sperm Motility ◽  
Sperm Quality ◽  
Bifidobacterium Longum ◽  
Fold Change ◽  
Sperm Chromatin ◽  
Computer Assisted ◽  
Sperm Analysis

The objective of this study is to analyse the effect of the ingestion of two selected antioxidant probiotics strains (Lactobacillus rhamnosus CECT8361 and Bifidobacterium longum CECT7347) on sperm quality parameters in asthenozoospermic males after three and six weeks of administration. Nine asthenozoospermic men without any medical treatment under similar diet conditions participated in the study. The quality of individual sperm samples was evaluated before (previous to ingestion), during (after 3 and 6 weeks of ingestion) and after probiotic administration (3 and 6 weeks after finishing the treatment). Sperm motility was evaluated by computer-assisted sperm analysis system, DNA fragmentation by sperm chromatin structure assay, cell viability by flow cytometry and measurement of intracellular H2O2 (reactive oxygen species; ROS) by flow cytometry using dichloro-dihydrofluorescein diacetate. Sperm motility was drastically improved after the treatment (approximately 6 fold change), DNA fragmentation was statistically reduced after probiotic administration from (approximately 1.2 fold change) and intracellular H2O2 level was decreased (approximately 3.5 fold change). Cell viability was not affected by the treatment. The significant improvement in sperm motility and the decrease in DNA fragmentation reported in this study provide preliminary evidence that probiotics could be administrated to improve motility and decrease DNA fragmentation and ROS levels in asthenozoospermic human males.

P–069 microfluidic sperm sorting vs density gradient to yield sperm with reduced DFI for patients undergoing IVF-ICSI

2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
D P Makwana ◽  
S Makwana ◽  
T Sen
Keyword(s):  
Density Gradient ◽  
Gradient Method ◽  
Fluid Dynamic ◽  
Sperm Concentration ◽  
Semen Sample ◽  
Semen Parameters ◽  
Sperm Chromatin ◽  
Dna Integrity ◽  
Sperm Preparation ◽  
Sperm Sorting

Abstract Study question To compare the effect of sperm preparation methods on the DFI of semen sample for patients undergoing ICSI. Summary answer On comparing the results, microfluidic sperm sorting yielded sperms with significantly less DFI as compared to density gradient method of sperm preparation. What is known already The DNA integrity of the sperm plays an important role to ensure formation of good quality embryos with increased potential of fertilization, growth and ultimately implantation.. Centrifugation has shown to add stress to the sperm and leading to DNA damage, therefore there is a need to develop techniques of sperm preparation which help in retrieving as many sperms with intact DNA from the unprocessed sample as possible. Microfludic is fluid dynamic based technique of sperm preparation. in this study, we evaluated if microfluidic sperm sorter can recover motile sperm with better DNA integrity compared to density gradient preparation method. Study design, size, duration Prospective randomized study conducted in 80 patients undergoing IVF-ICSI with normal semen parameters (based WHO criteria 2010). DFI was done using Sperm Chromatin Dispersion (SCD) test in split semen samples prepared by microfluidic sperm sorter and density gradient method. Sperm morphology and motility were also recorded and evaluated based on the WHO 2010 criteria. Participants/materials, setting, methods Semen parameters of the sample were assessed by microscopic examination. DFI of each unprocessed sample was carried out using SCD test, following that the sample was split and sperm preparation was done using microfluidic sperm sorter and density gradient. the recovered sperm were tested for DFI and the results were compared. Main results and the role of chance Mean DFI in unprocessed semen samples was 23%. the analysis of split semen samples post preparation showed that the DFI was significantly reduced with the use of microfluidic sperm sorter (mean DFI 0.6%) as compared to density gradient (mean DFI 9%). Limitations, reasons for caution A major limitation of the microfluidic sperm sorter is the use sperm concentration and motility of the semen sample. In oligospermic and asthenospermic samples, density gradient is the preferred method of preparation. Lack of data showing improvement in clinical outcomes with reduced DFI is also a major limitation. Wider implications of the findings: Microfluidics has shown to significantly reduce the DFI of the semen sample, it requires no extra equipment and cost and is relatively easy to pick up. Density gradient method of sperm preparation continues to be the preferred method due to its versatility and recovery of good quality sperm. Trial registration number Not applicable

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.

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