Cryopreservation media differentially affect sperm motility, morphology and DNA integrity

Physiological bases of the sperm selection processes within the female reproductive tract before they meet and fertilize the oocyte are unknown. The aim of this work was to determine if one of the keys of spermatozoa selection could be DNA integrity. It has been reported that sperm DNA damage does not impair in vitro fertilization (IVF). However, it has been suggested that the zona pelucida (ZP) is able to select spermatozoa with unfragmented DNA (Liu and Baker 2007 Hum. Reprod. 22, 1597–1602). In this work, DNA damage of spermatozoa was artificially induced by scrotal heat treatment (HT) (42°C, 30 min). Twenty-one days after the HT, spermatozoa were recovered from the epididymis caudae of CD1 mice and from the uterine horns near the cervix (Uc), from the uterine horns near the oviducts (Uo), and from the oviducts (Ov) of CD1 females 1–2 h after mating with HT and control males. In each region we determined numbers of spermatozoa, individual motility and sperm DNA integrity by COMET assay (% DNA in tail, tail length, and COMET moment was calculated). Also, females naturally mated either with HT or control males were killed at Day 14 of pregnancy, and number of foetuses and resorptions was recorded. Additionally, IVF was performed with epididymal sperm from HT or control males, Two hours after IVF attached and un-attached spermatozoa to the ZP were recovered and samples were evaluated for sperm motility (CASA), sperm zona-binding, and sperm DNA fragmentation (COMET). Also cleavage rate of fertilized oocytes with sperm from HT or control males was analyzed. One-way ANOVA was used to compare the results form each group. Epididymal sperm count (12*106 and 4.4*106 for control and HT respectively), sperm motility (75 and 21% respectively) and testis weight (133.90 and 68.76 mg, respectively) were significantly reduced after heat treatment (P < 0.001). For the heat treatment, COMET values decreased significantly during the transit from Uc to Uo and from Uo to Ov (Tail DNA: 25.7, 23.5, and 14.4% respectively, P < 0.01; Tail length: 38.4, 29.4, and 11.2 pixels, P < 0.001; COMET Moment: 12.5, 8.5, and 2 respectively, P < 0.001). Heat treatment reduced numbers of foetuses (7 ± 0.5 v. 5 ± 0.49, control and HT group, respectively), but number of resorptions was not altered. Spermatozoa bound per ZP in IVF experiments (55 ± 7 and 13 ± 6, control and HT, respectively) and cleavage rate (61 ± 1 v. 15 ± 6, control and HT, respectively) were significantly reduced in the HT group. Two hours after IVF, spermatozoa attached to the ZP in HT group showed a significant decrease in COMET parameters as in tail length (59.46 ± 2.895 v. 34.66 ± 3.531), and in tail moment compared with unattached spermatozoa. Our results indicate that DNA integrity sperm selection mechanisms are present in both the female tract and the ZP. We suggest that genital tract and sperm-ZP binding process plays an important role in selection of sperm with normal chromatin DNA.

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97 IMPROVED CRYOPRESERVATION OF DOMESTIC CAT SPERMATOZOA IN A SOY LECITHIN-BASED EXTENDER

Reproduction Fertility and Development ◽

10.1071/rdv23n1ab97 ◽

2011 ◽

Vol 23 (1) ◽

pp. 153 ◽

Cited By ~ 5

Author(s):

M. M. Vick ◽

H. L. Bateman ◽

W. F. Swanson

Keyword(s):

Sperm Motility ◽

Room Temperature ◽

Egg Yolk ◽

Tris Buffer ◽

Domestic Cat ◽

Dna Integrity ◽

Freezing And Thawing ◽

Soy Lecithin ◽

Acridine Orange Staining ◽

Acrosome Integrity

Development of a chemically defined, plant-based cryopreservation media would reduce extender variability and the potential for transmission of zoonotic pathogens compared with traditional egg-yolk-based extenders. The objective of this study was to compare effects of egg yolk- and soy lecithin-based cryopreservation media and the temperature of glycerol addition on sperm parameters following freezing and thawing of domestic cat spermatozoa. Fresh semen was collected by manual stimulation on 3 separate occasions from 4 adult male cats. Each ejaculate was washed to remove seminal plasma, divided into 4 equal aliquots, and extended at room temperature in one of the following treatments: 1) TEST-egg yolk (Irvine Scientific Inc., Santa Ana, CA, USA) medium with 4% glycerol (EYG); 2) TEST-egg yolk, with 4% glycerol added after cooling to 5°C (EY); 3) TES-Tris buffer with soy-lecithin (1%) and 4% glycerol (SLG); and 4) TES-Tris buffer with 1% soy-lecithin, and 4% glycerol added after cooling to 5°C (SL). Sperm progressive motility (%) and rate of progressive movement (scale of 0–5) were evaluated at 0, 1, 3, 6, and 24 h post-thaw. Sperm capacitation (chlortetracycline staining), acrosome integrity (FITC-PNA staining), and DNA integrity (acridine orange staining) were assessed at 15 min post-thaw. Data were exponentially transformed to achieve normal distribution and then subjected to GLM analysis to determine effects of media and temperature of glycerol addition on sperm traits. At 0 and 1 h post-thaw, acrosome integrity, DNA integrity and % sperm motility did not differ (P > 0.05) among treatments. However, % sperm motility was greater in the soy-based media compared to egg yolk-based media at 3, 6, and 24 h post-thaw (Table 1; P < 0.05). A higher percentage of uncapacitated spermatozoa were present in soy-based compared to egg-yolk based cryopreservation media (63.9 ± 9.3 v. 51.2 ± 11.5, respectively; P < 0.05), regardless of temperature of glycerol addition. Finally, addition of glycerol at 5°C resulted in higher % sperm motility compared to room temperature at 6 and 24 h post-thaw in both medium types (Table 1; P < 0.05). Our results suggest that use of a chemically defined, soy-based medium improves long-term motility and capacitation status of frozen–thawed domestic cat spermatozoa compared with cryopreservation in a traditional egg yolk-based extender. Table 1.Motile spermatazoa and motility score at 3, 6, and 24 h

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164 CARNITINE IMPROVES POST-THAWING SPERM MOTILITY BY INCREASING ADENOSINE TRIPHOSPHATE CONTENT IN BUFFALO (Bubalus bubalis)

Reproduction Fertility and Development ◽

10.1071/rdv29n1ab164 ◽

2017 ◽

Vol 29 (1) ◽

pp. 190

Author(s):

V. Longobardi ◽

G. Zullo ◽

G. Albero ◽

C. De Canditiis ◽

A. Salzano ◽

...

Keyword(s):

Adenosine Triphosphate ◽

Sperm Motility ◽

Critical Role ◽

Automated System ◽

Dna Integrity ◽

Ammonium Compound ◽

Dependent Manner ◽

Atp Content ◽

Freezing And Thawing ◽

Semen Extender

Semen cryopreservation plays a critical role for a wide application of both AI and in vitro embryo production in buffalo. In this species, spermatozoa are more susceptible to hazards during freezing and thawing than cattle spermatozoa, thus resulting in lower fertilizing potential (Andrabi et al. 2008 Anim. Reprod. Sci. 104, 427–433). Carnitine is a quaternary ammonium compound with antioxidant capacities, able to reduce the availability of lipids for peroxidation by transporting fatty acids into the mitochondria for β-oxidation to generate adenosine triphosphate (ATP) energy (Tanphaichitr and Leelahagul 1993 Nutrition 9, 246–54). It is known that cryopreservation processes decreases the intracellular concentration of carnitine in spermatozoa (Reyes-Moreno et al. 2000 J. Androl. 21, 876–86). In cattle, supplementation of semen extender with carnitine improves sperm motility and DNA integrity (Bucak et al. 2010 Cryobiology 61, 248–53). The aim of this study was to evaluate whether supplementation of semen extender with carnitine would increase ATP content in buffalo sperm and affect post-thawing motility. Eight ejaculates from 4 bulls were used for the trial. Each ejaculate was split into 3 equal aliquots and diluted at 37°C with BioXcell extender containing 0 (control), 2.5, and 7.5 mM carnitine to a final concentration of 30 × 106 spermatozoa/mL. After 4 h at 4°C, the straws were frozen in an automated system. At thawing, sperm motility was evaluated by phase contrast microscopy at 40× magnification (Gillan et al. 2008 Anim. Reprod. Sci. 103, 201–204). Adenosine triphosphate content was measured using a Colourimetric ATP Assay Kit (Biovision, Milpitas, CA, USA). Briefly, Percoll-separated spermatozoa were homogenised and then deproteinized using 10-kDa spin columns. Samples were incubated at RT for 30 min and absorbance was measured at 570 nM in a microplate reader. Differences in sperm motility and ATP content among groups were analysed by ANOVA. Both concentrations of carnitine increased post-thawing sperm motility compared with the control (44.4 ± 3.5, 53.1 ± 3.9, and 52.5 ± 3.6, respectively, with 0, 2.5, and 7.5 mM carnitine; P < 0.05). Interestingly, carnitine increased ATP content of buffalo frozen–thawed sperm in a dose-dependent manner (4.1 ± 0.1, 5.3 ± 0.1, and 8.2 ± 0.4 nM × 108 sperm, respectively, with 0, 2.5, and 7.5 mM carnitine; P < 0.01). In conclusion, the enrichment of semen extender with carnitine improved post-thawing motility of buffalo sperm by boosting mitochondrial ATP production, hence providing energy for use by spermatozoa.

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Pentoxifylline increase sperm motility in devitrified spermatozoa from asthenozoospermic patient without damage chromatin and DNA integrity

Cryobiology ◽

10.1016/j.cryobiol.2017.04.008 ◽

2017 ◽

Vol 76 ◽

pp. 59-64 ◽

Cited By ~ 8

Author(s):

Ali Nabi ◽

Mohammad Ali Khalili ◽

Farzaneh Fesahat ◽

Alireza Talebi ◽

Saeed Ghasemi-Esmailabad

Keyword(s):

Sperm Motility ◽

Dna Integrity

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Lycopene and resveratrol improve post-thaw bull sperm parameters: sperm motility, mitochondrial activity and DNA integrity

Andrologia ◽

10.1111/and.12301 ◽

2014 ◽

Vol 47 (5) ◽

pp. 545-552 ◽

Cited By ~ 40

Author(s):

M. N. Bucak ◽

M. B. Ataman ◽

N. Başpınar ◽

O. Uysal ◽

M. Taşpınar ◽

...

Keyword(s):

Sperm Motility ◽

Sperm Parameters ◽

Mitochondrial Activity ◽

Dna Integrity ◽

Bull Sperm

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

Animals ◽

10.3390/ani9110999 ◽

2019 ◽

Vol 9 (11) ◽

pp. 999 ◽

Cited By ~ 5

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.

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DNA integrity in fresh, chilled and frozen-thawed canine spermatozoa

Veterinární Medicína ◽

10.17221/5853-vetmed ◽

2012 ◽

Vol 57 (No. 3) ◽

pp. 133-142 ◽

Cited By ~ 11

Author(s):

P. Prinosilova ◽

R. Rybar ◽

A. Zajicova ◽

J. Hlavicova

Keyword(s):

Sperm Motility ◽

Sperm Count ◽

Semen Analysis ◽

Poor Quality ◽

Sperm Chromatin ◽

Dna Integrity ◽

Damage Level ◽

Chromatin Integrity ◽

Fertility Problems

Sperm chromatin status in fresh dog semen and the effect of long-term storage of chilled and frozen dog semen on sperm chromatin integrity was assessed by the sperm chromatin structure assay (SCSA). In the first experiment, the chromatin integrity of fresh semen from 60 dogs with differing histories of fertility was compared with other sperm parameters (total sperm count, sperm motility, viability, acrosomal integrity and sperm morphology). Except for 15 dogs that had never mated before, all were used in breeding as semen donors. Thirty-three of them were successful breeding males while in 12 repeated fertility problems were noted. Ejaculates were assigned to groups with good and poor quality, based on determined sperm motility and percentage of morphologically normal sperm. In the second experiment, chromatin status was measured in fresh and chilled spermatozoa (on day 0, 2, 4, 6, 8 and 10 of storage). Finally, in the third experiment, the chromatin status was measured in fresh and cryopreserved spermatozoa. Evaluating fresh dog semen, we observed that DNA fragmentation index (DFI) and percentage of cells with high DNA stainability (HDS) negatively correlated with total sperm count, percentage of total and progressively motile spermatozoa, sperm viability and percentage of morphologically normal spermatozoa, even with rather low correlation indices. Lower chromatin integrity was found in the group of dog ejaculates showing poor quality in comparison with the group of good quality ejaculates. All dogs with repeated fertility problems were classed in the group showing poor quality, and even though their DFI was significantly higher than the DFI of successful breeding males, the highest DFI we obtained was only just below 9%. We can assume that the chromatin damage level in any of the evaluated dogs was not high enough to have a significant effect on their fertility. Concerning the potential cause of reduced male fertility, the assessment of chromatin integrity in fresh dog ejaculates failed to add any additional information to the results obtained by other techniques of semen analysis. Thus, the current study indicates that neither 10-day preservation of canine sperm chilled in commercial extenders nor long-term cryopreservation in extenders recommended for canine sperm preservation produce adverse effects on sperm chromatin integrity.  

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21 Effect of egg yolk extracted low-density lipoprotein on cryopreserved Nguni bull semen

Reproduction Fertility and Development ◽

10.1071/rdv31n1ab21 ◽

2019 ◽

Vol 31 (1) ◽

pp. 136

Author(s):

M. M. Tshabalala ◽

K. A. Nephawe ◽

M. L. Mphaphathi ◽

C. M. Pilane ◽

T. L. Nedambale

Keyword(s):

Plasma Membrane ◽

Sperm Motility ◽

Sperm Quality ◽

Egg Yolk ◽

Semen Parameters ◽

Dna Integrity ◽

Low Density ◽

Bull Semen ◽

Frozen Semen ◽

Live Sperm

Egg yolk has been reported to have a beneficial effect on sperm quality following cryopreservation, and this led to its widespread use in semen extenders. However, egg yolk contains substances that inhibit respiration of sperm cells and diminish their motility rate. Moreover, it also contains low-density lipoproteins (LDL) that have a protective effect on sperm during the cryopreservation process. The objective of this study was improve cryopreservation of Nguni bull semen using egg yolk low-density protein. A total of 25 ejaculates were collected from 5 Nguni bulls aged 4 to 5 years using an electroejaculator during the natural breeding season. Collected raw semen samples were transported to the laboratory and evaluated for sperm motility, viability, plasma membrane, acrosome, and DNA integrity before dilution. Semen was randomly diluted with a sodium citrate extender supplemented with 20% egg yolk (control) and with 6, 8, 10, and 12% LDL concentrations. The diluted semen sample groups were equilibrated for 4h at 5°C. Following equilibration, semen was loaded into 0.25-mL straws and frozen in a controlled-rate programmable freezer. The groups of semen straws were then plunged into LN and transferred into LN tanks (−196°C) for storage. The frozen semen straws per treatment group were thawed at 37°C and evaluated for sperm motility, viability, plasma membrane, acrosome, and DNA integrity. Data were analysed with ANOVA using Stata V12 statistical software (StataCorp., College Station, TX), and treatment means were separated using Fisher’s protected t-test at the significant level of P<0.05. Sperm motility and membrane integrity were significantly higher (P<0.05) in frozen-thawed semen diluted with 8% LDL compared with the other concentrations. However, 6 and 8% LDL resulted in a significantly higher (P<0.05) live sperm, DNA, and acrosome integrity. Frozen-thawed semen diluted with 10 and 12% LDL resulted in the lowest percentages of sperm motility, live sperm, plasma membrane, acrosome, and DNA integrity following cryopreservation. In conclusion, extender containing 8% LDL resulted in improved Nguni bull semen parameters such as sperm motility, viability, plasma membrane, acrosome, and DNA integrity following cryopreservation. Further studies are required to determine the fertilizing capacity of semen diluted and frozen with LDL in vitro and in vivo.

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