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.

scholarly journals Correlation Between Semen Examination Based on the WHO Criteria and Sperm DNA Fragmentation Test

2021 ◽  
Author(s):  
Tsuyoshi Okubo ◽  
Noriyuki Onda ◽  
Teruaki Hayashi ◽  
Kenji omi ◽  
Tomoya Segawa
Keyword(s):  
Dna Fragmentation ◽  
Sperm Concentration ◽  
Qualitative Evaluation ◽  
Sperm Dna Fragmentation ◽  
Normal Morphology ◽  
Vitro Fertilization ◽  
Who Criteria ◽  
Fragmentation Test ◽  
Sperm Dna

Abstract Purpose: We performed and analyzed the DFI test and semen test based on the WHO criteria and compared the two using semen factors. Methods: DFI tests and general semen tests were conducted in accordance with the WHO criteria, and correlations between the two tests were investigated. The WHO criteria were set as the cutoff value for each of the following factors: fluid volume, concentration, motility, and normal morphology, and compared with the DFI results.Results: The subjects had a mean DFI of 15.6±11.5% and the DFI increased with age. In contrast, the concentration, motility, and normal morphology decreased as the DFI increased. Patients who satisfied the WHO criteria in terms of sperm concentration, motility, and normal morphology had a significantly lower DFI than the group that did not satisfy the criteria. Therefore, evaluation with a general semen test based on the WHO criteria should be regarded as a qualitative evaluation of all factors other than fluid volume.Conclusions: Male infertility due to sperm DNA fragmentation should be suspected when in vitro fertilization (IVF) results are poor despite normal semen findings based on the WHO criteria. The results of this study suggest that the DFI test may be an important evaluation in follow-up semen analyses.

scholarly journals Correlation between semen examination based on the WHO criteria and sperm DNA fragmentation test

2021 ◽  
Author(s):  
Tsuyoshi Okubo ◽  
Noriyuki Onda ◽  
Teruaki Hayashi ◽  
Kenji Omi ◽  
Tomoya Segawa
Keyword(s):  
Dna Fragmentation ◽  
Sperm Concentration ◽  
Qualitative Evaluation ◽  
Fluid Volume ◽  
Sperm Dna Fragmentation ◽  
Normal Morphology ◽  
Vitro Fertilization ◽  
Who Criteria ◽  
Sperm Dna

Abstract In this study, we performed and analyzed the DFI test and semen test based on the WHO criteria and compared the two using semen factors. DFI tests and general semen tests were conducted in accordance with the WHO criteria, and correlations between the two tests were investigated. The WHO criteria were set as the cutoff value for each of the following factors: fluid volume, concentration, motility, and normal morphology, and compared with the DFI results. The subjects had a mean DFI of 15.6 ± 11.5% and the DFI increased with age. In contrast, the concentration, motility, and normal morphology decreased as the DFI increased. Patients who satisfied the WHO criteria in terms of sperm concentration, motility, and normal morphology had a significantly lower DFI than the group that did not satisfy the criteria. Therefore, evaluation with a general semen test based on the WHO criteria should be regarded as a qualitative evaluation of all factors other than fluid volume. Male infertility due to sperm DNA fragmentation should be suspected when in vitro fertilization (IVF) results are poor despite normal semen findings based on the WHO criteria. The results of this study suggest that the DFI test may be an important evaluation in follow-up semen analyses.

scholarly journals Effect of sperm DNA fragmentation on clinical outcomes for Chinese couples undergoing in vitro fertilization or intracytoplasmic sperm injection

2016 ◽  
Vol 44 (6) ◽  
pp. 1283-1291 ◽  
Author(s):  
Lin-Tao Xue ◽  
Rui-Xue Wang ◽  
Bing He ◽  
Wei-Ying Mo ◽  
Li Huang ◽  
...  
Keyword(s):  
In Vitro Fertilization ◽  
Dna Fragmentation ◽  
Intracytoplasmic Sperm Injection ◽  
Fertilization Rate ◽  
Sperm Dna Fragmentation ◽  
Roc Curve Analysis ◽  
Vitro Fertilization ◽  
Fragmentation Rate ◽  
Sperm Dna

Objective To investigate the effect of sperm DNA fragmentation on the fertilization rate, embryo development and pregnancy outcome of in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) in a cohort of Chinese couples. Methods Infertile couples that had undergone assisted reproductive technology at our centre between January 2011 and December 2013 were included in this retrospective study. Fractions of prepared sperm samples were evaluated for sperm DNA fragmentation on the day of oocyte recovery. Results Of the 550 couples selected, 415 had undergone IVF and 135 ICSI. Sperm DNA fragmentation rate was significantly negatively correlated with the fertilization rate in the ICSI cycles but not the IVF cycles. No association was found between sperm DNA fragmentation and cleavage rate or good quality embryo formation rates in IVF or ICSI cycles. Receiver operating characteristic (ROC) curve analysis showed that the sperm DNA fragmentation rate was a statistically significant prognostic indicator of the clinical fertilization rate in ICSI cycles; a rate > 22.3% was associated with a lower fertilization rate following ICSI compared with a rate ≤ 22.3%. Conclusions High values of sperm DNA fragmentation were associated with a low fertilization rate following ICSI but were not associated with alterations in pregnancy or live birth rates in either ICSI or IVF in this cohort of Chinese couples.

339. THE RELATIONSHIP BETWEEN DNA FRAGMENTATION IN MOUSE OOCYTES AND GENITAL TRACT MICROORGANISMS

2010 ◽  
Vol 22 (9) ◽  
pp. 139
Author(s):  
J. E. Harris ◽  
E. S. Pelzer ◽  
J. A. Allan ◽  
E. Whiteside ◽  
L. De Boer ◽  
...  
Keyword(s):  
Dna Fragmentation ◽  
Virulence Factors ◽  
Follicular Fluid ◽  
Genital Tract ◽  
Oocyte Quality ◽  
Dna Integrity ◽  
Sperm Dna Fragmentation ◽  
Mouse Oocytes ◽  
Tract Infections

Colonising bacteria detected within the follicular fluid of women undergoing assisted reproductive technology cycles has been associated with decreased embryo transfer rates and decreased pregnancy rates. The bacteria isolated from the follicular fluid of these women, at the time of trans-vaginal oocyte retrieval include Gram-positive anaerobic rods, Gram-negative anaerobic rods, streptococci, staphylococci, and lactobacilli. Some of these bacteria are opportunistic pathogens in the female genital tract. The expression of virulence factors including hyaluronidase and DNase, could affect the structural integrity of the oocyte and induce DNA fragmentation and apoptosis. Hyaluronin is a major carbohydrate component of the cumulus oocyte complex that could be a target for bacterial hyaluronidase. Other virulence factors associated with bacteria detected in cases of genital tract infections include the hydrolytic enzymes sialidase, β-galactosidase and β-N-acetylhexosaminidase (Howe 1999). Production of reactive oxygen species as a result of the presence of bacteria and bacterial heat shock proteins have been suggested as mechanisms responsible for increased DNA fragmentation and apoptotic progression of male gametes in colonised semen. Previous studies have not confirmed a direct relationship between sperm morphology and the level of sperm DNA fragmentation. DNA fragmentation in oocytes in relation to the presence of bacteria remains to be investigated. Oocyte quality is only determined by the assessment of cumulus cell morphology prior to in-vitro fertilisation. In-vitro testing of oocyte DNA integrity under various conditions may further the understanding of oocyte quality. In this study, mouse oocytes were used to investigate the structural and DNA integrity of oocytes after in vitro exposure to Lactobacillus spp., Streptococcus spp., and Staphylococcus spp. and determine whether the morphological appearance of the oocyte was predictive of the level of DNA fragmentation and whether DNA fragmentation can be attributed to certain bacterial species found colonising follicular fluid. (1) Howe L (1999). Mucinase and sialidase activity of the vaginal microflora: implications for the pathogenesis of preterm labour. International Journal of STD and AIDS 10(7), 442–446.

Carryover effect of atrazine and its metabolite—from treated bovine spermatozoa to the embryo’s transcriptome†

2021 ◽  
Author(s):  
A Komsky-Elbaz ◽  
D Kalo ◽  
Z Roth
Keyword(s):  
Dna Fragmentation ◽  
Annexin V ◽  
Dna Integrity ◽  
Blastocyst Formation ◽  
Transcriptome Profile ◽  
Carryover Effect ◽  
Vitro Fertilization ◽  
Cellular Processes ◽  
Bovine Spermatozoa

Abstract Atrazine (ATZ) is an extensively used herbicide and ubiquitous environmental contaminant. ATZ and its metabolite, diaminochlorotriazine (DACT), cause several cellular and functional alterations in spermatozoa. We aimed to examine the effect of ATZ/DACT on spermatozoon DNA integrity, fertilization competence, embryonic development, and transcriptome profile of in vitro-produced embryos derived from fertilization with pre-exposed sperm. Bovine spermatozoa exposed to ATZ (0.1 or 1 μM) or DACT (1 or 10 μM) during in vitro capacitation were used for in vitro fertilization of untreated oocytes. Cleavage and blastocyst-formation rates were evaluated 42 h and 7 days postfertilization, respectively. The association between DNA fragmentation and apoptosis (annexin V kit) was determined. Fertilization competence of annexin-positive (AV+) and annexin-negative (AV−) spermatozoa was examined. Microarray analysis was performed for 7-day blastocysts. Intracytoplasmic sperm injection was performed with control (AV+, AV−) and DACT (AV+, AV−) spermatozoa. Cleavage rates did not differ between groups and blastocyst formation tended to be higher for AV− vs. AV+ in both control and DACT groups, suggesting that acrosome reaction, rather than DNA fragmentation, underlies the reduced cleavage. Transcriptomic analysis revealed 139 and 230 differentially expressed genes in blastocysts derived from ATZ- and DACT-exposed spermatozoa, respectively, relative to controls. Proteomic analysis shown differential expression of proteins in ATZ- or DACT-treated spermatozoa, in particular proteins related to cellular processes and biological pathways. Therefore, we assume that factors delivered by the spermatozoa, regardless of DNA fragmentation, are also involved. Overall, the current study reveals a deleterious carryover effect of ATZ/DACT from the spermatozoa to the developing embryo.

scholarly journals The Re-Addition of Seminal Plasma after Thawing Does Not Improve Buck Sperm Quality Parameters

Animals ◽  
2021 ◽  
Vol 11 (12) ◽  
pp. 3452
Author(s):  
Uchechi Linda Ohaneje ◽  
Uchebuchi Ike Osuagwuh ◽  
Manuel Alvarez-Rodríguez ◽  
Iván Yánez-Ortiz ◽  
Abigail Tabarez ◽  
...  
Keyword(s):  
Seminal Plasma ◽  
Sperm Quality ◽  
Membrane Integrity ◽  
Egg Yolk ◽  
Quality Parameters ◽  
Mitochondrial Activity ◽  
Dna Integrity ◽  
Vitro Fertilization

In order to achieve a higher post-thaw buck sperm quality, an approach in the thawing protocol of cryopreserved sperm doses under in vitro capacitation conditions mimicking the in vivo female environment was studied. Therefore, functional and kinetic characteristics of buck thawed sperm from males of different ages, the season of collection, and melatonin implanted males in the non-breeding season were assessed after 3 h of incubation in an in vitro fertilization (IVF) media with 20% of buck seminal plasma (SP). Previously, fresh ejaculates were collected via artificial vagina from eight males of the Cabra Blanca de Rasquera breed during two consecutive years in breeding and non-breeding periods. Prior to semen collection in non-breeding seasons, males were split into two groups: one group was implanted with melatonin, while the other was not. In each group, semen samples were pooled, centrifuged, and diluted in an extender containing 15% powdered egg yolk and 5% glycerol before freezing. After thawing, sperm were washed and incubated in three different media: (a) control media (modified phosphate-buffered saline (PBS), (b) IVF commercial media, and (c) IVF media + 20% SP. Sperm motility was evaluated by CASA, while plasma and acrosome membrane integrity, mitochondria activity, and DNA fragmentation were analysed by flow cytometer at 0 h and after 3 h incubation. A significant reduction in motility, mitochondrial activity, plasma, and acrosome membrane integrity were observed after incubation in the presence of SP, although similar to that observed in IVF media alone. DNA integrity was not affected under in vitro capacitation conditions, regardless of SP addition. In conclusion, the addition of SP failed to improve post-thaw buck sperm quality under in vitro conditions irrespective of male age, the season of collection, and melatonin implant.

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