Effect of quercetin or butylated hydroxytoluene on cooled or frozen-thawed ram sperm quality

Cooling and freezing processes cause physical and chemical damage to sperm by cold shock and oxidative stress. This study aimed to evaluate the effect of two antioxidants on sperm parameters of cooled and frozen-thawed ram semen diluted in an egg yolk-based extender. Semen was collected from 30 rams and processed in two consecutive experiments to test the inclusion of different concentrations of quercetin and butylated hydroxytoluene (BHT) in an egg yolk-based semen extender. Dimethyl sulfoxide (DMSO) was added as a solvent to the semen extender in a ratio of 1 mL DMSO for 90 mg of quercetin and 1 mL DMSO for 880 mg of BHT. After collection, semen was diluted at 200 × 106 motile sperm/mL (control) and split into different groups in each experiment. In experiment 1, semen was diluted with the extender containing quercetin (Q5, 5 μg/mL; Q10, 10 μg/mL; Q15, 15 μg/mL) or DMSO alone (DMSO1, 0.055 μL DMSO per mL; DMSO2, 0.165 μL DMSO per mL). In experiment 2, semen was diluted with the extender with BHT (BHT1, 0.5 μg/mL; BHT2, 1 μg/mL; BHT3, 1.5 μg/mL) or DMSO alone (DMSO3, 0.375 μL DMSO per mL; DMSO4, 1.125 μL DMSO per mL). After dilution, the semen was divided into two aliquots. Treated ram sperm samples were also subjected to different storage methods. The first set of samples was cooled at 5 °C for 24 h, whereas the second set of samples was frozen-thawed. Sperm motility parameters and plasma membrane integrity (PMI) were evaluated immediately after dilution (0h) and 24 h after cooling and in the frozen-thawed samples via computer-assisted sperm analysis and epifluorescence microscopy, respectively. The inclusion of quercetin or BHT did not affect sperm motility parameters or PMI of fresh, cooled, or frozen-thawed sperm in this study (P < 0.05). However, further studies are needed to test the effects of these antioxidants on the fertility of cryopreserved ram semen.

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

Polish Journal of Veterinary Sciences ◽

10.2478/v10181-010-0019-y ◽

2010 ◽

Vol 13 (4) ◽

pp. 571-579 ◽

Cited By ~ 5

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.

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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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Cryopreservation of collared peccary (Pecari tajacu L., 1758) epididymal sperm using extenders based on Tris and powdered coconut water (ACP®-116c)

Zygote ◽

10.1017/s0967199418000230 ◽

2018 ◽

Vol 26 (4) ◽

pp. 301-307 ◽

Cited By ~ 2

Author(s):

José A. B. Bezerra ◽

Andréia M. Silva ◽

Patrícia C Sousa ◽

Lívia B. Campos ◽

Érica C. G. Praxedes ◽

...

Keyword(s):

Sperm Quality ◽

Semen Analysis ◽

Membrane Integrity ◽

Egg Yolk ◽

Coconut Water ◽

Computer Assisted ◽

Epididymal Sperm ◽

Sperm Plasma Membrane ◽

Pecari Tajacu ◽

Functional Membrane

SummaryThe aim of this study was to establish a functional freezing–thawing protocol for epididymal sperm of collared peccaries (Pecari tajacu L., 1758) by comparing different extenders. The epididymal sperm from 12 sexually mature males was recovered by retrograde flushing using Tris-based or coconut water-based (ACP®-116c) extenders. After initial evaluation, samples were diluted and frozen with the same extenders to which 20% egg yolk and 6% glycerol were added. After 2 weeks, thawing was performed at 37°C/60 s and sperm motility, vigour, morphology, functional membrane integrity, sperm viability, sperm plasma membrane integrity, and a computer-assisted semen analysis (CASA) were assessed. In addition, to evaluate the survival of frozen–thawed sperm, a thermal resistance test (TRT) was executed. Samples preserved using Tris were in better condition compared with those preserved using ACP®, showing higher values for most assessments performed, including CASA and the TRT (P<0.05). After determining Tris to be the better of the two extenders, additional samples were thawed using different thawing rates (37°C/60 s, 55°C/7 s, 70°C/8 s). Sperm thawed at 37°C/60 s had the greatest preservation (P<0.05) of viability (54.1 ± 5.9%) and functional membrane integrity (43.2 ± 5.4%), and had higher values for various CASA parameters. In conclusion, we suggest the use of a Tris-based extender added to egg yolk and glycerol for the cryopreservation of epididymal sperm obtained from collared peccaries. In order to achieve better post-thawing sperm quality, we suggest that samples should be thawed at 37°C/60 s.

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

Czech Journal of Animal Science ◽

10.17221/122/2017-cjas ◽

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.

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The Quality of Frozen-thawed Canine Semen With Respect to Semen Extender Composition and Sequence of Ejaculate Collection in Dogs

Acta Universitatis Agriculturae et Silviculturae Mendelianae Brunensis ◽

10.11118/actaun201664010169 ◽

2016 ◽

Vol 64 (1) ◽

pp. 169-175

Author(s):

Jiří Šichtař ◽

Ondřej Šimoník ◽

Petra Folková ◽

Adéla Dokoupilová ◽

Radko Rajmon ◽

...

Keyword(s):

Plasma Membrane ◽

Membrane Integrity ◽

Egg Yolk ◽

Computer Assisted ◽

Time Interval ◽

Sperm Analysis ◽

Semen Collection ◽

Movement Characteristics ◽

Semen Extender

The aim of this study was to evaluate the effect of clarified egg yolk addition to semen extender, and the semen collection sequence on the quality of frozen-thawed semen in dogs. Semen was collected from 6 dogs in a time interval of 24 hours. As parameter of the quality of frozen-thawed (F-T) semen, the motility by computer assisted sperm analysis (CASA) and plasma membrane integrity by hypo-osmotic swelling test (HOS) were evaluated. All kinematic parameters of sperm motility were higher in F-T samples containing the whole in comparison to the clarified egg yolk. The sequence of semen collection affected sperm movement characteristics of native as well as F-T semen, but it was not possible to determine whether the fresh semen from the 1st or 2nd collection is of higher quality. All motility parameters of sperms frozen with extender containing the whole egg yolk were significantly higher in the case of the 2nd collection. The situation was not so clear in the case of clarified egg yolk addition, but the velocity values were higher in F-T samples from the 2nd collection. In contrast to proven differences in motility, the effect of the addition of clarified egg yolk and the sequence of semen collection were not projected at all on the quality of plasma membrane of canine sperms evaluated by HOS test.

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130 EFFECTIVENESS OF BUTYLATED HYDROXYTOLUENE AS EGG YOLK SUBSTITUTE FOR CRYOPRESERVATION OF BOAR SPERMATOZOA

Reproduction Fertility and Development ◽

10.1071/rdv19n1ab130 ◽

2007 ◽

Vol 19 (1) ◽

pp. 182 ◽

Cited By ~ 1

Author(s):

L. Rodriguez-Vilar ◽

M. Hernandez ◽

C. Lopez-Sanchez ◽

J. M. Vazquez ◽

E. A. Martinez ◽

...

Keyword(s):

Protective Effect ◽

Mixed Model ◽

Sperm Quality ◽

Membrane Integrity ◽

Egg Yolk ◽

Positive Control ◽

Negative Control ◽

Butylated Hydroxytoluene ◽

Main Effects ◽

Boar Spermatozoa

Butylated hydroxytoluene (BHT) has proven to be efficient as a supplement for cryopreservation boar spermatozoa (Roca et al. 2004 J. Androl. 25, 397–405). Moreover, it has been successfully used as an egg yolk substitute to cryopreserve goat spermatozoa (Khalifa and El-Saidy 2006 Anim. Reprod. Sci. 93, 303–315). The objective of this study was to evaluate the effectiveness of BHT as an egg yolk substitute for freezing boar spermatozoa. Nine sperm-rich ejaculate fractions were collected from 3 boars (3 ejaculates per boar) using the gloved-hand method. After centrifugation (2400g for 3 min), the sperm pellet of each ejaculate was split into 5 aliquots. The aliquots were diluted (to a final concentration of 1 � 109 sperm/mL) in a Tris-citric-glucose extender with 3% glycerol and supplemented with 20% egg yolk (positive control, PC aliquot) or BHT at the final concentrations of 0 (negative control, NC aliquot), 0.2, 0.4, and 0.8 mM. Diluted semen samples were dispensed into 0.5-mL straws, and frozen in a programmable cell freezer at 20�C min. Thawing was carried out in a water bath at 70�C for 8 s. Post-thaw sperm survival was assessed according to total sperm motility (TSM, %) using a CASA system (SCA�; Microptic, Barcelona, Spain), and plasma membrane integrity (PMI, %) and acrosome membrane integrity (AMI, %) using a flow cytometric procedure (SYBR-14/propidioum iodide/FITC-phycoerythrin), at 30 and 150 min post-thawing in diluted Beltsville thawing solution with spermatozoa held in a waterbath at 37�C (3 straws per ejaculate). Data were analyzed using a ANOVA mixed model including the main effects of aliquot, boar, post-thaw assessment time, and their interactions, with ejaculate and straw as random effects. All main effects had significant influence (P ≤ 0.01) in all post-thaw sperm assessments. However, no interactions (P ≥ 0.05) among main effects were shown. Data were combined for the 2 post-thaw assessment times. The best (P ≤ 0.05) post-thaw sperm quality (mean � SEM) was achieved in PC aliquots (47.11 � 3.10, 58.98 � 2.78, and 51.35 � 3.42 for TSM, PMI, and AMI, respectively). In NC aliquots, the percentage of TSM, PMI, and AMI were always below 1% (P ≤ 0.05). BHT has a beneficial (P ≤ 0.05) effect on post-thaw sperm assessments, and no differences (P ≥ 0.05) among concentrations were shown. The mean post-thaw sperm quality in the BHT aliquots was 8.50 � 0.80, 20.29 � 0.53, and 16.03 � 0.55 for TSM, PMI, and AMI, respectively. On the basis of these data, we can conclude that BHT has a protective effect for boar spermatozoa during the cryopreservation process. However, BHT alone is insufficient to replace the protective effect of egg yolk. This work was supported by CICYT (AGF2005-00706), Madrid, Spain.

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247 COMPARISON OF THE DNA FRAGMENTATION INDEX BETWEEN CRYOPRESERVED EJACULATED SPERM AND EPIDIDYMAL SPERM IN STALLIONS

Reproduction Fertility and Development ◽

10.1071/rdv25n1ab247 ◽

2013 ◽

Vol 25 (1) ◽

pp. 271

Author(s):

G. A. Monteiro ◽

C. P. Freitas-DellAqua ◽

P. N. Guasti ◽

Y. F. R. Sancler-Silva ◽

C. Ramires-Neto ◽

...

Keyword(s):

Dna Fragmentation ◽

Sperm Motility ◽

Epifluorescence Microscopy ◽

Sperm Chromatin ◽

Computer Assisted ◽

Epididymal Sperm ◽

Fragmentation Index ◽

Motion Parameters ◽

Motility Parameters ◽

Dna Fragmentation Index

The development of a reliable technique for freezing epididymal semen would provide a unique opportunity to preserve valuable genetic material from unexpectedly lost stallions. The semen analysis method with the best ability to predict fertility is an examination of the sperm chromatin structure. This test evaluates the susceptibility of spermatozoa DNA to denaturation. The ability of spermatozoal DNA to maintain an intact double-stranded configuration is determined by exposure to an acid environment. The aim of this study was to compare the DNA fragmentation index of sperm obtained from ejaculate (G1) and sperm from the cauda epididymis (G2). For G1, two ejaculates from each of seven stallions were collected and then subjected to cryopreservation using BotuCrioTM extender. One week after the last semen collection, the stallions underwent bilateral orchiectomy. Sperm from the cauda epididymis was harvested immediately after castration (G2) by retrograde flushing of the caudal portion of the epididymis using a skim milk-based extender (BotuSemenTM). The recovered sperm was then cryopreserved using BotuCrioTM extender. The sperm motility parameters were analysed by computer-assisted sperm analysis (HTM IVOS 12, Hamilton Thorne Inc., Beverly, MA, USA), and the DNA fragmentation index was estimated using acridine orange test epifluorescence microscopy. The samples were evaluated immediately (0 h) and 8 h after thawing. The total motility, progressive motility, and percentage of rapid cells of the G1 v. G2 samples at 0 h were, respectively, 62.3 ± 12.9a v. 72.6 ± 8.4a, 31.6 ± 9.2a v. 35.3 ± 10.32a, and 49.3 ± 14.33a v. 59.7 ± 13.59a. At 8 h, the results were 26.0 ± 21.6b v. 54.7 ± 12.2a, 6.1 ± 6.4b v. 17.4 ± 8.54a, and 13.7 ± 14.85b v. 37.6 ± 14.15a. Evaluation of the DNA fragmentation in the G1 and G2 samples yielded 6.7 ± 1.41a v. 5.7 ± 1.60a at 0 h and 8.3 ± 1.78b v. 7.2 ± 1.19b at 8 h for percentage of DNA fragmentation after thawing. At 0 h, no differences in the sperm parameters were observed between groups, but statistical differences were observed in the sperm motility parameters between the treatment groups after 8 h. For the DNA fragmentation index, no difference was found at 0 and 8 h between the groups. However, after thawing, a higher percentage of DNA fragmentation was observed in the ejaculated sperm (8 h) as compared with the epididymal sperm (0 h). On the basis of these results, we can conclude that frozen–thawed cauda epididymal sperm had similar or higher motion parameters than ejaculated sperm after thawing. In addition, incubating the sperm at 20°C for 8 h after thawing resulted in higher motion parameters and less DNA fragmentation of the epididymal sperm. This finding suggests that epididymal sperm are more resistant to the cold shock caused by cryopreservation. FAPESP for financial support and Botupharma for donation of BotuSemenTM and BotuCrioTM extender.

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180 ASSESSMENT OF BULL SEMEN QUALITY LOADED IN NEW SensiTemp STRAWS USING SEMEN AND IN VITRO PRODUCTION TECHNOLOGIES

Reproduction Fertility and Development ◽

10.1071/rdv28n2ab180 ◽

2016 ◽

Vol 28 (2) ◽

pp. 221

Author(s):

D. Le Bourhis ◽

S. Camugli ◽

P. Salvetti ◽

L. Schibler ◽

E. Schmitt

Keyword(s):

Sperm Motility ◽

Semen Quality ◽

Sperm Quality ◽

Semen Analysis ◽

Membrane Integrity ◽

Computer Assisted ◽

Cleavage Rate ◽

Fluid Medium ◽

And Control

SensiTemp, a new in vitro maturation (IMV) bull straw concept, presents the advantage of colour changing while the straw is thawed. The colour of frozen straws is blue and straws start to become white when the temperature reaches 33°C, with a complete change of colour at 37°C. The objective of this study is to assess sperm quality after thawing of semen frozen in SensiTemp from 2 bulls, by analysing, in experiment 1, sperm motility and membrane integrity using computer-assisted semen analysis (CASA) and flow cytometry (FC), and, in experiment 2, the in vitro embryo production (IVP) using IVP technologies [IVM, IVF, and in vitro culture (IVC)]. The ejaculates of 2 bulls, selected during preliminary experiments on high in vitro fertility, were harvested at CIA L’Aigle, France, and split ejaculates were frozen in experimental (SensiTemp) and conventional (control) straws. In experiment 1 after thawing semen from the 2 types of straws (5 pooled straws each; 2 replicates), motility was assessed using the IVOS CASA system (Hamilton Thorne Inc., Beverly, MA, USA) and membrane integrity was evaluated through FC with Cytosoft software (Millipore-Guava Technologies Inc., Hayward, CA, USA). In experiment 2, IVF was used to evaluate the non-toxicity of SensiTemp and control straws. Cumulus-oocyte complexes (COC; n = 1178; 4 replicates) collected from slaughterhouse ovaries were matured in IVM medium (TCM-199 with bicarbonate, Sigma-Aldrich, Saint Quentin Fallavier, France; 10 µg mL–1 FSH-LH, Reprobiol, Liège, Belgium; and 10% FCS, Thermo Fisher, Illkirch, France) for 22 h. After fertilization, presumptive zygotes of each group (SensiTemp and control for each bull) were cultured in synthetic oviduct fluid medium (SOF, Minitube, Tiefenbach, Germany) with 1% estrous cow serum (ECS) and 0.6% BSA (Sigma-Aldrich, France) up to 8 days. All cultures were conducted at 38.5C in 5% CO2, and 5% O2. The cleavage and blastocysts rates were evaluated on Days 3 and 7, respectively, for each group. Embryo quality was recorded on Day 7 according to the IETS evaluation. Data from each bull were analysed separately using the chi-squared test (P < 0.05). In experiment 1, neither sperm motility from bull 1 (61.2 and 60.5%) and bull 2 (66.2 and 66.5%) nor membrane integrity from bull 1 (58.6 and 52.2%) and bull 2 (61.0 and 61.9%) were different between SensiTemp and control, respectively. Results from experiment 2 showed no difference (P > 0.05) in cleavage rate between SensiTemp and control for the 2 bulls: 92.1 and 91.7% for bull 1 and 94.2 and 94.6% for bull 2 respectively. The blastocysts rate on Day 7 did not differ (P > 0.05) among groups (47.5, 47.1 and 51.3, 50.4% for SensiTemp and control bull 1 and bull 2, respectively) nor the quality of embryos retrieved in the different groups: 25.4, 23.3, and 30.8, 29.6% in grade 1 embryo for SensiTemp and control bull 1 and bull 2, respectively. Those results demonstrate, in vitro, that the new SensiTemp straws were non-toxic and did not affect the semen quality after thawing nor did the SensiTemp straws affect the ability of sperm cells to fertilize oocytes and produce 8-day-old embryos.

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Effect of Glutathione on Sperm Quality in Guanzhong Dairy Goat Sperm During Cryopreservation

Frontiers in Veterinary Science ◽

10.3389/fvets.2021.771440 ◽

2021 ◽

Vol 8 ◽

Author(s):

Jiahao Zou ◽

Lixuan Wei ◽

Dexian Li ◽

Yongtao Zhang ◽

Guang Wang ◽

...

Keyword(s):

Plasma Membrane ◽

Sperm Motility ◽

Sperm Quality ◽

Membrane Integrity ◽

Total Power ◽

Dairy Goat ◽

Control Group ◽

Membrane Function ◽

Motility Parameters

In the process of cryopreservation of dairy goat semen, it will face many threats such as oxidative damage, which will affect the motility and plasma membrane function of sperm. As an endogenous antioxidant in animals, glutathione (GSH) can significantly improve the quality of thawed sperm when added to the frozen diluent of semen of pigs and cattle. In this study, different concentration gradients of GSH [0 mmol/L (control), 1, 2, 3, 4 mmol/L] were added to the frozen diluent of Guanzhong dairy goat semen. By detecting the sperm motility parameters, acrosome intact rate and plasma membrane intact rate after thawing, the effect of GSH on the cryopreservation of dairy goat semen was explored. Sperm motility parameters were measured with the computer-aided sperm analysis (CASA) system (total power, TM; forward power, PM; linearity, LIN; average path speed, VAP; straight line speed, VSL; curve speed, VCL; beat cross frequency, BCF). The sperm acrosome integrity rate after thawing was detected by a specific fluorescent probe (isothiocyanate-labeled peanut agglutinin, FITC-PNA), and the sperm plasma membrane integrity rate after thawing was detected by the hypotonic sperm swelling (HOST) method. Reactive oxygen species (ROS) kit, malondialdehyde (MDA) kit, superoxide dismutase (SOD) kit, glutathione peroxidase (GSH-PX) kit were used to detect various antioxidant indicators of thawed sperm. in vitro fertilization experiment was used to verify the effect of adding glutathione on sperm fertilization and embryo development. The results showed that when the concentration of glutathione was 2 mmol/l, the sperm viability, plasma membrane intact rate, and acrosome intact rate were the highest after thawing, reaching 62.14, 37.62, and 70.87% respectively, and they were all significantly higher. In terms of antioxidant indexes; the values of SOD and GSH-PX were 212.60 U/ml and 125.04 U/L, respectively, which were significantly higher than those of the control group; The values of ROS and MDA were 363.05 U/ml and 7.02 nmol/L, respectively, which were significantly lower than the control group. The addition of 2 mmol/L glutathione significantly improves the fertilization ability of sperm. In short, adding 2 mmol/l glutathione to the semen diluent can improve the quality of frozen Guanzhong dairy goat sperm.

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73 EFFECT OF THE BUFFER SYSTEM, CRYOPROTECTANT, AND ANTIOXIDANT ON SPERM FROM ENDANGERED CATALONIAN SHEEP XISQUETA AND ARANESA AND GOAT BLANCA DE RASQUERA BREEDS DURING PRESERVATION

Reproduction Fertility and Development ◽

10.1071/rdv25n1ab73 ◽

2013 ◽

Vol 25 (1) ◽

pp. 184

Author(s):

M.-J. Palomo ◽

A. Tabarez ◽

W. Garcia ◽

M. Terre ◽

A. Ferrando ◽

...

Keyword(s):

Soybean Lecithin ◽

Sperm Quality ◽

Egg Yolk ◽

Reproductive Period ◽

Tris Buffer ◽

Butylated Hydroxytoluene ◽

Computer Assisted ◽

Buffer System ◽

Sperm Viability ◽

Sperm Analysis

In an attempt to increase the possibilities of a better sperm cryopreservation from these endangered small ruminant catalonian breeds, we are studying different strategies. One of them is to study the effect of the buffer system Tes-Tris (TEST) compared with the Tris and citric acid (TRIS) buffer system, testing simultaneously both systems in a 1% (w/v) soybean lecithin or in a 15% (v/v) powdered egg yolk–based media supplemented both with 5% glycerol and to assess also the inclusion of 5 mM of butylated hydroxytoluene (BHT) as an antioxidant on the cryopreservation media. However, the main objective of the present study was to test first the effect of the different media when the sperm were cultured 4 h at 5°C. Briefly, fresh ejaculates from 6 young bucks of Blanca de Rasquera breed (1 year old) were collected by an artificial vagina in favourable reproductive period and immediately mixed in equal quantities. Pooled ejaculates were split into 2 equal aliquots and washed by centrifugation (twice for 10 min at 600g) in TRIS- or TEST-based media without cryoprotectants and antioxidant. Afterward each pellet was split into 4 equal aliquots, re-suspended in TRIS or TEST, depending on the experimental group, soybean lecithin, or powdered egg yolk-based media, and supplemented or not with BHT and kept for 4 h at 5°C. Likewise, fresh ejaculates from 8 young rams (4 rams of Xisqueta and 4 rams of Aranesa breed, 1 year old) were collected and processed as buck semen samples. Sperm survival before cryopreservation was determined by eosine-nigrosine stain, and sperm motion parameters were analysed by a computer-assisted sperm analysis system (ISAS®). Six replications were performed in both species, and General Lineal Model (SAS®, Cary, NC, USA) was used for the statistical analysis. The highest sperm viability percentage (mean ± SE) on goat sperm cultured 4 h at 5°C was observed in the extender with TRIS buffer system in powdered egg yolk-based media supplemented with BHT (81.1 ± 2.8), not showing significant differences with the others extenders, except with the viability of the samples in the extenders with TEST buffer system in soybean lecithin-based media supplemented (56.5 ± 2.9; P < 0.001) or not with BHT (60.1 ± 5.1; P < 0.001). On the other hand, no significant differences on sperm viability were observed on ram sperm between treatments. Nevertheless, the sperm quality motion characteristics (data not shown) were quite different between all the treatments in both species. Considering that the present results are still preliminary, we suggest that more analysis should be made to explain how the different composition of the extenders affect sperm quality during the cryopreservation process. Supported by INIA (RZ2009-00008-00-00), Generalitat de Catalunya (2009SGR0621 and CUR-DIUE), and FSE and Fundacion Carolina.

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