scholarly journals Improvement of Post-Thaw Sperm Kinematics and DNA Integrity of Cross-Bred Bovine Sperm by Incorporating DGC as Selection Method Prior to Cryopreservation

2017 ◽  
Vol 9 (13) ◽  
pp. 24
Author(s):  
Nur Hilwani Ismail ◽  
Khairul Osman ◽  
Farida Zuraina Mohd Yusof ◽  
Syarifah Faezah Syed Mohamad ◽  
Farah Hanan Fatihah Jaafar ◽  
...  
Keyword(s):  
Treatment Group ◽  
Sperm Quality ◽  
Gradient Centrifugation ◽  
Control Group ◽  
Dna Integrity ◽  
Sperm Selection ◽  
Sperm Analysis ◽  
Fragmentation Index ◽  
Bovine Spermatozoa ◽  
Kinematics Parameters

The aim of this study was to assess post-thaw sperm quality following initial sperm selection using density gradient centrifugation (DGC) prior to cryopreservation. Ejaculates from four mature Charolais cross Kedah-Kelantan bulls were collected using artificial vagina at IBVK Pahang, Malaysia. The ejaculates were aliquoted into 3 groups: non-cryopreserved group (NC); control group of cryopreserved sperm without DGC (ND) and treatment group of sperm undergoing DGC sperm selection before cryopreservation (CDGC). Prior to analysis, samples from both cryopreserved groups were thawed at 37 °C for 30 sec. All samples were analysed for kinematics parameters, viability and compromise in DNA integrity (evaluated as DNA Fragmentation Index, DFI). All kinematics parameters were analysed using computer aided sperm analysis (CASA). Results indicated significant (p < 0.05) kinematics parameter changes for all parameters of velocity (VCL, VSL, VAP) and progression (WOB, LIN, ALH and BCF). Unfortunately, changes in spermatozoa straightness were insignificant (STR) F(2, 68) = 1.004, p = 0.371. Spermatozoa viability had increased by 26.2% (p < 0.01) following the treatment. DFI revealed the treatment group recorded a significant reduction in DFI value (0.17% fragmented DNA). In conclusion, DGC sperm selection prior to cryopreservation reduced the effects of cryodamage and showed an improvement in post-thaw sperm quality, thus reducing the occurrence of asthenozoospermia in populations of frozen-thawed cross-bred bovine spermatozoa.

97 DIFFERENCES IN SPERM CHROMATIN STRUCTURE AMONG GOOD AND BAD BOAR SPERM FREEZERS

2006 ◽  
Vol 18 (2) ◽  
pp. 157
Author(s):  
M. Hernández ◽  
J. Roca ◽  
J. Ballester ◽  
J. M. Vázquez ◽  
E. A. Martínez ◽  
...  
Keyword(s):  
Chromatin Structure ◽  
Sperm Quality ◽  
Egg Yolk ◽  
Sperm Chromatin ◽  
Dna Integrity ◽  
Computer Assisted ◽  
Sperm Analysis ◽  
Fragmentation Index ◽  
Sperm Dna ◽  
Multivariate Pattern

Inter- and intra-boar differences in sperm freezability are observed independent of the sperm quality before freezing, the breed, or the genetic line. This study aimed to determine whether boars with different post-thaw sperm quality also show differences in sperm DNA integrity. Sperm-rich fractions (3 to 10 ejaculates per boar) from 19 fertile mature boars were extended in Beltsville thawing solution (BTS) and cooled to 17�C for 16 h. Then, samples were centrifuged at 2400g for 3 min, extended in freezing extender (lactose/egg yolk/glycerol/Equex STM; Nova Chemical Sales, Inc., Scituate, MA, USA) to a final concentration of 1 � 109 spermatozoa/mL, dispensed into 0.5 mL straws, and frozen in a programmable cell freezer at a rate of -20�C min. Thawing was carried out in a water bath at 37�C for 20 s. Frozen-thawed spermatozoa were evaluated for progressive sperm motility (PSM) using a computer-assisted sperm analysis (CASA) system, and sperm viability (PMI) using flow cytometry. All data generated were used for a multivariate pattern analysis (PATN; CSIRO, Canberra, Australia) which objectively classified all boars into two groups, categorized as good (n = 10; >60% PSM and PMI) or bad (n = 9; <40% PSM and PMI) based on their sperm freezability. Post-thaw sperm quality was consistent for different ejaculates within boars (P < 0.05). The DNA-integrity of frozen-thawed spermatozoa was evaluated according to the sperm chromatin structure assay (SCSA; Evenson et al. 1980 Science 210, 1131-1133). All SCSA variables (X mean, DNA fragmentation index (DFI), and the standard deviation of the DFI), were significantly higher for bad freezers (P < 0.001). The percentage of spermatozoa with abnormal chromatin structure ranged from 1.06 to 3.42% for good and 3.06 to 6.04% for bad freezers. Although these differences exist between good and bad sperm freezers, and can only to some extent be the product of cryopreservation, the levels of affected spermatozoa can not explain the differences on post-thaw sperm survival seen in the two categories of sires. This work was supported by CICYT, AGL05-0471 (Spain), SLF and Formas (Sweden).

scholarly journals Improvement of Sperm Quality in Hyperviscous Semen following DNase I Treatment

2019 ◽  
Vol 2019 ◽  
pp. 1-8
Author(s):  
Effrosyni Nosi ◽  
Angelos D. Gritzapis ◽  
Konstantinos Makarounis ◽  
Georgios Georgoulias ◽  
Vasilios Kapetanios ◽  
...  
Keyword(s):  
Density Gradient ◽  
Sperm Motility ◽  
Density Gradient Centrifugation ◽  
Sperm Quality ◽  
Sperm Count ◽  
Gradient Centrifugation ◽  
Dnase I ◽  
Control Group ◽  
First Case ◽  
Dnase Treatment

Semen hyperviscosity impairs sperm motility and can lead to male infertility. This prospective study aimed at assessing the ability of exogenous DNase in improving sperm quality, taking into consideration that DNase has been found in the seminal plasma of several species and that neutrophils release chromatin in order to trap bacteria. A total of seventy-seven semen samples with high seminal viscosity (HSV) as the study group and sixty-two semen samples with normal seminal viscosity (NSV) as the control group were compared in this analysis. These semen samples were divided into three groups of receiving treatment (a) with DNase I at 37°C for 15 min, (b) by density gradient centrifugation, and (c) with a combination of the above two methods. Following a fifteen-minute treatment of hyperviscous semen, the motility of spermatozoa in 83% of semen samples increased to a statistically significant degree. On the contrary, DNase treatment of semen with normal viscosity had no such effects. The above treatment was also accompanied by a significant increase in the percentage of normal spermatozoa, resulting in a major decrease of the teratozoospermia index. Comparison between semen samples that underwent density gradient centrifugation following DNase I treatment, to those collected after density gradient treatment alone, showed that in the first case the results were more spectacular. The evaluation of each preparation in terms of yield (% total progressively motile sperm count after treatment in relation to the initial total sperm count) revealed that the combined approach resulted in 29.8% vs. 18.5% with density treatment alone (p=0.0121). DNase I treatment results in an improvement of sperm motility and morphology and could be beneficial to men with hyperviscous semen in assisted reproduction protocols.

The effects of chronic administration of ghrelin on rat sperm quality and membrane integrity

2009 ◽  
Vol 59 (2) ◽  
pp. 159-168 ◽  
Author(s):  
Arash Kheradmand ◽  
Majid Taati ◽  
Homayoon Babaei
Keyword(s):  
Plasma Membrane ◽  
Treatment Group ◽  
Sperm Motility ◽  
Sperm Quality ◽  
Sperm Concentration ◽  
Membrane Integrity ◽  
Chronic Administration ◽  
Control Group ◽  
Plasma Membrane Integrity ◽  
Significant Difference

AbstractAlthough ghrelin acts as a modulator of feeding behavior and energy metabolism in the central nervous system, recent studies have implicated the peripheral actions of ghrelin in reproductive tissues. Here, we investigated the effects of chronic administration of ghrelin on the motility, plasma membrane integrity and concentration of rat spermatozoa. 45-d male Wistar rats were scheduled for the study and were divided into control and treatment groups. In the treatment group, 1 nmol of ghrelin was administered as sc injection for 10 consecutive days or vehicle (physiological saline) to the control rats. Sperm collection was achieved by killing of the rats on days 15, 25 and 50 after first injection. Total sperm motility and forward progressive movement did not exhibit significant difference during the experiment, although, there was a tendency for greater motion rate on d 15 and 25 in the treated rats compared to the control group. Plasma membrane integrity (HOS-reacted spermatozoa) was significantly higher in the treated animals, especially on day 15 as well as day 25, because of possible antioxidant properties of ghrelin. This value was statistically higher on day 15 than that of day 25 (P <0.05). Likewise, there was a significant correlation between the FPM (P <0.0001, r = 0.79) and TSM (P <0.01, r = 0.52) with the HOS test percentage in the treatment group. It was not observed statistically difference in the sperm concentration between groups during all of the experimental days. In conclusion, chronic administration of ghrelin (similar to induced by energy deficiency such as fasting) increased the integrity of sperm membrane, however, the sperm motility and concentration did not display any alterations.

Association between chronic pain and the sperm motion characteristics

2015 ◽  
Vol 8 (1) ◽  
pp. 48-48 ◽  
Author(s):  
F. Dardmeh ◽  
H. Alipour ◽  
H.I. Nielsen ◽  
S. Rasmussen ◽  
J.T. Yousefi ◽  
...  
Keyword(s):  
Chronic Pain ◽  
Sperm Quality ◽  
Total Concentration ◽  
Gonadal Hormones ◽  
Prognostic Indicators ◽  
Control Group ◽  
Sperm Analysis ◽  
Significant Difference ◽  
Chronic Pain Conditions ◽  
Sperm Motion

Abstract Aims Sex hormones play an important role in pain in many chronic pain conditions. Relationship between chronic pain and sperm quality has not been investigated thoroughly and may provide an insight to better understanding, management and treatment of cases where chronic pain and male sub-fertility co-exist. Methods Neat (fresh semen) and processed sperm from 15 males with orthopedic chronic pain (CP) were assessed and compared with 15 healthy age matched controls. Sperm analysis was performed using the SCA computer-aided sperm analyzer. Results There was no significant difference in any parameters of the neat semen between the pain and control group. However the percentage of non-progressive motile spermatozoa (type B) was significantly higher in the pain group (27.96) compared to the control group (15.96). Straight line trajectories including linearity, straightness, wobble and beat cross frequency were also significantly higher in the processed sample of the CP group. Conclusions This study demonstrated that chronic pain does not affect the sperm morphology, total concentration and motility based on conventional analysis but has significant influence at the level of sperm motion kinetics which could prove to be clinically valuable, prognostic indicators of successful fertilization. Maturation of sperm motility occurs during their transit through the epididymis and vas deferens regulated by androgens. As male gonadal hormones haveaninhibitory, adaptive effectonthe behavioral and neuronal responses to repeated nociceptive stimulation, it can be speculated that the observed difference in sperm kinematic parameters could be related to the alterations in serum sex hormone levels emanating from the chronic pain. Further studies are required to explain the possible mechanism of actionof chronic pain on male fertility.

scholarly journals Melatonin improves rate of monospermic fertilization and early embryo development in a bovine IVF system

PLoS ONE ◽  
2021 ◽  
Vol 16 (9) ◽  
pp. e0256701
Author(s):  
Juan Carlos Gutiérrez-Añez ◽  
Heiko Henning ◽  
Andrea Lucas-Hahn ◽  
Ulrich Baulain ◽  
Patrick Aldag ◽  
...  
Keyword(s):  
Embryo Development ◽  
Sperm Quality ◽  
Hierarchical Cluster ◽  
Early Embryo ◽  
Developmental Competence ◽  
Control Group ◽  
Computer Assisted ◽  
Sperm Analysis ◽  
Flow Cytometry Data

The developmental competence of male and female gametes is frequently reduced under in vitro conditions, mainly due to oxidative stress during handling. The amino-acid derived hormone melatonin has emerged as a potent non-enzymatic antioxidant in many biological systems. The goal of the present study was to evaluate the effects of melatonin on post-thaw sperm quality, fertilizing ability, and embryo development and competence in vitro after in vitro fertilization. Frozen-thawed bovine spermatozoa were incubated either in the presence of 10−11 M melatonin (MT), or its solvent (ethanol; Sham-Control), or plain Tyrode’s Albumin Lactate Pyruvate medium (TALP, Control). Computer-Assisted Sperm Analysis (CASA) and flow cytometry data after 30 min, 120 min, and 180 min incubation did not reveal any significant effects of melatonin on average motility parameters, sperm subpopulation structure as determined by hierarchical cluster, or on the percentage of viable, acrosome intact sperm, or viable sperm with active mitochondria. Nevertheless, in vitro matured cumulus-oocyte-complexes fertilized with spermatozoa which had been preincubated with 10−11 M melatonin (MT-Sperm) showed higher (P < 0.01) rates of monospermic fertilization, reduced (P < 0.05) polyspermy and enhanced (P < 0.05) embryo development compared to the Control group. Moreover, the relative abundance of MAPK13 in the in vitro-derived blastocysts was greater (P < 0.05) than observed in the Control group. In conclusion, adding melatonin to the sperm-preparation protocol for bovine IVF improved proper fertilization and enhanced embryonic development and competence in vitro.

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.

scholarly journals Analysis of Bovine Spermatozoa CSP-Gene Expression using qPCR and Relative Quantification Method as Biodiagnostic Tool for Fertilizing Capacity

2019 ◽  
Vol 7 (4.14) ◽  
pp. 127
Author(s):  
N H Ismail ◽  
S F Syed Mohamad ◽  
F H F Jaafar ◽  
K Osman ◽  
F Z Mohd Yusof ◽  
...  
Keyword(s):  
Gene Expression ◽  
Treatment Group ◽  
Sperm Quality ◽  
Cold Shock Protein ◽  
Protective Mechanism ◽  
Relative Quantification ◽  
Sperm Selection ◽  
Homogeneous Population ◽  
Expression Studies ◽  
Gene Expression Studies

Gene expression studies enable for real-time relative quantification of expressed genes. However, the incorporation of a primary discerning tool before amplification for specific sequences of genes of interest is yet to be implemented. The amplification of gene sequences from discriminate sample groups further enhances the results following qPCR and provides an absolute exclusive measurement for the defined sample. Cryopreserved spermatozoa have characteristics of compromised sperm quality as the cells are exposed to a rapid temperature downshift not normally encountered by such cells in vivo. Thus, the use of a separation system before cryopreservation produces a refined outcome in gene expression studies. The proliferation of Cold Shock Protein (CSP) can relatively be quantified by conducting qPCR following sperm selection and cryopreservation. CSPs are indicators of cells adapting to the decrease in temperature and indirectly acts as an intracellular protective mechanism for the cell. The current study compares the effects of sperm selection system in isolating a homogeneous population of spermatozoa for cryopreservation followed by quantification of CSP gene. Method of molecular assessment involved quantifying the amplified sequences via qPCR and gene expression following sperm isolation was significant in increasing spermatozoa viability by 26.2% (P< 0.05). The relative fold expression of CSP gene for treatment group increased from 1.00 fold to 1.38 fold. The Cq values for treatment group had recorded an earlier point of amplification (Cq= 24.87) as compared to control Cq= 25.83. Our findings suggest that the use of a sperm isolation system before cryopreservation would increase the probability of obtaining higher amplifications of CSP genes that would confer protection against extremely low temperatures during cryopreservation. This would increase the likelihood of in vitro fertilization using cryopreserved spermatozoa by implementing qPCR as a potential biomolecular diagnostic tool to ascertain the fertilizing potential of the spermatozoa.  

scholarly journals Sperm phenotypes in varicocele

2019 ◽  
Vol 20 (4) ◽  
pp. 24-33
Author(s):  
O. B. Zhukov ◽  
E. E. Bragina ◽  
A. V. Levin
Keyword(s):  
Surgical Treatment ◽  
Treatment Group ◽  
Conservative Treatment ◽  
Control Group ◽  
Sperm Ultrastructure ◽  
Study Objective ◽  
Sperm Analysis ◽  
Before And After ◽  
Group 2 ◽  
Group 1

The study objective is to microscopically evaluate the morphology of sperm in patients with varicocele after surgical treatment and therapy with Prostatilen® AC.Materials and methods. The treatment group included 20 males between 26 and 45 years of age (mean age 31.6 ± 6.1 years) with various stages of varicocele and fertility problems; among them, 10 patients with subclinical stages of varicocele received conservative treatment with Prostatilen® AC (group 1) and were examined before and after the treatment; 10 males with infertility caused in part by varicocele of the spermatic cord veins were examined 6 months to 3 years after surgical treatment (group 2). Standard clinical and lab tests, sperm analysis, electron microscopy of the ejaculate were performed. The control group included 65 fertile males whose sperm samples were obtained from a bank of reproductive cells and tissues and used for comparison in microscopic examination.Results. In patients who received conservative treatment the number of sperm with immature chromatin decreased (p = 0.045) compared to the control group. This characteristic differed in patients after varicocelectomy and patients after conservative treatment (p = 0.037). Compared to control, the number of sperm with excess residual cytoplasm in the head and neck was higher in patients after varicocelectomy (p = 0.011). After conservative treatment, the number of sperm with excess residual cytoplasm was close to the control number and lower than in patients after varicocelectomy (р = 0.028).Conclusion. In patients with subclinical varicocele, conservative treatment with Prostatilen® AC leads to significant improvement in sperm ultrastructure compared to patients who underwent surgery to treat this pathology. 

371 DOES Percoll™ GRADIENT CENTRIFUGATION CAUSE HYPERACTIVATION ON CRYOPRESERVED BOVINE SPERMATOZOA?

2010 ◽  
Vol 22 (1) ◽  
pp. 342
Author(s):  
L. Z. Oliveira ◽  
R. P. Arruda ◽  
E. C. C. Celeghini ◽  
A. F. C. Andrade ◽  
A. C. Lucio ◽  
...  
Keyword(s):  
Density Gradient ◽  
Calcium Influx ◽  
Ph Monitoring ◽  
Semen Analysis ◽  
Gradient Centrifugation ◽  
Control Group ◽  
Computer Assisted ◽  
Head Displacement ◽  
Bovine Spermatozoa

The density difference of 0.06% between X- and Y-bearing bovine spermatozoa has the potential to provide for separation X-bearing spermatozoa on specific gradient solutions of Percoll™ (Hossepian de Lima VFM et al. PCT/BR2004/000009). The question remains whether Percoll™ density gradient centrifugation induces the hyperactivation of spermatozoa, because it is believed that some decapacitating proteins are removed from the sperm surface during the process. Thus, the objective of this study was to evaluate if Percoll™ centrifugation causes hyperactivation of cryopreserved bovine spermatozoa. Semen doses were collected from six bulls of different breeds, including three taurine and three Zebu animals, and four ejaculates per bull were evaluated. The semen samples were thawed and evaluated before (control) and after (sexed group) centrifugation (500 g) in discontinuous Percoll™ density gradient (Hossepian Lima VFM et al. PCT/BR2004/000009). Sperm motility was assessed by Computer-Assisted Semen Analysis (CASA, Hamilton Thorne Biosciences, Beverly, MA, USA) and the results (mean ± SEM) were submitted to ANOVA. Higher (P < 0.0001) total and progressive sperm motilities were observed in sexed (85.0 ± 1.8 and 75.9 ± 1.7%, respectively) than in the control group (69.9 ± 1.7 and 59.2 ± 1.6%, respectively). The average path velocity, straight-line velocity and curvilinear velocity (VCL) were lower (P < 0.01) in sexed (91.3 ± 1.1, 79.8 ± 0.8, and 137.6 ± 3.5 (im/s, respectively) than in control group (107.6 ± 4.8, 91.1 ± 4.1, and 173.2 ± 8.9 (im/s, respectively). The amplitude of lateral head displacement (ALH) was higher (P < 0.0001) in control (6.7 ± 0.3 μm) than in sexed semen (5.1 ± 0.2 μm) and beat cross frequency was higher (P < 0.05) in sexed (35.9 ± 0.9 Hz) than in control semen (33.1 ± 1.0 Hz). The straightness and linearity (LIN) were higher (P < 0.01) in sexed (87.4 ± 0.7 and 61.4 ± 1.4%, respectively) than in control semen (84.4 ± 0.7 and 55.5 ± 1.2%, respectively). Regarding the results obtained in the present study, it was not possible to infer that the method used induced sperm hyperactivation. According to Marquez and Suarez (2007 Biol. Reprod., 76, 660-665), an increase in pH and intraflagellar calcium plays a key role in the axoneme changes, promoting an increase in ALH (from 9 to 13 μm) and VCL (from 200 to 315 μm/s), as well as a decrease in LIN (from 57 to 25%), which characterizes the presence of hyperactivated cells in the sample. In the present study, centrifuged spermatozoa presented significantly lower ALH and VCL and higher LIN compared to control semen samples. Therefore, the presence of glucose in the Percoll™ stock solution and constant pH monitoring could have prevented alkalinization of the medium, thereby preventing the occurrence of intraflagelar calcium influx and the consequent triggering of in vitro sperm hyperactivation.

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

Animals ◽  
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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