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

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.

12 FERTILITY OF FROZEN EQUINE SPERM IN SYSTEMS FOR CRYOPRESERVATION

2012 ◽  
Vol 24 (1) ◽  
pp. 117
Author(s):  
R. R. D. Maziero ◽  
P. N. Guasti ◽  
I. D. P. Blanco ◽  
I. Martin ◽  
G. A. Monteiro ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Liquid Nitrogen ◽  
Membrane Integrity ◽  
Skim Milk ◽  
Egg Yolk ◽  
Automated System ◽  
Epifluorescence Microscopy ◽  
Computer Assisted ◽  
Plasma Membrane Integrity ◽  
Sperm Analysis

Optimizing cryopreservation of equine sperm will facilitate genetic banking and propagation of important horse strains through assisted reproduction. This study aimed to evaluate the motility pattern using computer-assisted sperm analysis (CASA) and plasma membrane integrity by epifluorescence microscopy of equine semen frozen in 0.5 mL straws at different freezing rates; also, a fertility trial was performed according to the freezing protocol. Three ejaculates from four stallions of various breeds (Mangalarga Marchador, Westfallen, Hanovarian and Arabian) and ages (5 to 20 years) were collected and processed for cryopreservation. The stallions were housed at the CERBEQ, Reproduction Centre of the Department of Animal Reproduction and Veterinary Radiology, UNESP. The ejaculates were filtered and submitted to analysis by CASA (HTM IVOS 12, Hamilton Thorne Research, USA). In addition, the plasma membrane integrity was determined by fluorescent probes. After evaluation, the ejaculates were diluted at 1:1 (extender:semen) with skim milk extender Botu-Semen™ and centrifuged at 600 × g for 10 min. The supernatant was removed and the pellet resuspended to a final concentration of 100 × 106 sperm mL–1 with milk-egg yolk freezing extender (Botu-Crio™). Semen was packaged in 0.5-mL straws (IMV, LAigle, France) and was placed in nitrogen for 20 min and then from room temperature to 5°C and then frozen in two different cooling systems: an isothermic box (42 cm × 28 cm × 12.5 cm) was placed upon racks suspended 6 cm above liquid nitrogen or other 20 min then immersed into nitrogen and automated system Mini Digitcool™ (IMV Technologies, France), cooling at a –40°C min–1 rate. All straws were stored in liquid nitrogen until thawing and analysis. The straws were thawed in a water bath at 46°C for 20 s and the samples were evaluated for progressive motility, angular progressive velocity, progressive velocity, track speed, percentage of rapid sperm and percentage of sperm with plasma membrane integrity. For the fertility trial, 65 clinically healthy mares had their oestrous cycle monitored by ultrasound and inseminated postovulation with sperm into the uterus. Ovulation was induced with 1 mL of deslorelin acetate (GnRH) injected IM when a 35-mm follicle was detected. Thirty-six hours later, mares were monitored every 6 h until ovulation was detected. When it was detected, mares were inseminated with 800 × 106 total sperm. Pregnancy was confirmed via ultrasound examination 15 days after ovulation. Pregnancy rate was 52.2% using the isothermic box and 60% using the automated machine. Statistical analysis from the frozen–thawed semen evaluated parameters was performed using the statistics software Proc. MIXED of SAS 9.1 and for the fertility trial, logistic regression using the Proc GENMOD from SAS 9.1. The conventional method using the isothermic box was similar to the automated machine with a fast freezing rate. Additionally, AI with 800 × 106 sperm frozen in the isothermic box or automated system resulted in similarly acceptable conception rates.

scholarly journals Changes in Quality of Native and Frozenthawed Semen in Relation to Two Collections Performed in a 24-hour Interval and Adition of Clarified Egg Yolk to Extender

2016 ◽  
Vol 47 (2) ◽  
pp. 60-67
Author(s):  
P. Folková ◽  
J. Šichtař ◽  
O. Šimoník ◽  
A. Dokoupilová ◽  
R. Rajmon
Keyword(s):  
Sperm Motility ◽  
Egg Yolk ◽  
Semen Collection ◽  
Semen Extender

Abstract The aim of the study was to evaluate the effect of repeated semen collection and the substitution of normal egg yolk with clarified egg yolk to commercially produced semen extender on qualitative parameters of frozen-thawed canine semen. Two semen collections were scheduled in a 24-hour interval and in each of six dogs, three 1st and three 2nd collections were performed. The frozen-thawed sperm samples were prepared either with clarified or normal egg yolk and motility and viability were evaluated. The effect of the sequence of semen collection was demonstrated by significant differences in motility and also in viability of sperms both in native and frozen-thawed ejaculate. The percentage of viable sperms was significantly higher in samples from the 2nd compared to the 1st collection. This trend was the same also in motility except in native ejaculate. The addition of clarified egg yolk was beneficial for higher survival of sperms immediately after thawing and also after 30 min of incubation, compared to samples with normal egg yolk. Sperm motility evaluated after thawing was higher in samples with clarified egg yolk, without an apparent connection with semen collection sequence. The decrease of values of the qualitative parameters of sperms observed in the period of 30 min of incubation was significantly slowed down when clarified egg yolk was used. This was especially obvious in samples from the 2nd collection.

362 STORAGE OF BOVINE SPERM FOR 20 H BETWEEN SEMEN COLLECTION AND SEXING

2010 ◽  
Vol 22 (1) ◽  
pp. 337
Author(s):  
J. L. Anema ◽  
J. K. Graham ◽  
R. W. Lenz ◽  
G. E. Seidel
Keyword(s):  
Flow Cytometry ◽  
Egg Yolk ◽  
Computer Assisted ◽  
Ph Measurements ◽  
Sperm Analysis ◽  
Bovine Sperm ◽  
Semen Collection ◽  
Dairy Bulls ◽  
And Control ◽  
Better Than

The objective of this study was to optimize bovine sperm storage for up to 20 h between semen collection and sex sorting followedby cryopreservation. Two successive ejaculates were obtained from mature dairy bulls (Holstein, n = 5; Jersey, n = 3) via artificial vagina. Treatments were then applied to the neat semen to which antibiotics were added as recommended by Certified Semen Services (Columbia, MO). Nothing further was added to the control samples until staining with Hoechst 33342 for sorting. For Treatment 1, semen was diluted 9:1 with a MOPS solution resulting in 24 mM MOPS and similarly, Treatment 2 resulted in 24 mM MOPS +2% egg yolk. A subsample of each treatment and control was sorted by flow cytometry shortly after collection, and sperm then were frozen following standard processing procedures. The other subsample was stored at 15-18°C and sorted 20 h after collection followed by cryopreservation. pH measurements were made before staining samples for sorting. Samples were evaluated post-thaw for subjective progressive and total sperm motility, by computer-assisted sperm analysis (CASA, Berkeley, CA, USA), and by flow cytometry for sperm viability using propidium iodide and SYBR-14. Treatment 1 performed better than the control (Table 1), while results for Treatment 2 were similar to the control. Second ejaculates were superior to first ejaculates. pH measurements showed that addition of MOPS kept the pH about 0.2 units higher than the control, but pH declined similarly over time in all groups. While responses for the 20 h sort were numerically lower than the 0 h sort (P > 0.1), the majority of responses were acceptable for most, but not all bulls. In conclusion, storing sperm in 24 mM MOPS was beneficial. Surprisingly, 2% egg yolk negated the beneficial effect of MOPS, possibly due to increasing osmolarity by ∼15mOsM/kg due to pH adjustment. Addition of MOPS provided better results than the control for both the 0 h and 20 h sorts. Table 1.Main effect means of semen characteristics

scholarly journals Effect of the addition of sodium caseinate on the viability of cryopreserved buffalo semen

2020 ◽  
pp. 2209-2218
Author(s):  
Fernando Evaristo da Silva ◽  
Jaqueline Candido Carvalho ◽  
Camila de Paula Freitas Dell'Aqua ◽  
Frederico Ozanam Papa ◽  
Marc Roger Jean Marie Henry ◽  
...  
Keyword(s):  
Cell Damage ◽  
Membrane Integrity ◽  
Sodium Caseinate ◽  
Egg Yolk ◽  
Freezing Process ◽  
Computer Assisted ◽  
Semen Cryopreservation ◽  
Sperm Analysis ◽  
Frozen Semen ◽  
Buffalo Semen

The use of cooled semen in artificial insemination operations results in higher pregnancy rates than the use of frozen semen. This result seems to be related to the more severe damage triggered by the freezing process than that observed during refrigeration. Due to its ability to bind to sperm-binding proteins and calcium ions, sodium caseinate has been studied as a substance capable of preventing early sperm capacitation, a significant cause of the decreased pregnancy rate resulting from the use of frozen semen. The first objective of this study was to evaluate whether a commercial egg yolk diluent developed for frozen bovine semen could be used for buffalo semen cryopreservation; the second objective was to investigate the effect of this diluent in combination with sodium caseinate during the procedures of buffalo sperm cryopreservation using flow cytometry and computer-assisted sperm analysis. In the first part of the study, comparing the results of spermatic kinetics and plasma and acrosomal membrane integrity, it was observed that the freezing process resulted in more cell damage than the cooling process. In the second part of the study, no effects of the addition of sodium caseinate to the egg yolk diluent were observed. From the results of the present study, it was possible to conclude that the egg yolk-based diluent was suitable for buffalo semen cryopreservation and that the addition of sodium caseinate did not decrease the harmful effects related to seminal cryopreservation.

scholarly journals Effect of two freezing extenders on characteristic of fresh and frozen-thawed semen in endangered Old Kladruber stallions – A pilot study

2017 ◽  
Vol 62 (No. 6) ◽  
pp. 227-233 ◽  
Author(s):  
J. Šichtař ◽  
A. Nehasilová ◽  
O. Šimoník ◽  
F. Bubeníčková
Keyword(s):  
Egg Yolk ◽  
Quality Parameters ◽  
Computer Assisted ◽  
Kinematic Parameters ◽  
Fluorescence Staining ◽  
Sperm Analysis ◽  
Sperm Characteristic ◽  
Computer Assisted Sperm Analysis ◽  
Fresh Semen

The aim of the study was to evaluate the effect of two different extenders on sperm characteristic before equilibration and post-thaw in the endangered Old Kladruber stallions. Also, the response of individual stallions to the extenders used was tested. Semen was collected from six stallions every other day within one week. After centrifugation of the collected sperm-rich fraction, the supernatant was removed and sperm pellets were divided to two aliquots; these were diluted either with Gent (Minitube, Germany) or privately manufactured lactose-EDTA-egg yolk extender (Lact). Three cryopreserved insemination doses (IDs) from each extender (Gent and Lact) were prepared for each stallion from one collection (108 samples from six stallions in total). As a parameter of quality, the motility (computer assisted sperm analysis), viability (fluorescence staining), and morphology (eosin/nigrosine staining) were evaluated after dilution with freezing extenders (fresh) and after thawing (frozen-thawed). The different effects of chosen extenders on the quality of fresh semen were only manifested in higher kinematic parameters of sperm when the Lact extender was used. However, in frozen-thawed samples, the Gent extender yielded significantly better results in all of the evaluated parameters. The representation of sperm subpopulation was significantly influenced by extender in fresh as well as frozen-thawed samples; moreover, we found a significant effect of freezing on the distribution of these subpopulations. The response of individual stallions to chosen extenders was evident in the different quality of fresh as well as frozen-thawed IDs; Gent extender yielded better frozen-thawed IDs. Based on our results, among others describing quality parameters of ejaculate in endangered Old Kladruber stallions, we can recommend using Gent extender for the production of frozen-thawed IDs.  

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 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.

scholarly journals How to Increase Post-Thaw Semen Quality in Poor Freezing Stallions: Preliminary Results of the Promising Role of Seminal Plasma Added after Thawing

Animals ◽  
2019 ◽  
Vol 9 (7) ◽  
pp. 414 ◽  
Author(s):  
Jiří Šichtař ◽  
Filipa Bubeníčková ◽  
Jitka Sirohi ◽  
Ondřej Šimoník
Keyword(s):  
Plasma Membrane ◽  
Seminal Plasma ◽  
Semen Quality ◽  
Membrane Integrity ◽  
Control Group ◽  
Computer Assisted ◽  
Kinematic Parameters ◽  
Prolonged Incubation ◽  
Sperm Analysis ◽  
Spermatozoa Motility

The aim of this study was to evaluate the effect of the addition of two types of seminal plasma (SP) after thawing on the functional characteristics of frozen–thawed (F–T) spermatozoa of poor freezing stallions during prolonged incubation periods. Seminal plasma from stallions with 35–40% (standard seminal plasma, (S-SP)) and 60–70% (above standard seminal plasma, (A-SP)) progressively motile spermatozoa after thawing was used. The motility, kinematic parameters (Computer Assisted Sperm Analysis), distribution of spermatozoa into subpopulations, integrity (carboxyfluorescein diacetate/propidium iodide staining), and functionality (hypo-osmotic swelling (HOS) test) of the spermatozoa plasma membrane were evaluated after thawing (T0) and after 30 min (T30) of incubation at 37 °C. There was no effect of SP addition on spermatozoa motility, but there was a significant positive effect on the kinematic parameters at T0 and T30. The addition of SP significantly increased the percentage of spermatozoa in the fast subpopulation at T0 as well as at T30. Plasma membrane integrity was not affected by the treatment, but functionality significantly decreased by 5% compared to the control group when samples were incubated for 30 min with A-SP. In conclusion, generally, the post-thaw addition of seminal plasma positively affected the post-thaw quality of semen from poor freezing stallions.

95 EFFECT OF CRYOPROTECTANT ADDITION AND REMOVAL AT 4°C ON MOTILITY AND PLASMA MEMBRANE INTEGRITY OF BOVINE CAUDAL EPDIDYMAL SPERMATOZOA

2006 ◽  
Vol 18 (2) ◽  
pp. 156
Author(s):  
C. Guerrero ◽  
S. Leibo ◽  
D. Paccamonti ◽  
B. Eilts ◽  
K. Bondioli ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Semen Analysis ◽  
Membrane Integrity ◽  
Egg Yolk ◽  
Single Step ◽  
Freezing Process ◽  
Computer Assisted ◽  
Chemical Toxicity ◽  
Plasma Membrane Integrity ◽  
Epididymal Spermatozoa

Cryopreservation of spermatozoa harvested from the epididymides would be a means of salvaging germplasm from genetically valuable males that die unexpectedly from injury, disease, or poaching. It is well known that the addition of cryoprotective agents (CPAs) is essential for sperm survival following the freezing process. However, CPAs can cause loss in sperm viability due to osmotic damage or chemical toxicity. The objective of this study was to determine the effects of single-step addition and/or removal of glycerol (GLY) or ethylene glycol (EG) on motility and plasma membrane integrity of bovine epididymal spermatozoa. Paired testes were obtained from mature bulls (n = 10) at a local abattoir and transported to the laboratory at 25–28°C within 4–6 h post-mortem. Epididymal spermatozoa were harvested by multiple incisions from the caudae epididymides of each bull, pooled, and washed in Brackett-Oliphant medium by centrifugation for 5 min at 500g. Pellets were resuspended in egg yolk-Tris-glucose-citric acid monohydrate medium (EYT-GC) at a concentration of 120 × 106 cells/mL and cooled to 4°C at a rate of 0.1°C/min. Specimens were allocated to each of five treatment groups: control (no CPA), 7% GLY, and 14% GLY, 7% EG, 14% EG. Then, replicate samples were diluted 1:1 in EYT-GC medium containing twice the final desired concentration of CPA. After being exposed for 10 min, each sample was diluted directly into EYT-GC at 4°C. Motility was assessed by means of a computer assisted semen analysis system and plasma membrane integrity was determined by SYBR 14 and propidium iodide staining followed by fluorescence microscopy. Differences among treatments were analyzed using one way ANOVA (P < 0.05). The results (Table 1) show that maximum survival, as assessed by measurements of motility and membrane integrity, was achieved with spermatozoa exposed to 7% EG. Almost identical intermediate levels of survival were observed with spermatozoa exposed to 7% GLY or 14% EG. The lowest survival was observed for spermatozoa exposed to 14% GLY. The results indicate that the use of EG as a cryoprotectant may minimize toxicity and osmotic damage to fresh bovine epididymal spermatozoa. Its efficacy as a CPA is currently being determined. Table 1. Sperm motility and membrane integrity (mean ± SEM) after addition of CPA to epididymal sperm

Insights into soy lecithin and egg yolk-based extenders for chilling canine spermatozoa

Zygote ◽  
2018 ◽  
Vol 27 (1) ◽  
pp. 17-24 ◽  
Author(s):  
Andressa Dalmazzo ◽  
Daniel de Souza Ramos Angrimani ◽  
João Diego A. Losano ◽  
Carolina C. Rocha ◽  
Carlos A. B. Sobrinho ◽  
...  
Keyword(s):  
Lipid Peroxidation ◽  
Membrane Integrity ◽  
Egg Yolk ◽  
Control Group ◽  
Mitochondrial Activity ◽  
Computer Assisted ◽  
Compensatory Mechanism ◽  
Soy Lecithin ◽  
Sperm Analysis ◽  
Tbars Assay

SummaryThe aim of this study was to compare different concentrations of soy lecithin (LEC0.01%, LEC0.05% and LEC0.1%) with egg yolk (Control) in cooling extenders during the storage of semen at 5ºC for 5 days. Twelve dogs (n = 12) were selected, and semen was cooled and assessed after 2, 24, 48, 72, 96 or 120 h. At each time point, sperm were analyzed for kinetic patterns (using computer-assisted sperm analysis), mitochondrial activity (3′3- diaminobenzidine assay), lipid peroxidation (TBARS assay), DNA fragmentation (SCSA®) and plasma and acrosome membrane integrity (eosin/nigrosin and fast green/rose Bengal stains, respectively). The Control group (1814.4 ± 197.2) presented the highest rates of lipid peroxidation at 120 h. Conversely, progressive motility (42.8 ± 4%), linearity (45.4 ± 1%), and VAP (88 ± 3%) were higher in the Control group. In addition, there was lower mitochondrial activity in the Control group at 72 h. Therefore, our data show that lecithin used at these concentrations was not able to maintain sperm viability at as high qualities as would egg yolk. Moreover, the decrease in high mitochondrial activity and the persistence of sperm motility may indicate a compensatory mechanism in canine spermatozoa (i.e., glycolytic pathway). Furthermore, these higher lipid peroxidation indexes could indicate the necessity for future therapy using extenders and antioxidants over a long cooling time for dog sperm.

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