scholarly journals Effect of Trehalose and Sucrose in Post-thaw Quality of Crassostrea angulata Sperm

2021 ◽  
Vol 12 ◽  
Author(s):  
Catarina Anjos ◽  
Ana Luísa Santos ◽  
Daniel Duarte ◽  
Domitília Matias ◽  
Elsa Cabrita
Keyword(s):  
Plasma Membrane ◽  
Sperm Quality ◽  
Artificial Seawater ◽  
Live Cells ◽  
Dna Integrity ◽  
Computer Assisted ◽  
Sperm Cryopreservation ◽  
Sperm Analysis ◽  
Crassostrea Angulata ◽  
And Function

Sperm cryopreservation can be a helpful tool in reproductive management and preservation of biodiversity. However, the freezing methodologies lead to some damage in structure and function of cells that may compromise post-thaw sperm activity. Cryoprotectant supplementation with sugars proved to be a successful strategy to reduce cryodamage in sperm of several species, once allowing to stabilize the plasma membrane constituents. Therefore, this study intends to understand the effects of sugars in the plasma membrane, DNA integrity, and oxidative response during Portuguese oyster sperm cryopreservation. Three cryoprotectants solutions with an initial concentration of 20% dimethyl sulfoxide (DMSO) and 20% DMSO complemented with 0.9 M trehalose or sucrose in artificial seawater were employed. Sperm samples of mature males were individually collected and diluted 1:10 (v/v) in artificial seawater followed by addition of cryoprotectants [1:1 (v/v)]. Thereafter, sperm was loaded into 0.5 ml straws, maintained at 4°C for 10 min, frozen in a programmable biofreezer at −6°C/min from 0 to −70°C, and stored in liquid nitrogen. Samples were thawed in a 37°C bath for 10 s. Several techniques were performed to evaluate post-thaw quality. Sperm motility and DNA integrity were analyzed by using computer-assisted sperm analysis (CASA) software and comet assay. Flow cytometry was employed to determine membrane and acrosome integrity and to detect intracellular reactive oxygen species (ROS) and apoptosis activity. Lipid peroxidation was determined by malondialdehyde (MDA) detection by using spectrophotometry. Sperm antioxidant capacity was evaluated through glutathione peroxidase, glutathione reductase, and superoxide dismutase. Motility was not affected by the extenders containing sugars; these compounds did not reduce the DNA damage. However, both the trehalose and sucrose protected plasma membrane of cells by increasing cell viability and significantly reducing MDA content. The same finding was observed for the ROS, where live cells registered significantly lower levels of ROS in samples cryopreserved with sugars. The activity of antioxidant enzymes was higher in treatments supplemented with sugars, although not significant. In conclusion, the addition of sugars seems to play an important role in protecting the Crassostrea angulata sperm membrane during cryopreservation, showing potential to improve the post-thaw sperm quality and protect the cells from cryoinjuries.

scholarly journals Efficiency of Commercial Egg Yolk-Free and Egg Yolk-Supplemented Tris-Based Extenders for Dromedary Camel Semen Cryopreservation

Animals ◽  
2019 ◽  
Vol 9 (11) ◽  
pp. 999 ◽  
Author(s):  
Ayman Abdel-Aziz Swelum ◽  
Islam M. Saadeldin ◽  
Hani Ba-Awadh ◽  
Mohsen G. Al-Mutary ◽  
Abdullah F. Moumen ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Sperm Motility ◽  
Sperm Quality ◽  
Membrane Integrity ◽  
Egg Yolk ◽  
Dna Integrity ◽  
Computer Assisted ◽  
Dromedary Camel ◽  
Plasma Membrane Integrity ◽  
Semen Cryopreservation

This study compared the efficiency of commercial egg yolk-free (AndroMed, OPTIXcell) and egg yolk-supplemented (Triladyl, Steridyl) Tris-based extenders for semen cryopreservation in seven adult dromedary camels. The camel-specific extender SHOTOR was used as control. The collected semen samples were evaluated and diluted with SHOTOR, Triladyl, Steridyl, AndroMed, or OPTIXcell. The diluted semen was gradually cooled and equilibrated for two hours before liquid nitrogen freezing. Semen was evaluated prior to freezing and after freeze-thawing cycles for motility, kinetics, vitality, abnormality, plasma membrane integrity, and DNA fragmentation using computer-assisted sperm analysis. In pre-freezing evaluation, progressive sperm motility was higher in SHOTOR-diluted samples (21.54 ± 1.83) than in samples diluted with Steridyl, OPTIXcell, or AndroMed (15.76 ± 1.80, 17.43 ± 1.10, and 13.27 ± 1.07, respectively). Moreover, Triladyl and SHOTOR resulted in significantly (p < 0.05) better sperm vitality and DNA integrity than all other diluents, but Triladyl resulted in a significantly (p < 0.05) better plasma membrane integrity (87.77 ± 0.31) than SHOTOR (85.48 ± 0.58). In the post-thawing evaluation, Triladyl led to significantly (p < 0.05) higher sperm motility (38.63 ± 0.81%; p < 0.05) when compared to SHOTOR, Steridyl or AndroMed (35.09 ± 1.341%, 34.4 ± 0.84%, and 31.99 ± 1.48%, respectively), with OPTIXcell being the least efficient (28.39 ± 0.86%). Progressive sperm motility was the highest when using Triladyl. Post-thawing curvilinear, straight line and average path sperm velocities were highest with Triladyl and lowest with AndroMed. Triladyl led to the highest linearity coefficient and straightness sperm coefficient, while SHOTOR to the highest DNA and plasma membrane integrity. OPTIXcell and AndroMed resulted in poor post-thawing sperm vitality, while Steridyl was less efficient than Triladyl. The highest rate of sperm abnormalities was recorded with OPTIXcell and the lowest with SHOTOR or Triladyl. In conclusion, SHOTOR, Triladyl, Steridyl, AndroMed, and OPTIXcell can all be used for camel semen cryopreservation; however, SHOTOR and Triladyl provided the best post-thawing sperm quality. Based on our findings, Triladyl is the best commercially available extender for dromedary camel semen cryopreservation to date.

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 Post-Thaw Sperm Quality and Functionality in the Autochthonous Pig Breed Gochu Asturcelta

Animals ◽  
2021 ◽  
Vol 11 (7) ◽  
pp. 1885
Author(s):  
José Néstor Caamaño ◽  
Carolina Tamargo ◽  
Inmaculada Parrilla ◽  
Felipe Martínez-Pastor ◽  
Lorena Padilla ◽  
...  
Keyword(s):  
Genetic Resource ◽  
Sperm Quality ◽  
Genetic Material ◽  
Mitochondrial Activity ◽  
Computer Assisted ◽  
Kinematic Parameters ◽  
Linear Mixed Effects ◽  
Sperm Analysis ◽  
Computer Assisted Sperm Analysis ◽  
Analysis System

Genetic resource banks (GRB) preserve the genetic material of endangered, valuable individuals or genetically relevant breeds. Semen cryopreservation is a crucial technique to reach these goals. Thus, we aimed to assess the sperm parameters of semen doses from the native pig breed Gochu Asturcelta stored at the GRB of Principado de Asturias (GRB-PA, Gijón, Spain), focusing on intrinsic and extrinsic (boar, season) factors. Two straws per boar (n = 18, 8–71 months of age) were thawed, pooled, and assessed after 30 and 150 min at 37 °C by CASA (computer-assisted sperm analysis system; motility and kinematic parameters) and flow cytometry (viability, acrosomal status, mitochondrial activity, apoptosis, reactive oxygen species, and chromatin status). The effects of age, incubation, and season on post-thawing quality were determined using linear mixed-effects models. Parameters were on the range for commercial boar breeds, with chromatin status (SCSA: fragmentation and immaturity) being excellent. Incubation decreased sperm quality and functionality. The boar age did not have a significant effect (p > 0.05), but the between-boar variability was significant (p < 0.001). The season significantly affected many parameters (motility, kinematics, viability, acrosomal status, mitochondrial activity), especially after 150 min of incubation. In general, samples collected in spring and summer showed higher quality post-thawing, the lowest in winter. In conclusion, the sperm doses from the Gochu Asturcelta breed stored at the GRB-PA showed excellent chromatin status and acceptable characteristics after thawing. Therefore, boar and seasonal variability in this autochthonous breed could be relevant for cryobank management.

Effect of transfection and co-incubation of bovine sperm with exogenous DNA on sperm quality and functional parameters for its use in sperm-mediated gene transfer

Zygote ◽  
2016 ◽  
Vol 25 (1) ◽  
pp. 85-97 ◽  
Author(s):  
María Elena Arias ◽  
Esther Sánchez-Villalba ◽  
Andrea Delgado ◽  
Ricardo Felmer
Keyword(s):  
Gene Transfer ◽  
Sperm Quality ◽  
Computer Assisted ◽  
Exogenous Dna ◽  
Sperm Analysis ◽  
Functional Parameters ◽  
Transgenic Embryos ◽  
Fertilization Capacity ◽  
Motility Parameters

SummarySperm-mediated gene transfer (SMGT) is based on the capacity of sperm to bind exogenous DNA and transfer it into the oocyte during fertilization. In bovines, the progress of this technology has been slow due to the poor reproducibility and efficiency of the production of transgenic embryos. The aim of the present study was to evaluate the effects of different sperm transfection systems on the quality and functional parameters of sperm. Additionally, the ability of sperm to bind and incorporate exogenous DNA was assessed. These analyses were carried out by flow cytometry and confocal fluorescence microscopy, and motility parameters were also evaluated by computer-assisted sperm analysis (CASA). Transfection was carried out using complexes of plasmid DNA with Lipofectamine, SuperFect and TurboFect for 0.5, 1, 2 or 4 h. The results showed that all of the transfection treatments promoted sperm binding and incorporation of exogenous DNA, similar to sperm incorporation of DNA alone, without affecting the viability. Nevertheless, the treatments and incubation times significantly affected the motility parameters, although no effect on the integrity of DNA or the levels of reactive oxygen species (ROS) was observed. Additionally, we observed that transfection using SuperFect and TurboFect negatively affected the acrosome integrity, and TurboFect affected the mitochondrial membrane potential of sperm. In conclusion, we demonstrated binding and incorporation of exogenous DNA by sperm after transfection and confirmed the capacity of sperm to spontaneously incorporate exogenous DNA. These findings will allow the establishment of the most appropriate method [intracytoplasmic sperm injection (ICSI) orin vitrofertilization (IVF)] of generating transgenic embryos via SMGT based on the fertilization capacity of transfected sperm.

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.

17 Increased inbreeding levels negatively affect sperm kinetics and motility in Purebred Spanish horses

2021 ◽  
Vol 33 (2) ◽  
pp. 116
Author(s):  
Y. Pirosanto ◽  
A. Molina ◽  
M. Valera ◽  
J. Dorado ◽  
E. Terán ◽  
...  
Keyword(s):  
Sperm Motility ◽  
Sperm Quality ◽  
Reproductive Traits ◽  
Study Groups ◽  
Inbreeding Coefficient ◽  
Computer Assisted ◽  
Effective Population ◽  
Mean Velocity ◽  
Sperm Analysis ◽  
Head Displacement

Reproductive performance is one of the key factors in livestock production. It is well known that reproductive traits are influenced by several genetic factors, such as the increase of individual inbreeding levels, which are associated with changes in sperm motility and shape in several species. In horses, the increase in inbreeding is a common problem because of the reduction in effective population size and the increase in selection intensity observed in several breeds. However, studies assessing the effect of high levels of inbreeding on the sperm quality of stallions are scarce. In the present study, we aimed to determine the effect of increased inbreeding levels and age on the sperm motility patterns of Purebred Spanish horses (PRE). We performed kinetic characterisation of 557 sperm samples of 82 PRE stallions aged between 3 and16 years, using computer-assisted sperm analysis (Androvision™, Minitube). We evaluated 5 parameters in 6 different fields per sample: curved line velocity (VCL, µm/s), velocity average path (VAP, µm/s), velocity straight line (VSL, µm/s), amplitude of lateral head displacement (ALH, µm), and beat-cross frequency (BCF, Hz). We determined the pedigree-based inbreeding coefficient (Fped) based on ∼300,000 PRE pedigree records to evaluate the inbreeding effect. Individuals were separated into 2 groups: highly inbred (n=339) and lowly inbred (n=218) according to an F value of 12.5%. Differences between groups were analysed using a generalized linear model. The analysis did not show significant differences (P&gt;0.05) in the variables analysed with respect to the age of stallions. However, VAP, VCL, and AHL were lower in highly inbred than in lowly inbred animals (P&lt;0.05), suggesting less velocity and amplitude of head displacement. In the case of BCF, no significant differences (P&gt;0.05) were observed between the two study groups. In conclusion, age did not affect sperm quality parameters in the age group of stallions analysed. In addition, we demonstrated that high inbreeding coefficient reduced the mean velocity and trajectory pattern of spermatozoa in PRE.

scholarly journals Effects of chilled storage and pH of activating solution on different motility parameters in burbot (Lota lota) sperm

2018 ◽  
Vol 63 (No. 11) ◽  
pp. 429-434
Author(s):  
Zoltán Bokor ◽  
Balázs Csorbai ◽  
Levente Várkonyi ◽  
Zsolt Szári ◽  
Ferenc Fodor ◽  
...  
Keyword(s):  
Sperm Motility ◽  
Sperm Quality ◽  
Computer Assisted ◽  
Sperm Analysis ◽  
Chilled Storage ◽  
Straight Line ◽  
H 26 ◽  
Computer Assisted Sperm Analysis ◽  
Motility Parameters ◽  
Activating Solution

The effects of a simple saline solution prepared using two different pH (4.4 and 8.5) on sperm motility in burbot were investigated. Results were recorded during a 96-hour chilled storage (4°C) in 24-hour intervals. Measurements were focused on the detailed characteristics of motility using 12 parameters obtained from the Computer-assisted Sperm Analysis (CASA). Significantly higher progressive motility (pMOT), distance average path (DAP), distance curved line, distance straight line (DSL), average path velocity (VAP), curvilinear velocity, straight line velocity, and beat cross frequency (BCF) were observed with the activating solution buffered at pH 8.5 in comparison with pH 4.4. Already after 24 h a significant reduction was measured in pMOT (0 h: 49 ± 24%, 24 h: 12 ± 7%). Similar decreasing tendency was recorded only after 72 h in DAP (0 h: 26 ± 4 µm/s, 72 h: 19 ± 9 µm/s), DSL (0 h: 21 ± 5 µm/s, 72 h: 17 ± 8 µm/s), VAP (0 h: 59 ± 9 µm/s, 72 h: 43 ± 21 µm/s), and BCF (0 h: 28 ± 2 Hz, 72 h: 18 ± 10 Hz). The response of different investigated CASA parameters to different treatments varied in our experiments. According to our studies, numerous burbot sperm motility parameters are sensitive to chilled storage and to low pH of the activating solution. Our results could support the effective sperm quality assessment and successful artificial propagation process in burbot.

Effect of platelet activating factor (PAF) supplementation in semen extender on viability and ATP content of cryopreserved canine spermatozoa

2010 ◽  
Vol 13 (4) ◽  
pp. 571-579 ◽  
Author(s):  
W. Kordan ◽  
M. Lecewicz ◽  
R. Strzeżek ◽  
A. Dziekońska ◽  
L. Fraser
Keyword(s):  
Plasma Membrane ◽  
Sperm Motility ◽  
Mitochondrial Function ◽  
Sperm Quality ◽  
Membrane Integrity ◽  
Platelet Activating Factor ◽  
Computer Assisted ◽  
Atp Content ◽  
Plasma Membrane Integrity ◽  
Semen Extender

Effect of platelet activating factor (PAF) supplementation in semen extender on viability and ATP content of cryopreserved canine spermatozoa The aim of this study was to investigate the effect of platelet activating factor (PAF) on the quality characteristics of cryopreserved canine spermatozoa. Cryopreserved semen of 5 mixed-breed dogs was treated with different concentrations of exogenous PAF (1 × 10-3M, 1 × 10-4M, 1 × 10-5M and 1 × 10-6M) and examined at different time intervals (0, 30, 60 and 120 min). Cryopreserved semen treated without PAF was used as the control. Sperm quality was evaluated for motility (computer-assisted semen analysis, CASA), mitochondrial function (JC-1/PI assay) and plasma membrane integrity (SYBR-14/PI assay and Hoechst 33258). Also, ATP content of spermatozoa was determined using a bioluminescence assay. Treatment of cryopreserved semen with 1 × 10-3 M PAF at 120 min of incubation resulted in significantly higher total sperm motility compared with the control. It was observed that PAF-improved total sperm motility was concurrent with enhanced sperm motility patterns after treatment of cryopreserved semen. Treatment of cryopreserved semen with PAF did not improve either sperm mitochondrial function or plasma membrane integrity, as monitored by different fluorescent membrane markers. Furthermore, ATP content of cryopreserved spermatozoa was significantly higher when PAF was used at a concentration of 1 × 10-3 M compared with the control and other PAF treatments, regardless of the incubation time. The findings of this study indicated that treatment with 1 × 10-3 M PAF at 120 min of incubation rendered better quality of cryopreserved canine semen, which was associated with improved sperm motility parameters and ATP content. It can be suggested that exogenous PAF addition is beneficial as a supplement for canine semen extender used for.

scholarly journals Sperm quality assessments for endangered razorback suckers Xyrauchen texanus

Reproduction ◽  
2011 ◽  
Vol 141 (1) ◽  
pp. 55-65 ◽  
Author(s):  
Jill A Jenkins ◽  
Bruce E Eilts ◽  
Amy M Guitreau ◽  
Chester R Figiel ◽  
Rassa O Draugelis-Dale ◽  
...  
Keyword(s):  
Sperm Quality ◽  
Natural Populations ◽  
Reproductive Capacity ◽  
Dna Integrity ◽  
Computer Assisted ◽  
Progressive Motility ◽  
Isotonic Buffer ◽  
Quality Assessments ◽  
Sperm Motion

Flow cytometry (FCM) and computer-assisted sperm motion analysis (CASA) methods were developed and validated for use with endangered razorback suckersXyrauchen texanuscollected (n=64) during the 2006 spawning season. Sperm motility could be activated within osmolality ranges noted during milt collections (here 167–343 mOsm/kg). We hypothesized that sperm quality of milt collected into isoosmotic (302 mOsm/kg) or hyperosmotic (500 mOsm/kg) Hanks' balanced salt solution would not differ. Pre-freeze viabilities were similar between osmolalities (79%±6 (s.e.m.) and 76%±7); however, post-thaw values were greater in hyperosmotic buffer (27%±3 and 12%±2;P=0.0065), as was mitochondrial membrane potential (33%±4 and 13%±2;P=0.0048). Visual estimates of pre-freeze motility correlated with total (r=0.7589; range 23–82%) and progressive motility (r=0.7449) by CASA and were associated with greater viability (r=0.5985;P<0.0001). Count (FCM) was negatively correlated with post-thaw viability (r=−0.83;P=0.0116) and mitochondrial function (r=−0.91;P=0.0016). By FCM-based assessments of DNA integrity, whereby increased fluorochrome binding indicated more fragmentation, higher levels were negatively correlated with count (r=−0.77;P<0.0001) and pre-freeze viabilities (r=−0.66;P=0.0004). Fragmentation was higher in isotonic buffer (P=0.0234). To increase reproductive capacity of natural populations, the strategy and protocols developed can serve as a template for use with other imperiled fish species, biomonitoring, and genome banking.

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