78 DIFFERENT EXTENDERS TO HARVEST EQUINE EPIDIDYMAL SPERM AND THEIR INFLUENCE ON FREEZABILITY

2013 ◽  
Vol 25 (1) ◽  
pp. 186
Author(s):  
R. F. Soares ◽  
F. O. Papa ◽  
L. C. O. Magalhães ◽  
G. A. Monteiro ◽  
I. Martin ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Semen Analysis ◽  
Membrane Integrity ◽  
Freezing Process ◽  
Computer Assisted ◽  
Epididymal Sperm ◽  
Traumatic Injuries ◽  
Plasma Membrane Integrity ◽  
Quarter Horse ◽  
Pipette Tip

Harvesting and freezing epididymal sperm is a technology that enables the preservation of the gene pool from animals that had died either unexpectedly or because of colic conditions. This technique may also be employed in animals that have to be euthanized because of traumatic injuries. Therefore, the objective of the present study was to improve the process of freezing epididymal sperm using a freezing extender without the prior centrifugation of the samples. Twelve stallions aging between 3 and 6 years and of different breeds were used (Quarter Horse, Mangalarga, and Brazilian Jumping Horse). Stallions were castrated, and the cauda epididymides were isolated from the testis. The connective tissue was carefully dissected, and the cauda epididymides were straightened. A 200-µL pipette tip was attached to a 20-mL syringe, and the cauda epididymides were flushed using 40 mL of either (A) BotuSemen® (Nidacon, Mölndal, Sweden) or (B) BotuCrio® (Nidacon). They were then immediately processed at room temperature (25°C). Samples flushed with B were randomly subjected to either of the 2 procedures: B1) directly loaded into 0.5-mL straws or B2) centrifuged at 600g for 10 min, the supernatant was discarded, and the pellet was resuspended with B and loaded into 0.5-mL straws. The straws were kept at 5°C for 20 minutes followed by another 20 min at 6 cm above liquid nitrogen before immersion. After thawing at 46°C for 20 s the samples were analyzed by computer-assisted semen analysis (HTM – IVOS 12) and plasma membrane integrity was assessed using fluorescent probes (carboxyfluorescein diacetate and propidium iodide). Data were analyzed by ANOVA followed by the Tukey test (P < 0.05). No differences were observed for the values of total motility (A: 57.1 ± 12.35, B1: 46.3 ± 10.0, B2: 47.2 ± 12.84), progressive motility (A: 25.5 ± 9.05, B1: 21.7 ± 9.02, B2: 20.7 ± 7.20), percentage of rapids (A: 40.6 ± 15.92, B1: 30.7 ± 10.51, B2: 32.6 ± 12.39), and plasma membrane integrity (A: 47.8 ± 9.56, B1: 45.0 ± 13.81, B2: 41.3 ± 8.74). It was found that the fluid derived from epididymal secretions, which composes seminal plasma, had no influence on sperm parameters, because there was no difference among freezing protocols. Therefore, flushing equine epididymal cauda with B and freezing the samples without centrifuging can be successfully used. Both extenders (A and B) were efficient in protecting epididymal sperm throughout the freezing process.

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

118 Semen quality and fertilization ability of myostatin-knockout boars

2020 ◽  
Vol 32 (2) ◽  
pp. 186
Author(s):  
M.-F. Xuan ◽  
S.-Z. Han ◽  
B.-H. Quan ◽  
X.-J. Yin ◽  
J.-D. Kang
Keyword(s):  
Plasma Membrane ◽  
Semen Quality ◽  
Semen Analysis ◽  
Membrane Integrity ◽  
Mitochondrial Activity ◽  
Human Consumption ◽  
Computer Assisted ◽  
Plasma Membrane Integrity ◽  
Sperm Plasma Membrane ◽  
Student’S T

Myostatin-knockout (MSTN−/−) pigs may provide a source of healthy lean protein for human consumption. However, little is known about the effect of this knockout on semen quality, which is important if these pigs are used for production. The purpose of this study is to evaluate the semen quality and fertility of MSTN−/− boars. We generated MSTN−/− boars from Duroc-Landrace-Yorkshire hybrid pig cell lines by somatic cell nuclear transfer, and all 12 boars showed sexual maturation with an obvious “double muscling” phenotype. Semen was collected randomly from three MSTN−/− boars using the gloved-hand technique by one technician and then tested by computer-assisted semen analysis. Semen acrosomal integrity and deformity were measured using Coomassie blue- and eosin-stained smears, respectively. Sperm plasma membrane integrity and mitochondrial activity were evaluated by Hoechst 33342, propidium iodide, and JC-1 multiple staining. The reproductive performance of MSTN−/− boars was evaluated by IVF and by AI. All data were analysed by Student's t-tests. The results showed that the semen color, odor, and pH had no abnormalities. The concentration, motility, plasma membrane integrity, deformity, acrosome integrity, and mitochondrial activity of the semen presented no significant differences from those of the control semen (Duroc). The ejaculation volume of the MSTN−/− boars was significantly lower than that of the control (168.78±6.70 and 223.11±21.21mL, respectively), although the total sperm number was not significantly different. The rate of cleavage and blastocyst formation (247 to 254 oocytes per boar) was not significantly different from those of the control (69.1±0.7 vs. 65.2±1.6%, and 20.2±1.2 vs. 22.8±1.4%, respectively). Seventeen healthy offspring were successfully produced from three sows through AI using semen from one MSTN−/− boar. However, the genotype of piglets has not been tested at present. Thus, MSTN−/− boar may be used as sires, and these pigs are expected to be developed to provide new super-lean meat varieties in the future.

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.

217 QUALITY CHANGES IN POST-THAW SPRINGBOK (ANTIDORCAS MARSUPIALIS) EPIDIDYMAL SPERM MAINTAINED IN FERTILIZATION MEDIUM

2006 ◽  
Vol 18 (2) ◽  
pp. 216
Author(s):  
F. P. Chatiza ◽  
G. M. Pieterse ◽  
P. Bartels
Keyword(s):  
South Africa ◽  
Plasma Membrane ◽  
Liquid Nitrogen ◽  
Membrane Integrity ◽  
Water Bath ◽  
Quality Changes ◽  
Epididymal Sperm ◽  
Plasma Membrane Integrity ◽  
Free Ranging ◽  
Over Time

The availability of gametes from the cropping of excess wildlife species provides the opportunity for the advancement of knowledge into assisted reproductive technology for possible future conservation measures. Little is known about the longevity of springbok (Antidorcas marsupialis) spermatozoa maintained in fertilization medium. The aim of this project was to determine the quality changes of post-thawed springbok spermatozoa incubated in fertilization medium by measuring plasma membrane integrity over time. Testes (n = 12) were obtained from two geographically distinct free-ranging springbok populations in South Africa. Spermatozoa were flushed from the cauda epididymides within three hours of the animals' death. Samples from an individual male were pooled, diluted to 400 × 106 sperm/mL with Biladyl (Minitüb, Tiefenbach, Germany) fraction A (no glycerol) and equilibrated in a water bath for 6 h at 4°C. An equal volume of Biladyl fraction B (containing 12% glycerol) was added to the sample to make a final concentration of 200 × 106 sperm/mL. Samples were loaded into 0.25-mL straws and frozen in liquid nitrogen vapor (5 cm above the liquid nitrogen level) for 20 min after which they were plunged into liquid nitrogen. Straws from each sample were thawed for 20 s at 36°C in a water bath. Thawed spermatozoa (100 μL) was added to 1 mL IVF-TALP medium containing heparin and PHE (Vajta et al. 1996 Theriogenology 45, 683–689) in 2-mL Nunc tubes (AEC, Amersham, South Africa) and incubated at 38.7°C, in humidified 5% CO2 balance air for 30 h. Aliquots were extracted from the incubating spermatozoa to determine plasma membrane integrity at 6-h intervals. Propidium iodide (Sigma, South Africa) at 50 ng/mL (10 min at RT) was used to evaluate membrane integrity under fluorescence microscopy at ×400, with a 450-nm excitation filter, a 510-nm dicroic beam splitter, and a 520-nm barrier filter. Cells with damaged plasma membrane have nuclei that fluorescence red. Eosin/nigrosin was also used to evaluate membrane integrity under ×400 bright-field microscopy. Cells with damaged plasma membrane stain purple-red, whereas the balance of cells remain translucent. The average post-thaw motility of spermatozoa in populations A and B was 69% (n = 6) and 68% (n = 6), respectively. Plasma membrane integrity of post-thawed springbok spermatozoa deceased steadily in IVF-TALP medium over the 30-h period (Table 1). Cryopreserved epididymal sperm derived from free-ranging springbok populations survive in IVF-TALP media and may be useful in future conservation activities where an isolated gene pool requires genetic supplementation through one or more assisted reproduction techniques such as IVF or AI. Further research is required to confirm and extend these findings. Table 1. Percentage plasma membrane integrity of post-thawed springbok sperm over time

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.

68 DIFFERENCE IN THAWING CURVE OF STALLION FROZEN SEMEN DILUTED IN 2 EXTENDERS

2015 ◽  
Vol 27 (1) ◽  
pp. 127
Author(s):  
C. P. Freitas-Dell'aqua ◽  
C. Ramires Neto ◽  
Y. F. R. Sancler-Silva ◽  
P. M. Papa ◽  
J. A. Dell'aqua ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Sperm Quality ◽  
Membrane Integrity ◽  
Freezing Process ◽  
Freezing And Thawing ◽  
Becton Dickinson ◽  
Plasma Membrane Integrity ◽  
Frozen Semen ◽  
Group 2 ◽  
Artificial Vagina

Commercial freeze extenders have different composition and ratio of cryoprotectors; freezing and thawing protocols are different for each extender. The aim of this experiment was to observe the effect of thawing curve in stallion frozen semen with 2 commercial extenders. Two ejaculates from each of 9 stallions of different breeds (Quarter Horses and Mangalarga Marchador) were used. Semen was collected using an artificial vagina, and the ejaculate was divided into 2 groups following the manufacture's protocol: group 1 (INRA), in which the semen was diluted 1 : 1 with the extender INRA 96TM (IMV, Paillette Crista, France) and group 2 (BC), in which the semen was diluted (1 : 1) with the extender Botu-SemenTM (Botupharma, Brazil). The samples of the 2 groups were centrifuged at 600 × g for 10 min, the supernatant was discarded, and the pellet was resuspended with INRA FreezeTM (group INRA, IMV) and with BotucrioTM (group BC, Botupharma) at the concentration of sperm 100 × 106 sperm mL–1. After this, the semen was packaged in 0.5-mL straws. For each group the freezing process was carried out according to the manufacturer's instructions. The straws were thawed in a water bath with 3 different thawing curves: 37°C for 30 s (37/30), 46°C for 20 s (46/20), and 75°C for 7 s (75/7) before analysis. The aim of these rates is to keep the semen in 37°C post-thaw. The sperm kinetic analysis was performed by computerized method (CASA, HTM-IVOS, IMV, USA) and the analysis of plasma membrane integrity by flow cytometer (BD LSR Fortessa, Becton Dickinson, Mountain View, CA, USA). Data of sperm kinetic and of plasma membrane integrity were compared among the 3 thawing curves for one extender using analysis of variance. Differences were considered significant at a probability level of 5%. No differences were observed in total motility (%, BC 37/30 = 72.8 ± 14.4; BC 46/20 = 70.0 ± 14.2; BC 75/7 = 70.3 ± 12.0 v. INRA 37/30 = 57.2 ± 19.1; INRA 46/20 = 50.0 ± 21.9; BC 75/7 = 58.8 ± 20.8), progressive motility (%, BC 37/30 = 36.9 ± 8.2; BC 46/20 = 34.4 ± 10.5; BC 75/7 = 33.6 ± 7.8 v. INRA 37/30 = 25.3 ± 12.7; INRA 46/20 = 21.9 ± 13.9; BC 75/7 = 28.9 ± 14.8), rapid sperm (%, BC 37/30 = 59.7 ± 16.4; BC 46/20 = 56.8 ± 17.1; BC 75/7 = 58.1 ± 14.9 v. INRA 37/30 = 38.3 ± 20.9; INRA 46/20 = 35.3 ± 22.9; BC 75/7 = 44.4 ± 23.8), and plasma membrane integrity (%, BC 37/30 = 49.1 ± 14.8; BC 46/20 = 43.1 ± 13.1; BC 75/7 = 46.7 ± 11.8 v. INRA 37/30 = 32.2 ± 10.7; INRA 46/20 = 29.6 ± 10.1; BC 75/7 = 37.4 ± 9.1) among the 3 thawing curves for INRA and BC groups. In this study, we can conclude there is no influence of the 3 tested thawing curves in sperm quality for stallion frozen semen with INRA Freeze and Botucrio extenders.

Plasma membrane changes during the liquid storage of boar spermatozoa: A comparison of methods

2010 ◽  
Vol 58 (1) ◽  
pp. 105-116 ◽  
Author(s):  
Dariusz Gączarzewicz ◽  
Małgorzata Piasecka ◽  
Jan Udała ◽  
Barbara Błaszczyk ◽  
Tomasz Stankiewicz ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Sperm Motility ◽  
Semen Analysis ◽  
Membrane Integrity ◽  
Live Cells ◽  
Lower Percentage ◽  
Loss Of Function ◽  
Plasma Membrane Integrity ◽  
Sperm Plasma Membrane ◽  
Semen Preservation

Studies were performed on boar semen routinely used at the local artificial insemination (AI) centre. The semen was stored in a Safe Cell Plus commercial extender at 17 °C for nine days. The aim of our research was focused on changes in sperm plasma membrane integrity. The integrity of the sperm plasma membrane and acrosome as well as sperm motility decreased after dilution and during storage of the semen. The highest percentage of live sperm was identified by the eosin-nigrosin method, a lower percentage by the SYBR-14/PI test, and the lowest percentage of live cells was discovered by the hypoosmotic swelling (HOS) test (P < 0.01). There were significant differences between the results of staining methods and sperm motility (P < 0.01). No significant differences were found between the HOS test results and sperm motility. The plasma membrane integrity parameters positively correlated (P < 0.001) with each other and with sperm motility but negatively with aspartate aminotransferase activity. Our findings confirmed that the boar sperm aging changes, which increased during liquid semen preservation, were connected with the loss of function and integrity of the sperm plasma membrane. The employed complementary tests are comprehensive indicators of sperm membrane integrity during long-term semen preservation, and they can help establish the actual number of ‘healthy’ cells. The assays may be used in AI laboratories and should be incorporated into the routine of semen analysis.

65 ARE “BAD FREEZER” STALLIONS ALSO “BAD COOLER” STALLIONS?

2015 ◽  
Vol 27 (1) ◽  
pp. 125
Author(s):  
C. Ramires Neto ◽  
M. M. B. Castro-Chaves ◽  
Y. F. R. Sancler-Silva ◽  
R. C. Uliani ◽  
P. V. L. Oliveira ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Seminal Plasma ◽  
Membrane Integrity ◽  
Computer Assisted ◽  
Cooling Process ◽  
Freezing Resistance ◽  
Plasma Membrane Integrity ◽  
Progressive Motility ◽  
Frozen Semen ◽  
Fresh Semen

Several factors can interfere with sperm cryopreservation resistance, especially the genetic factors and those related to the plasma membrane composition of the sperm and seminal plasma. However, it is still unclear if the same factors that confer freezing resistance will perform the same role during the cooling process. Thus, the aim of this study was to determine the relation between the resistance to freezing and cooling processes in stallions. Two ejaculates from each of 75 stallions were used. All animals showed good quality of fresh semen (total motility higher than 60% and plasma membrane integrity higher than 50%). After collection, the semen was diluted 1 : 1 with commercial skim milk-based extender (Botu-SemenTM, Botupharma, Brazil) and then a part was designed to cooling and the another to freezing. The cooled semen was divided into 2 groups: Group PS, in which the semen was diluted with Botu-SemenTM at a concentration of 50 × 106 sperm mL–1, and Group SPS, which was subjected to a centrifugation at 600 × g for 10 min and resuspended with Botu-SemenTM at 50 × 106 sperm mL–1. Semen samples from both groups were placed in the same cooling passive system for a period of 24 h/5°C. To accomplish the freezing process, the semen sample was subjected to centrifugation at 600 × g for 10 min. The supernatant was discarded, and the pellet was re-suspended in a Botu-CrioTM. The straws were frozen according to the manufacture. The sperm parameters from fresh semen, cooled semen for 24 h with and without seminal plasma, and frozen semen were evaluated for kinetics by computer-assisted semen analysis and for plasma membrane integrity (IMP%) by epi-fluorescence microscopy. The animals were classified in relation to their resistance to cooling and freezing processes as follow: “bad coolers” – reduction in sperm total motility and in plasma membrane integrity higher than 35% after 24 h of cooling in samples with seminal plasma; “good coolers” – reduction in sperm total motility and in plasma membrane integrity lower than 35% after 24 h of cooling in samples with seminal plasma; “bad freezer” – sperm total motility lower than 40% and progressive motility lower than 20% in seminal sample after thawing; “good freezer” – sperm total motility higher than 60% and progressive motility higher than 30% in seminal sample after thawing. The comparison between the resistance to cooling and freezing processes was performed by Fisher's exact test. The level of significance was 5%. No difference (P < 0.05) between the resistance to cooling and freezing processes was observed. The percentage of stallions “good freezer” and “good cooler” was 54%, “good freezer” and “bad cooler” was 22.6%, “bad freezer” and “good cooler” was 12%, and “bad freezer” and “bad cooler” was 10.6%. Within stallions classified as “good freezer” and “bad cooler,” 52.9% also were “good cooler” when the seminal plasma was removed before the cooling process, and 47.1% remained as “bad cooler.” The result of this study demonstrates that there is a strong relation between the resistance to cooling and freezing processes in stallions. In stallions categorized as “bad cooler,” the seminal plasma presents a major influence on the quality and longevity of cooled semen.

scholarly journals Cooling of ejaculated and epididymal stallion sperm

2013 ◽  
Vol 65 (3) ◽  
pp. 681-686 ◽  
Author(s):  
G.A. Monteiro ◽  
P.N. Guasti ◽  
F.P. Hartwig ◽  
J.A. Dellaqua Jr. ◽  
M.A. Alvarenga ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Sudden Death ◽  
Kinetic Parameters ◽  
Genetic Material ◽  
Membrane Integrity ◽  
Epididymal Sperm ◽  
Cooling Process ◽  
Plasma Membrane Integrity ◽  
Bilateral Orchiectomy ◽  
Serious Injury

After a serious injury or sudden death, epididymis cauda sperm recovery and cryopreservation may present as the last opportunity to obtain genetic material from a valuable stallion. This study evaluated the viability of cooled equine sperm collected by three different methods: sperm of ejaculated (G1), sperm recovered from the epididymal cauda immediately after orchiectomy (G2) and sperm recovered from the epididymal cauda after storage for 24 hours at 5°C (G3). To obtain G1 sperm, two ejaculates were collected. After 1 week, all stallions underwent a bilateral orchiectomy, and one of the removed epididymides was flushed to obtain G2 sperm. The contralateral epididymis was stored at 5°C for 24 hours before being flushed to obtain G3 sperm. The sperm samples were evaluated immediately after the addition of the refrigeration extender, and after 24 and 48 hours of storage at 5°C. After 24 and 48 hours of storage, the epididymal sperm demonstrated higher motility traits when compared to the ejaculated sperm (P<0.05). These results indicate that sperm recovered from the epididymal cauda of stallions are more resistant to the cooling process, with higher kinetic parameters and plasma membrane integrity when compared to the ejaculated sperm.

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