54 CRYOPRESERVATION OF DOMESTIC CAT EPIDIDYMAL SPERM IN A DEFINED EXTENDER WITHOUT ANIMAL OR PLANT PROTEINS

2012 ◽  
Vol 24 (1) ◽  
pp. 139
Author(s):  
J. R. Saenz ◽  
C. Dumas ◽  
B. L. Dresser ◽  
M. C. Gómez ◽  
R. A. Godke ◽  
...  
Keyword(s):  
Membrane Integrity ◽  
Egg Yolk ◽  
Domestic Cat ◽  
The Other ◽  
Initial Assessment ◽  
Computer Assisted ◽  
Plant Proteins ◽  
Epididymal Sperm ◽  
Sperm Suspension ◽  
Sperm Survival

Previously, we have shown that survival of cat sperm is maintained in both non-egg yolk, semi-defined extenders and in extenders with greatly reduced levels of egg yolk (2%). Usually, cryoprotectant is added to extended samples after gradual cooling to 4°C, but recent reports have shown that satisfactory sperm survival can be obtained after addition at 22°C. Here, our objectives were to examine sperm survival after (1) cryopreservation from 22°C vs after gradually cooling to 4°C or (2) cryopreservation in a completely defined extender without animal or plant proteins vs extender + 2% egg yolk. Epididymides from local veterinary clinics were dissected in HEPES 199 medium (He199). The sperm suspension was filtered (40 μ), layered onto a density gradient column and centrifuged at 650 × g for 20 min. Then, the sperm pellet was resuspended in 1 mL of He199 and centrifuged for 5 min at 800 × g and the subsequent pellet was extended in TEST Buffer with either 0% (0% EY) or 2% egg yolk (2% EY). Next, 0% EY samples were further split into 2 groups—either gradually cooled to 4°C before 12% glycerol (1:1) was added (4C-0%EY) or 12% glycerol (1:1) was added at 22°C without cooling (22C-0%EY). Control samples extended in 2% EY were cooled to 4°C before addition of 12% glycerol (1:1) (4C-2%EY). Samples were loaded into 0.25-mL straws and placed in a –80°C freezer for 20 min before storage in LN2. Sperm samples were thawed in air (22°C) for 5 s and immersed in a 60°C water bath for 5 s. After a 7-step addition of He199, samples were centrifuged at 800 × g for 5 min and pellets resuspended in He199. Sperm samples were evaluated for motility (Mot; computer-assisted semen analysis, 37°C) at 0 h (initial assessment), after cooling to 4°C (PC) and at 0-h (0-PT) and 3-h post-thaw (3-PT) incubation at 37°C. Membrane integrity (MI; SYBR 14-PI) and acrosomal status (AS; FITC-PNA) were analysed at the initial assessment, 0-PT and 3-PT. Results are shown in Table 1. At 4°C (PC), sperm extended in 0% EY and 2% EY maintained 92 and 91%, respectively, of their initial motility (66%). At 0-PT and 3-PT, motility in the 3 groups had decreased by >50% and >70%, respectively. Motility at 3-PT in the 22C-0%EY treatment was less than the other 2 treatments (P < 0.05; 1-way ANOVA). At 0-PT, samples in the 4C-2%EY group had a higher membrane integrity value (P < 0.05) than did the 22C-0%EY group, whereas that of the 4C-0%EY group was not different from the other 2 groups. However, at 3-PT, both groups cooled to 4°C before cryopreservation had higher membrane integrity values (P < 0.05) than the group cryopreserved at 22°C. At 0-PT and 3-PT, the percentage of sperm with intact acrosomes ranged from 69% (4C-2%EY) to 59% (22C-0%EY) and from 55% (4C-2%EY) to 43% (22C-0%EY) of the initial value (89%), respectively. In summary, we demonstrated that cat epididymal sperm could be frozen successfully in a completely defined TEST-buffered extender. Furthermore, we confirmed that addition of cryoprotectant (i.e. glycerol) after gradual cooling to 4°C is beneficial to post-thaw survival. Table 1.Motility (Mot), membrane integrity (MI) and acrosomal status (AS) of cat epididymal sperm before and after cryostorage

89 SURVIVAL OF CAT EPIDIDYMAL SPERM AFTER TEMPORARY COOL STORAGE OR CRYOPRESERVATION IN DEFINED EXTENDERS

2011 ◽  
Vol 23 (1) ◽  
pp. 150
Author(s):  
J. R. Saenz ◽  
C. Dumas ◽  
B. L. Dresser ◽  
M. C. Gómez ◽  
R. A. Godke ◽  
...  
Keyword(s):  
Membrane Integrity ◽  
Egg Yolk ◽  
Epididymal Sperm ◽  
Sperm Analysis ◽  
Santa Ana ◽  
Cool Storage ◽  
Sperm Suspension ◽  
Sperm Survival ◽  
Analysis System

A general objective of our studies on cat sperm is to enhance methods for both short- (+4°C) and long-term (–196°C) cryostorage, with particular focuses on improving compatibility with sex sorting and conforming to regulations for international shipment. Here, our specific aims were to a) determine the ability of cat sperm to survive during temporary cool storage in defined extenders (Exp. 1), and b) compare sperm survival after cryopreservation in the optimal defined extender v. TEST buffered extender + 2% egg yolk (TYB, Exp. 2). Testes from local veterinary clinics were transported in HEPES saline. Epididymides were dissected in HEPES 199 medium (He199), repeatedly sliced, and held at 37°C for ∼20 min. The sperm suspension was filtered (40 μm), layered onto a density gradient column (Isolate®, Irving Scientific, Santa Ana, CA, USA), and centrifuged at 650 × g for 20 min. Then, the sperm pellet was resuspended in 1 mL He199 and centrifuged for 5 min at 250 × g. In Exp. 1 (5 replicates), aliquots of the sperm pellet were extended in either of 2 defined extenders, Bioxcell® (BXC; IMV, Minneapolis, MN, USA) or HypoThermosol®-FSR (HTS; BioLife Solutions Inc., Bothell, WA, USA) or in TYB. Motility (Mot, Hamilton Thorne Sperm Analysis System CEROS 12, 37°C), membrane integrity (M.I., SYBR 14-PI), and acrosomal status (A.S., FITC-PNA) were evaluated at days 0, 1, 2, and 3 (Exp. 1), or after cooling (4°C) and post-thawing (p.t.), after 0 and 3 h incubation at 37°C (Exp. 2). In Exp. 2 (10 replicates), the sperm pellet was extended in BXC or TYB and gradually cooled to 4°C. Then, BXC or TYB + 12% glycerol was added (1:1) using a modified fixed osmolarity method (1995 Hum. Reprod. 10, 1109). Samples were loaded into 0.25-mL straws and frozen on a dry ice block (–80°C) for 20 min before storage in LN2. Straws were thawed in air (∼22°C) for 5 s and immersed in a 60°C water bath for 5 s. Samples were diluted by addition of He199 in 7 steps, centrifuged at 800 × g for 5 min, and pellets resuspended in He199. In Exp. 1, sperm in TYB, BXC, and HTS maintained 93, 69, and 56%, respectively, of initial motility (71%) after 3 days at 4°C (TYB > BXC and HTS; P < 0.05, 1-way ANOVA). Initially, 75 and 86% of sperm had membrane integrity and intact acrosomes, respectively. At 72 h, ∼80% of membrane intact sperm retained integrity in the two defined extenders v. nearly 90% in TYB (P > 0.05). At 24 h, all groups had high percentages of sperm with intact acrosomes (87 to 93%), but at 72 h, there was a difference between HTS (96%) and BXC (79%; P < 0.05). In Exp. 2 (Table 1), motility in TYB and BXC at 0 h p.t. was 77 and 70% of pre-freeze values – 77% (TYB) and 73% (BXC), respectively. Motility at 3 h p.t. was similar (BXC = 35% v. TYB = 37%). Membrane integrity and acrosomal status at 3 h p.t. ranged from 60% (BXC) to 72% (TYB) and from 65% (BXC) to 68% (TYB) of pre-freeze values, respectively. At 3 h p.t. M.I. of sperm in TYB was higher (P < 0.05) than in BXC. In summary, we have shown that cat epididymal sperm can be stored temporarily and cryopreserved successfully in a defined extender without animal proteins. Table 1.Motility, membrane integrity, and acrosomal status of cat epididymal sperm after cryo-storage

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

Zygote ◽  
2018 ◽  
Vol 26 (4) ◽  
pp. 301-307 ◽  
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.

76 SURVIVAL AND IN VITRO FUNCTIONALITY OF DOMESTIC CAT EPIDIDYMAL SPERMATOZOA FOLLOWING CRYOPRESERVATION IN EXTENDERS WITH OR WITHOUT EGG YOLK

2009 ◽  
Vol 21 (1) ◽  
pp. 138
Author(s):  
J. R. Saenz ◽  
C. Dumas ◽  
B. L. Dresser ◽  
M. C. Gómez ◽  
R. A. Godke ◽  
...  
Keyword(s):  
Membrane Integrity ◽  
Egg Yolk ◽  
Human Sperm ◽  
Domestic Cat ◽  
Blastocyst Development ◽  
Santa Ana ◽  
Sperm Suspension ◽  
Epididymal Spermatozoa ◽  
One Way Anova

Our purpose was to compare in vitro survivability and functionality of cat epididymal spermatozoa cryopreserved in TEST egg-yolk buffered extender (TYB) with that obtained by use of clear Tris-citrate and HEPES-buffered extenders containing BSA. Testes were transported to the lab in HEPES saline; epididymides were dissected in HEPES-199 medium (HE-199) and repeatedly sliced. The sperm suspension was filtered (40 μm), layered onto a density gradient column (Isolater, Irving Scientific, Santa Ana, CA), and centrifuged at 600g for 20 min. Aliquots of the sperm pellet were extended in TYB, Human Sperm Preservation Medium (HSPM), or Tris-citrate + 10% BSA (TCBSA). After cooling to 4°C, samples were diluted 1:1 with extender + 12% glycerol in 4 steps as modified from Gao DY et al. 1995 Hum. Reprod. 10, 1109–1122. Then, samples were loaded into 0.25-mL straws, sealed, and frozen on a dry ice block (–80°C) for 20 min before storage in LN2. Straws were thawed by exposure to air (22°C) for 5 s and immersion in a 60°C water bath for 5 s. Samples were diluted by addition of HE-199 in 7 steps as modified from Gao DY et al. 1995 Hum. Reprod. 10, 1109–1122, centrifuged at 200g for 10 min and pellets resuspended in HE-199. Motility (MOT, phase contrast, 37°C), membrane integrity (MI, SYBR 14–PI), and acrosomal status (AS, FITC–PNA) were evaluated at 0 h, after gradual cooling to 4°C, and after freezing at 0 h and 3 h post-thaw (37°C). Cumulus oocyte complexes (COC) were placed in modified TCM-199 and cultured for 24 h in 5% O2, 5% CO2, and 90% N2 at 38°C (IVM). For IVF, COC were co-incubated with spermatozoa frozen in either TYB or HSPM in droplets (1 million sperm mL–1) of IVF medium under 5% CO2 in air at 38°C. After 18 h, oocytes were rinsed and cultured using a 3-step system (Pope CE et al. 2006 Theriogenology 66, 59–71) until blastocyst development was evaluated (Day 8). There were no treatment differences at any time/temperature point for the 3 sperm parameters evaluated (one-way ANOVA; P > 0.05). As shown in Table 1, sperm motility in TCBSA and HSPM decreased by 20% after cooling to 4°C and another 20% after freezing, whereas motility in TYB was maintained after cooling and decreased <30% after freezing. Membrane integrity and acrosomal status values were 12 to 15% greater at collection, at 4°C and at 0 h post-thaw, and 25% greater at 3 h post-thaw than were the motility values. Cleavage frequency and blastocyst development rate of 203 IVM oocytes after IVF using sperm frozen in TYB and HSPM was 36 v. 33% and 50 v. 44%, respectively. In summary, we have shown that cat epididymal spermatozoa can be frozen successfully in cryoprotectant solutions that do not contain egg yolk. Table 1.Motility, membrane integrity and acrosomal status of cat epididymal sperm after cryo-storage

112 EFFECT OF EGG YOLK CONCENTRATION IN TEST BUFFERED EXTENDER ON SURVIVAL AND IN VITRO FUNCTIONALITY OF DOMESTIC CAT EPIDIDYMAL SPERMATOZOA FOLLOWING CRYOPRESERVATION

2010 ◽  
Vol 22 (1) ◽  
pp. 214
Author(s):  
J. R. Saenz ◽  
C. Dumas ◽  
B. L. Dresser ◽  
M. C. Gómez ◽  
R. A. Godke ◽  
...  
Keyword(s):  
Membrane Integrity ◽  
Egg Yolk ◽  
Domestic Cat ◽  
Blastocyst Development ◽  
Santa Ana ◽  
Sperm Suspension ◽  
Epididymal Spermatozoa ◽  
One Way Anova ◽  
Cryoprotectant Solution

Our purpose was to examine the effect of egg yolk concentration (EY; 2, 5, or 10%) on in vitro survivability and functionality of cat epididymal spermatozoa cryopreserved in TEST-buffered extender (TYB). Testes were transported in HEPES saline; epididymes were dissected in HEPES 199 medium (He199) and repeatedly sliced. The sperm suspension was filtered (40 μ), layered onto a density gradient column (Isolate®, Irving Scientific, Santa Ana, CA, USA), and centrifuged at 650 g for 20 min. Aliquots of the sperm pellet were extended in TYB containing 2, 5, or 10% EY. After cooling to 4°C, samples were diluted 1:1 with TYB containing 2, 5, or 10% EY + 12% glycerol in 4 steps as modified from Gao DY et al. Hum. Reprod. 1995 10, 1109-1122. Then, samples were loaded into 0.25-mL straws, sealed, and frozen on a dry ice block (-80°C) for 20 min before storage in LN2. Straws were thawed by exposure to air (˜22°C) for 5 s and immersion in a 60°C water bath for 5 s. Samples were diluted by addition of He199 in 7 steps as modified from Gao DY et al. Hum. Reprod. 1995 10, 1109-1122 centrifuged at 200g for 10 min, and pellets resuspended in He199. Motility (Mot, phase contrast, 37°C), membrane integrity (M.I., SYBR 14-PI), and acrosomal status (A.S., FITC-PNA) were evaluated at 0 h, after gradual cooling to 4°C and after freezing at 0 and 3 h post-thaw (37°C). Ten replicates were done. Cumulus oocyte complexes (COC) were placed in modified TCM-199 and cultured for 24 h in 5% O2, 5% CO2, and 90% N2 at 38°C (IVM). For IVF, COC were co-incubated with spermatozoa frozen in TYB + 2% egg yolk or HSPM (no egg yolk) in droplets (1 million sperm/mL) of IVF medium under 5% CO2 in air at 38°C. After 18 h, oocytes were rinsed and cultured until blastocyst development was evaluated (Day 8). There were no treatment differences at any time or temperature point for the 3 sperm characteristics evaluated (one-way ANOVA; P > 0.05). As shown in the Table 1, at 0 h post-thawing, sperm in each group retained ˜70% of their initial pre-freeze motility. After 3 h of post-thaw incubation, motility decreased to ˜50% of the pre-freeze value. Cooling to 4°C did not affect membrane integrity or acrosomal status, but post-thaw values were reduced by 30-35% as compared with pre-freeze. Cleavage frequency and blastocyst development of 284 IVM oocytes after IVF using sperm frozen in TYB + 2% EY and HSPM were 53 v. 52% and 42 v. 38%, respectively (P > 0.05). In summary, we have shown that cat epididymal spermatozoa can be frozen successfully in a cryoprotectant solution containing minimal egg yolk (2%). Table 1.Motility, membrane integrity, and acrosomal status of cat epididymal sperm after cryo-storage

scholarly journals Characteristics of the post-thawed Balinese bull semen extended in three different extenders and equilibration times

2019 ◽  
Vol 44 (2) ◽  
pp. 135
Author(s):  
A. S. Amal ◽  
R. I. Arifiantini ◽  
M. A. Setiadi ◽  
S. Said
Keyword(s):  
Semen Analysis ◽  
Membrane Integrity ◽  
Egg Yolk ◽  
Computer Assisted ◽  
Equilibration Time ◽  
Osmotic Swelling ◽  
Sperm Analysis ◽  
Bull Semen ◽  
Sperm Survival ◽  
Computer Assisted Sperm Analysis

The objectives of the present study were to compare and determine the best post-thawed characteristics of balinese bull sperm cryopreserved in three different extenders; animal based (Tris-clarified egg yolk (Tris-cEY)), and non-animal based extenders (Bioxcell® (lecithin based) and Optixcell® (liposome based)) in combination with three different equilibration times (30 minutes, 2 hours, 4hours). Thirty six ejaculates were collected from six Balinese bulls and frozen in three extenders (Tris-cEY, Bioxcell® and Optixcell®) after equilibration in three different times (30 minutes, 2hours and 4hours). Computer-assisted sperm analysis (CASA), hypo-osmotic swelling test (HOST) and eosin nigrosin staining were used in the post-thawed semen analysis. There was a significant interaction between equilibration time and extender type for sperm motility, viability and membrane integrity. Thirty minutes equilibration time had the lowest values (P<0.05) for all the evaluated parameters independent of extender type. Overall, semen extended in Tris-cEY, Bioxcell® and Optixcell® were similarly better when equilibrated at 4 hours (P>0.05). Moreover, post-thawed semen which were extended in Optixcell® for 2 hours equilibration showed a better motility compared with the other extenders (P<0.05). In conclusion, two hours equilibration of semen with Optixcell® is sufficient for semen freezing. Four hours equilibration has the best sperm survival, independent of the extender type.

240 FUNCTIONAL TRAITS OF CAT SPERM DURING DISTINCT MATURATION STATUS

2011 ◽  
Vol 23 (1) ◽  
pp. 218
Author(s):  
E. G. A. Perez ◽  
M. Nichi ◽  
C. A. Baptista Sobrinho ◽  
P. A. A. Góes ◽  
A. Dalmazzo ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Lipid Peroxidation ◽  
Structural Damage ◽  
Membrane Integrity ◽  
Domestic Cat ◽  
Lower Percentage ◽  
Mitochondrial Activity ◽  
Computer Assisted ◽  
Suitable Model ◽  
Sperm Analysis

Sperm recovery from the caudae epididymides can be advantageous for preserving semen of endangered animal species. In this context, the domestic cat is a suitable model for the study of sperm physiology in endangered feline species and the research on epididymal sperm preservation combined with the use of reproductive biotechnologies including intracytoplasmic sperm injection (ICSI). The aim of the present study was to examine the sperm collected from the cauda and caput of the cat epididymis using functional tests. Testicles and epididymides from 5 adult tomcats were collected by orchiectomy and maintained at 4°C for 4 h, until semen collection. Semen samples were collected from the epididymal tail and head by careful dissection. Samples were then analysed for motility by computer assisted sperm analysis (CASA; only for the caudal sperm). The 3-3′ diaminobenzidine stain was used as an index of mitochondrial activity, the eosin nigrosin stain as an index of membrane integrity, the simple stain (fast green/Bengal rose) as an index of acrosome integrity, and the measurement of thiobarbituric acid reactive substances (TBARS) as an index of lipid peroxidation. Statistical analysis was performed using the SAS System for Windows (SAS Institute Inc., Cary, NC, USA; least significant differences test and Spearman correlation; P < 0.05). No motility was observed in samples collected from the epididymal head, whereas samples from the tail showed 50.0 ± 4.2% motile spermatozoa. Surprisingly, more spermatozoa with high mitochondrial activity were found in the epididymal head than in samples from the tail (74.0 ± 3.5 v. 50.0 ± 4.3%, respectively). Similarly, samples collected from the head showed a higher susceptibility against the attack of ROS (31.9 ± 5.5 v. 16.3 ± 7.1 ng of TBARS/106 sperm, respectively). Furthermore, epididymal head sperm showed a lower percentage of sperm with intact membrane and a higher percentage of sperm with intact acrosome (44.9 ± 3.3 and 78.4 ± 1.8 v. 66.4 ± 4.2 and 56.7 ± 4.4%, respectively). Our results demonstrate that, during maturation, feline sperm are subjected to high oxidative stress, as shown by the lipid peroxidation assay, which would lead to structural damage to biomolecules, DNA, lipids, carbohydrates and proteins, as well as other cellular components, such as mitochondria, and acrosomal impairment. Similar results were found in humans, in which higher levels of oxidative stress occurred in the post-testicular environment. The plasma membrane seems to be more resistant to damages. This may be due to the described rearrangement in the lipid profile occurring during maturation, but studies to test this hypothesis are still underway.

90 EFFECT OF EGG YOLK ON THE KINEMATICS AND ACROSOME MEMBRANE INTEGRITY OF COOLED-REWARMED CANINE SPERMATOZOA

2010 ◽  
Vol 22 (1) ◽  
pp. 204
Author(s):  
J. Dorado ◽  
M. J. Galvez ◽  
M. R. Murabito ◽  
S. Demyda ◽  
L. J. De Luca ◽  
...  
Keyword(s):  
Semen Analysis ◽  
Membrane Integrity ◽  
Egg Yolk ◽  
Computer Assisted ◽  
Mean Values ◽  
Head Displacement ◽  
Healthy Dogs ◽  
Acrosome Integrity ◽  
Digital Manipulation ◽  
Lateral Head

Tris-egg yolk-based diluents provide adequate cryoprotection for the sperm of most species. This study was conducted to compare the ability of Tris-glucose extender containing 2 different concentrations of egg yolk to maintain sperm motility and acrosome integrity of canine spermatozoa during 72 h of preservation. For this purpose, a total of 20 ejaculates from 4 clinically healthy dogs (2 Spanish Greyhound, 1 German Pointer, and 1 Crossbreed) were collected by digital manipulation. The sperm-rich fraction of each ejaculate was divided into 2 aliquots. Then, they were diluted in Tris-based extender and centrifuged at 700g for 8 min. Sperm pellets were resuspended in either Tris buffer added to 20% (EY20) or 10% centrifuged egg yolk (EY10) and cooled to 5°C over 72 h. The effects of these extenders on motility and acrosome integrity were assessed objectively using a computer-aided semen analyzer (Sperm Class Analyzer, Microptic SL, Spain) and Spermac® staining, respectively. Each cooled-rewarmed semen sample was evaluated after 24, 48, and 72 h of preservation. Sperm motion parameters shown by computer-assisted semen analysis (CASA) are progressively motile (PMS) and motile spermatozoa (MS), curvilinear velocity (CLV), average path velocity (APV), progressive speed (SLV), and lateral head displacement (LHD). Data were statistically analysed by ANOVA. Dependent variables expressed as percentages were arsine-transformed before analysis. Differences between mean values were evaluated by the Duncan method. Data were presented as mean ± SEM. Differences were considered significant when P < 0.05. Analyses were performed using the statistical package SPSS 12.0. A total of 98 172 motile sperm trajectories were analyzed by CASA: 52 259 in EY20 and 45 913 in EY10. After 24, 48, and 72 h of preservation, MS and PMS were statistically higher (P < 0.01) in EY20. No significant differences were found for LHD using either extender over a 72-h period. No significant differences were observed for CLV using either extender during the first 2 days. At Day 3, CLV data were significantly higher (P < 0.01) in EY20. Similarly, from Day 2, APV was significantly higher (P < 0.001) in EY20. After 24 h of preservation, SLV was statistically higher (P < 0.001) in EY10, whereas the opposite tendency was found at Day 3. No significant differences were observed for SLV using either extender after 48 h of preservation. During the first 2 days, acrosome integrity was statistically higher (P < 0.001) in EY20. At hour 72, higher acrosome integrity (P < 0.001) was observed in EY10. In conclusion, we have observed that the EY20 extender provided higher motility after 72 h of chilled preservation; however, the acrosome membrane integrity was better preserved in EY10.

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 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.

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.

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