The correlation between male age, sperm quality and sperm DNA fragmentation in 320 men attending a fertility center

Abstract Study question Is it useful to avoid sperm centrifugation in laboratory routine work to improve sperm quality and reproductive outcome in Assisted Reproduction Techniques (ART)? Summary answer Exclusion of sperm centrifugation for sperm selection using neat sperm samples (IO-lix), increases sperm quality in the collected subpopulation decreasing miscarriage rate after using ICSI. What is known already Inclusion of sperm centrifugation in ART is an aggressive intervention for sperm selection with ineludible production iatrogenic damage affecting sperm integrity. The application of IMSI, PICSI or microfluidic devices avoid sperm centrifugation and may improve the quality of the subsample obtained. However, these methodologies may result time consuming, expensive or producing poor results when the quality of the sperm is limited. We have already shown that a modified swim-up avoiding centrifugation (called IO-lix) is a low-cost and efficient alternative to microfluidic devices, recovers 100 times more concentration and reduces sperm DNA fragmentation with no significant differences to other methodologies. Study design, size, duration This is a retrospective study from 2018 to 2020 which includes patients with an average of age of 38.2 years using their own oocytes with ICSI as fertilization technique. Two aleatory groups of patients were made: Group 1: 88 cycles with 503 fertilized oocytes and 206 blastocysts were obtained with sperm samples processed by IO-lix and Group 2: 303 cycles, 1451 fertilized oocytes and 591 blastocysts using a standard “swim up” technique to process sperm. Participants/materials, setting, methods A total of 391 ICSI cycles were included in this retrospective study. The male factor was similar in both groups and they showed altered SDF previously to the cycle. We compared data of the motility and SDF of sperm samples before and after applying IO-lix and we analyzed by X2 contingence test differences on miscarriage rates between groups 1 and 2. Main results and the role of chance General sperm parameter changes after IO-lix showed that averaged sperm concentration observed in neat ejaculated samples was 62M/SD=46.4. Values obtained after IO-lix in the same samples were 12.3M/SD8.0. Averaged sperm motility in neat samples was 54%/SD=9.3 and 70.9%/SD=13.2 after IO-lix. Finally, sperm DNA fragmentation in neat samples was 35.8%/SD17.3, while these values decreased to 9.2%/SD=3.9 after IO-lix. About reproductive outcome results, significant differences were not obtained on the development to blastocyst stage rate comparing both groups (X2=0.003; p value = 0.954; Alpha 0.05). In the case of IO-lix processed samples, the pregnancy rate was 59.42% in Group 1 and 44.72% in Group 2 (X2=0.651; p value =0.419; Alpha 0.05). A total of 9 miscarriages of 41 clinical pregnancies (21.95%) were observed after IO-lix, while this number increases to 59 out of 123 clinical pregnancies, which means the 47.96% of the embryo transfers, when “swim-up” was used. In this case significant differences were obtained (X2=3.935; p value = 0.0.047; Alpha 0.05). Limitations, reasons for caution Being a pilot study aimed to understand the results of IO-lix in ART, correlations have not been stablished between the levels of sperm improvement after IO-lix and paired results of ART. This study would be necessary, specially to identify the possible origin of miscarriage associated to the male factor. Wider implications of the findings: Elimination of sperm centrifugation using a combined strategy of gradients and “swim-up” for sperm isolation, reduce miscarriage rate and produce equivalent results of blastocyst development to those obtained with “swim-up”. Being a cost-effective and improving laboratory workload, its use for sperm selection is recommended. Trial registration number Not applicable

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Induced lipid peroxidation in ram sperm: semen profile, DNA fragmentation and antioxidant status

Reproduction ◽

10.1530/rep-15-0403 ◽

2016 ◽

Vol 151 (4) ◽

pp. 379-390 ◽

Cited By ~ 23

Author(s):

Thais Rose dos Santos Hamilton ◽

Letícia Signori de Castro ◽

Juliana de Carvalho Delgado ◽

Patrícia Monken de Assis ◽

Adriano Felipe Perez Siqueira ◽

...

Keyword(s):

Lipid Peroxidation ◽

Dna Fragmentation ◽

Sperm Quality ◽

Ovis Aries ◽

Sperm Dna Fragmentation ◽

Mitochondrial Potential ◽

Sperm Analysis ◽

Sperm Dna ◽

Significant Difference ◽

Ram Sperm

Action of reactive oxygen species, protamination failures and apoptosis are considered the most important etiologies of sperm DNA fragmentation. This study evaluated the effects of induced lipid peroxidation susceptibility on native semen profile and identified the mechanisms involved in sperm DNA fragmentation and testicular antioxidant defense on Santa Ines ram sperm samples. Semen was collected from 12 adult rams (Ovis aries) performed weekly over a 9-week period. Sperm analysis (motility, mass motility, abnormalities, membrane and acrosome status, mitochondrial potential, DNA fragmentation, lipid peroxidation and intracellular free radicals production); protamine deficiency; PRM1, TNP1 and TNP2 gene expression; and determination of glutathione peroxidase (GPx), glutathione reductase, catalase (CAT) and superoxide dismutase activity and immunodetection in seminal plasma were performed. Samples were distributed into four groups according to the sperm susceptibility to lipid peroxidation after induction with ascorbate and ferrous sulfate (low, medium, high and very high). The results were analyzed by GLM test and post hoc least significant difference. We observed an increase in native GPx activity and CAT immunodetection in groups with high susceptibility to induced lipid peroxidation. We also found an increase in total sperm defects, acrosome and membrane damages in the group with the highest susceptibility to induced lipid peroxidation. Additionally, the low mitochondrial membrane potential, susceptible to chromatin fragmentation and the PRM1 mRNA were increased in the group showing higher susceptibility to lipid peroxidation. Ram sperm susceptibility to lipid peroxidation may compromise sperm quality and interfere with the oxidative homeostasis by oxidative stress, which may be the main cause of chromatin damage in ram sperm.

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P–091 Magnetic Activated Cell Sorting (MACS) improves euploid blastocysts rate in pre-implantation genetic testing cycles with high levels of sperm DNA fragmentation and advanced paternal age

Human Reproduction ◽

10.1093/humrep/deab130.090 ◽

2021 ◽

Vol 36 (Supplement_1) ◽

Author(s):

F Scarselli ◽

E Cursio ◽

A Colasante ◽

V Zazzaro ◽

P Andrea ◽

...

Keyword(s):

Genetic Testing ◽

Dna Fragmentation ◽

Sperm Quality ◽

Annexin V ◽

Paternal Age ◽

Control Group ◽

Sperm Dna Fragmentation ◽

Sperm Dna ◽

Number Of Patients ◽

Blastocyst Rate

Abstract Study question Can MACS increase euploid blastocyst rate in Pre-implantation Genetic Testing (PGT) cycles for AMA-APA (Advanced Maternal-Paternal Age) in patients with high sperm DNA fragmentation (SDF)? Summary answer A slight increase in euploid blastocyst rate was found using MACS in infertile patients with high SDF undergoing PGT cycles compared to the control group. What is known already Many authors have shown a close correlation between the presence of apoptotic markers on spermatozoa and the failure of assisted reproduction treatments. In normal physiological conditions, apoptotic spermatozoa with phosphatidylserine (PS) residues externalized on the plasma membrane, are eliminated along female genital tract, preventing oocyte fertilization. MACS eliminates apoptotic sperm whit PS residues using superparamagnetic microbeads conjugated with annexin V. This technique reduces the proportion of sperm with high rates of SDF and can be used to maximize ART procedures results. MACS application improves sperm quality, fertilization, cleavage and pregnancy rates reducing miscarriage rate. Study design, size, duration From June to November 2020, 10 couples in which MACS was applied to select non-apoptotic spermatozoa, were randomly enrolled in our study (MACS group) and 8 couples without MACS were considered as controls (No-MACS Group). All couples in both groups underwent a PGT cycle and had high sperm DNA Fragmentation (> 20%). A higher rate of euploid and diploid-euploid mosaic blastocysts were obtained in the MACS group compared to the control group. Participants/materials, setting, methods Patients with severe oligoastenoteratozoospermia were excluded. MACS protocol was performed as follows: semen sample was analyzed (WHO 2010) and washed with buffered medium; pellet was removed and a swim-up was performed. Retrieved spermatozoa were washed with a binding buffer (Miltenyi Biotec), centrifuged (400 g x 4 minutes) and supernatant discarded. Pellet was covered with Annexin-V and re-suspended. After 15 minutes incubation at room temperature, the sample was eluted through the column and collected for ICSI. Main results and the role of chance In MACS group, female and male mean age ± SD were 41.6 ± 2.1 and 43.5 ± 7.3, respectively. Female and male mean age ± SD were 41.7 ± 2.8 and 44.6 ± 8.1 in the No-MACS group, respectively. In MACS and No-MACS groups, injected oocytes were 44 and 35, fertilized oocytes were 32 (72.3%) and 27 (77.1%) (NS), blastocyst formation rates were 71.8% (23/32) and 48.1% (13/27) (NS), respectively. In No-MACS group, only 1 euploid and 1 diploid-euploid mosaic blastocysts were obtained (1/13 = 8%) (NS). In MACS group, 4 euploid blastocysts were formed (4/23 = 17.4%) whereas mosaic diploid-euploid blastocysts were 3/23 (13.0%) (NS). Aneuploid blastocysts were 16/23 (69.6%) in MACS group and 11/13 (84.6%) in No-MACS group (NS). Limitations, reasons for caution AMA and APA of couples enrolled should be considered as a limit of the study. A larger number of patients and biopsied blastocysts are needed to analyze clinical results and perform a robust statistical analysis establishing if MACS is useful to improve transferable blastocyst rate in patients with high SDF. Wider implications of the findings: MACS is useful to select non apoptotic sperms; although fertilization, cleavage and blastocyst rates are not improved, aneuploid blastocysts rate slightly decreases using MACS. It I possible that, selecting spermatozoa free from PS residues, MACS allows to choose spermatozoa with a better DNA packaging, thus affecting the embryo ploidy. Trial registration number non applicable

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The aging male: Relationship between male age, sperm quality and sperm DNA damage in an unselected population of 3124 men attending the fertility centre for the first time

Archivio Italiano di Urologia e Andrologia ◽

10.4081/aiua.2018.4.254 ◽

2019 ◽

Vol 90 (4) ◽

pp. 254-259 ◽

Cited By ~ 5

Author(s):

Alessandro Colasante ◽

Maria Giulia Minasi ◽

Filomena Scarselli ◽

Valentina Casciani ◽

Vincenzo Zazzaro ◽

...

Keyword(s):

Dna Damage ◽

Sperm Morphology ◽

Semen Quality ◽

Sperm Quality ◽

World Health ◽

Semen Parameters ◽

Sperm Dna Fragmentation ◽

Sperm Dna Damage ◽

Male Age ◽

Sperm Dna

Objective: the aim of our study was to put forward insights to treat any possible correlation among sperm quality, sperm DNA damage and male age as they may have fertility implications for men who choose to delay fatherhood. Materials and methods: Our study is a non-interventional retrospective analysis of 3124 semen samples from patients that were investigated for the conventional semen parameters. Tunel test assay was set up for the evaluation of the sperm DNA fragmentation index (DFI). We applied the Kappa index to compare both the 1999 and the 2010 World Health Organization (WHO) reference criteria to evaluate the competence of such semen parameters categorization during the standard routine of our laboratory. Results: With regards to our findings, it is possible to underline a significant relationship between aging and semen volume (p = 0.001), motility (p = 0.009), semen viscosity (p < 0.003) and sperm DNA damage (p < 0.009). We found a trend when focusing on the semen concentration (p = 0.05). The analysis of sperm morphology did not show any influence with advancing age (p = 0.606). When comparing both the 1999 and the 2010 WHO scales we found no accordance in the appraisal of sperm morphology but a very good one in the evaluation of the other parameters. Conclusions: Conventional semen analysis represents the opportunity to draw up a proxy insight on the male fertility status even if semen quality can only indirectly assess the probability of pregnancy. Several studies have verified a decay in the male reproductive system, sperm quality and fertility with advancing age although the reported results are not yet conclusive. Our results substantially agree with those findings outlined in the literature. Moreover we find that the discrepancy between the two WHO reference scales would eventually lead to an improper diagnosis of infertility.

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Short communication. Stallion sperm quality after combined ejaculate fractionation and colloidal centrifugation

Spanish Journal of Agricultural Research ◽

10.5424/sjar/2015134-8108 ◽

2015 ◽

Vol 13 (4) ◽

pp. e04SC02 ◽

Cited By ~ 2

Author(s):

Francisco Crespo ◽

Jaime Gosálvez ◽

Stephen D. Johnston ◽

Joaquina De la Torre

Keyword(s):

Dna Fragmentation ◽

Sperm Quality ◽

Sperm Dna Fragmentation ◽

Fragmentation Index ◽

Sperm Dna ◽

Rate Of Increase ◽

Sperm Sample ◽

Artificial Vagina ◽

Sperm Dna Fragmentation Index ◽

Dna Fragmentation Index

<p>This study investigated the possible additive benefit of ejaculate fractionation and colloidal centrifugation on stallion sperm quality. Using an open-end artificial vagina, the sperm-rich fraction (FRAC-1) was separated from the rest of the ejaculate (FRAC-2) and a third sperm sample representing the combined ejaculate was reconstituted post-ejaculation (RAW). Each semen sample was processed for colloidal centrifugation. The percentage of abnormal spermatozoa was 17.8 ± 7.0% in RAW and 14.6 ± 9.5% in FRAC-1 but decreased to 11.4 ± 4.7% and 9.6 ± 6.9% respectively, after colloidal centrifugation. A sperm DNA fragmentation index of 10.9 ± 5.1% was observed in RAW and 7.5 ± 2.4% in FRAC-1 semen collected with the AV but this decreased to 7.8 ± 2.8% and 5.2 ± 2.3% after colloidal centrifugation. The rate of increase in sperm DNA fragmentation during the first 6 h of incubation at 37 ºC was 1.8 ± 0.9% per hour in RAW semen and 2.0 ± 2.0% per hour in FRAC-1 but this significantly decreased to 1.3 ± 1.4% and 0.9 ± 0.8% respectively after colloidal centrifugation. While stallion seminal characteristics can be improved using colloidal centrifugation, further enhancement is possible if the ejaculate is initially fractionated.</p>

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Effect of bacterial infection on sperm quality and DNA fragmentation in subfertile men with Leukocytospermia

BMC Molecular and Cell Biology ◽

10.1186/s12860-021-00380-8 ◽

2021 ◽

Vol 22 (1) ◽

Author(s):

Fatemeh Eini ◽

Maryam Azizi Kutenaei ◽

Fayegheh Zareei ◽

Zeinolabedin Sharifian Dastjerdi ◽

Maryam Hosseinzadeh Shirzeyli ◽

...

Keyword(s):

Bacterial Infection ◽

Dna Fragmentation ◽

Bacterial Infections ◽

Pathogenic Bacteria ◽

Seminal Fluid ◽

Sperm Quality ◽

Sperm Concentration ◽

Dna Integrity ◽

Sperm Dna Fragmentation ◽

Sperm Dna

Abstract Background Although bacterial infections have been recognized as a possible cause of male infertility, the effect of bacterial infections on sperm quality and sperm DNA fragmentation remains controversial. The current study aimed to investigate the prevalence rate of bacterial infection in subfertile men and its effect on semen quality. Seminal fluid was collected from 172 male members of infertile couples attending the andrology infertility center and a group of 35 fertile subjects as a control. Sperm parameters and DNA fragmentation were evaluated based on the type of bacteria in all ejaculates. Results From the 172 patients investigated for infertility, 60 (34.88%) patients had a positive culture for pathogenic bacteria of different species. Leukocytospermia was significantly higher in infected samples in comparison with non-infected samples (p < 0.05). Sperm concentration and motility and morphology were significantly lower in infected than non-infected samples. Moreover, sperm DNA fragmentation was significantly higher in infected than non-infected samples. Besides, our results showed that sperm DNA fragmentation was correlated significantly with leukocytospermia (R: 0.22, p < 0.01). Conclusion The present study suggested that bacterial infection significantly correlated with leukocytospermia could impair male fertility potential through decreasing sperm motility, morphology, and DNA integrity.

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P–042 Impact of semen parameters, sperm DNA fragmentation and sperm aneuploidy in male infertility

Human Reproduction ◽

10.1093/humrep/deab130.041 ◽

2021 ◽

Vol 36 (Supplement_1) ◽

Author(s):

M Maia ◽

C Almeida ◽

M Cunha ◽

A Gonçalves ◽

S S Soares ◽

...

Keyword(s):

Male Infertility ◽

Dna Fragmentation ◽

Sperm Concentration ◽

Semen Parameters ◽

Sperm Dna Fragmentation ◽

Male Age ◽

Fertility Status ◽

Sperm Aneuploidy ◽

Sperm Dna ◽

Aneuploidy Testing

Abstract Study question Should sperm aneuploidies and sperm DNA fragmentation (sDNAfrag) be included as valid tests in the routine investigation of male infertility? Summary answer Sperm DNA fragmentation was associated with male age, oligozoospermia (OZ), oligoteratozoospermia (OT), astenoteratozoospermia (AT) and oligoastenoteratozoospermia (OAT). Sperm aneuploidies were associated with OT and OAT. What is known already Semen parameters assist male infertility diagnosis and treatment, but sDNAfrag and aneuploidy analysis could add useful information, as abnormal values compromise fertility. To include these tests in the routine diagnosis it should be determined if behave as informative parameter and add information regarding the fertility status. For that, further studies comparing these tests to semen parameters are needed, since previous results are not consensual. Additionally, standardization of a sDNAfrag cut-off is needed, as different sample sizes and techniques originate distinct results. Also, until a standardization of the protocol is missing, a cut-off value should be defined for each laboratory. Study design, size, duration A retrospective and prospective investigation was performed, within a 12 years period (April 2007-December 2019). A total of 835 infertile males with a normal karyotype (46,XY) were included. Karyotyping and evaluation of sDNAfrag and sperm aneuploidies were made at a public Genetic unit. All normozoospermic (NZ) patients with a born child and patients whose infertility treatments were done due to female factors were selected from our database and used as controls (60 individuals). Participants/materials, setting, methods Semen analysis followed WHO–2010 guidelines. sDNAfrag was evaluated using the TUNEL assay. Sperm aneuploidies were detected using FISH (chromosomes 13, 18, 21, X, Y). Several tests were applied: correlations for linear associations between numerical variables, ANOVA for comparisons between means, Dunn-test for post-hoc comparisons. To determine the sDNAfrag cut-off value, the area under the ROC curve, sensitivity and specificity, were calculated, with the Youden-Index used to find a threshold that maximizes both sensitivity and specificity. Main results and the role of chance Regarding male age, it was observed a positive correlation with sperm concentration, a negative correlation with sperm vitality (VT) and hypoosmolality, and a positive correlation with sDNAfrag. Regarding sDNAfrag, it was observed negative correlation with sperm concentration, total progressive motility (TPM), morphology, VT and hypoosmolality. Regarding sperm aneuploidies, both total sperm aneuploidy and total sperm disomy exhibited a negative association with sperm concentration, TPM and morphology. It was also investigated whose groups of individuals could be indicated for sDNAfrag or sperm aneuploidy testing. The NZ group evidenced significant lower sDNAfrag, total sperm aneuploidy and total sperm disomy in relation to the non-NZ group. In the NZ group, sDNAfrag was significantly lower in relation to the OZ, OT, AT and OAT groups. The NZ group presented significant lower percentages of sperm aneuploidy in relation to the OT and OAT groups, and significant lower percentages of sperm disomy in relation to the OAT group. Additionally, sDNAfrag was positively correlated with total sperm aneuploidy and total sperm disomy. From the present large population, ROC curve analysis allowed estimating a cut-off value of 18.8% for the TUNEL-assay (sDNAfrag), with 0.658 of area under the curve, 53.9% sensitivity and 76.7% specificity. Limitations, reasons for caution Although presenting a high number of cases and strict controls, the present study was unable to include as controls healthy men with proven fertility. Additionally, the present study did not take into account life-style factors and male associated pathologies besides infertility. Wider implications of the findings: Semen parameters were shown to be negatively correlated with sDNAfrag and sperm aneuploidies. As sDNAfrag testing and sperm aneuploidy testing were associated with semen abnormalities and male age, it is suggested their inclusion in the routine evaluation of infertile men, thus adding important complementary information about the fertility status. Trial registration number Not Appliable

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Role of increased male age in IVF and egg donation: is sperm DNA fragmentation responsible?

Fertility and Sterility ◽

10.1016/j.fertnstert.2012.11.024 ◽

2013 ◽

Vol 99 (1) ◽

pp. 30-36 ◽

Cited By ~ 45

Author(s):

Kathryn C. Humm ◽

Denny Sakkas

Keyword(s):

Dna Fragmentation ◽

Egg Donation ◽

Sperm Dna Fragmentation ◽

Male Age ◽

Sperm Dna

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Our experience of application of low-level laser therapy in red spectrum in male idiopathic secretory infertility

Urologicheskie vedomosti ◽

10.17816/uroved41756 ◽

2020 ◽

Vol 10 (3) ◽

pp. 209-216

Author(s):

Maria K. Potapova ◽

Sergey Yu. Borovets ◽

Margarita N. Slesarevskaya ◽

Salman Kh. Al-Shukri

Keyword(s):

Laser Therapy ◽

Dna Fragmentation ◽

Sperm Quality ◽

Low Level Laser Therapy ◽

Sperm Dna Fragmentation ◽

Low Level ◽

Idiopathic Infertility ◽

Low Level Laser ◽

Level Laser ◽

Sperm Dna

Introduction. Due to insufficient efficacy of medical therapy of idiopathic secretory male infertility there is a need to develop and apply more resultative methods of treatment. The aim of our study was to assess the efficacy of low-level laser therapy (LLLT) in red spectrum on sperm parameters, sperm DNA fragmentation (SDNAF) and MAR-test rate in men with idiopathic infertility. Patients and methods. 85 men with idiopathic infertility were examined, 50 of them (first group) were treated with LLLT and 35 (second group) didnt have course of LLLT. Average age of patients of the first and second groups was 31.4 1.3 and 32.3 1.4 years. LLLT was performed in red spectrum on a Rubin-C device (Russia). 10 laser therapy procedures were carried out every two days with active luminescence for 10 minutes of both testicles. Male hormone panel, semen analysis, MAR-test, SDNAF were assessed before and after the treatment. Results. LLLT in patients of the first group resulted in significant increase in ejaculate volume, sperm viability, number of morphologically normal sperm forms (by an average of 11, 9 and 23% respectively), and active mobile sperm forms by an average of 14 and 19% one and two months after the therapy respectively. In 12 patients of the first group with initially increased SDNAF its level became normal after the treatment. Significant decrease of MAR-test we searched out in patients of the first group with its initial level less than 30%. As a result of the treatment, pregnancies developed in 10 (20%) out of 50 couples in the first group. Conclusions. LLLT in red spectrum leads to significant improvement of sperm quality in patients with idiopathic infertility, normalizes sperm DNA-fragmentation level and decreases MAR-test level.

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A Modified Flotation Density Gradient Centrifugation Technique Improves the Semen Quality of Stallions with a High DNA Fragmentation Index

Animals ◽

10.3390/ani11071973 ◽

2021 ◽

Vol 11 (7) ◽

pp. 1973

Author(s):

Muhammad Umair ◽

Heiko Henning ◽

Tom A. E. Stout ◽

Anthony Claes

Keyword(s):

Dna Fragmentation ◽

Semen Quality ◽

Sperm Quality ◽

Gradient Centrifugation ◽

Quality Parameters ◽

Sperm Dna Fragmentation ◽

Fragmentation Index ◽

Sperm Dna ◽

Cushion Layer ◽

Dna Fragmentation Index

Sperm DNA fragmentation compromises fertilization and early embryo development. Since spermatozoa lack the machinery to repair DNA damage, to improve the likelihood of establishing a healthy pregnancy, it is preferable to process ejaculates of stallions with a high sperm DNA fragmentation index (DFI) before artificial insemination or intracytoplasmic sperm injection. The aim of this study was to examine a modified flotation density gradient centrifugation (DGC) technique in which semen was diluted with a colloid solution (Opti-prepTM) to increase its density prior to layering between colloid layers of lower and higher density. The optimal Opti-prepTM solution (20–60%) for use as the bottom/cushion layer was first determined, followed by a comparison between a modified sedimentation DGC and the modified flotation DGC technique, using different Opti-prepTM solutions (20%, 25% and 30%) as the top layer. Finally, the most efficient DGC technique was selected to process ejaculates from Friesian stallions (n = 3) with high sperm DFI (>20%). The optimal Opti-prepTM solution for the cushion layer was 40%. The modified sedimentation technique resulted in two different sperm populations, whereas the modified flotation technique yielded three populations. Among the variants tested, the modified flotation DGC using 20% Opti-prepTM as the top layer yielded the best results; the average sperm recovery was 57%; the DFI decreased significantly (from 12% to 4%) and the other sperm quality parameters, including progressive and total motility, percentages of spermatozoa with normal morphology and viable spermatozoa with an intact acrosome, all increased (p < 0.05). In Friesian stallions with high sperm DFI, the modified flotation DGC markedly decreased the DFI (from 31% to 5%) and significantly improved the other semen quality parameters, although sperm recovery was low (approximately 20%). In conclusion, stallion sperm DFI and other sperm quality parameters can be markedly improved using a modified flotation DGC technique employing a 40% Opti-prepTM cushion and a 20% top layer.

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