scholarly journals 92THE ADDITION OF SEMINAL PLASMA FROM INDIVIDUAL BOARS TO FREEZING EXTENDER CAN IMPROVE THE POST-THAW SPERM SURVIVAL

2004 ◽  
Vol 16 (2) ◽  
pp. 167 ◽  
Author(s):  
T. Cremades ◽  
G. Carvajal ◽  
M. Hernandez ◽  
J.M. Vazquez ◽  
E.A. Martinez ◽  
...  
Keyword(s):  
Seminal Plasma ◽  
Membrane Integrity ◽  
Egg Yolk ◽  
Potential Effect ◽  
Conclusive Evidence ◽  
Sperm Cryopreservation ◽  
Low Concentrations ◽  
Sperm Survival ◽  
And Control ◽  
Boar Spermatozoa

Contradictory results have been reported about the effect of seminal plasma (SP) on the freezability of mammalian spermatozoa. In pigs, current methods for sperm cryopreservation involve removing seminal plasma. Therefore, no conclusive evidence of the potential effect of SP on the freezability of boar spermatozoa has been reported. In this study, we evaluate the effect of the addition of low concentrations of SP from individual boars to the freezing extender on post-thaw sperm survival. Sperm cryopreservation procedure included: dilution of sperm-rich fraction in Beltsville Thaw Solution extender (BTS), cooling to 17°C for 16h, centrifugation at 2400g for 3min, dilution in lactose/egg-yolk/glycerol/Equex Stem (freezing extender) to a final concentration of 1×109 spermmL−1, dispensing into 0.5-mL straws, and freezing in a programmable cell freezer at 20°Cmin−1. Thawing was carried out in a waterbath at 37°C for 20s. Post-thaw sperm survival was assessed by progressive sperm motility (PSM) using a CASA system (SCA); plasma membrane integrity (PMI) and acrosome membrane integrity (AMI) were assessed by flow cytometric procedures (SYBR-14/PI and FITC-PNA/PI, respectively) at 30 and 150min post-thawing in BTS-diluted thaw spermatozoa held in a waterbath at 37°C. Four individual seminal plasma donors (SP1 to SP4) were selected in a preliminary study in which 48 ejaculates from 12 boars (4 ejaculates/boar) were cryopreserved. Then the boars were classified into 3 groups (good, moderate and bad freezers) based on their post-thaw sperm survival. SP1 and SP2 were good freezers (>60% PSM and PMI), SP3 was a moderate freezer (40–60% PSM and PMI) and SP4 was a bad freezer (<40% PSM and PMI). In the main experiment, pooled sperm-rich fractions collected from 9 mature hybrid boars were divided into five aliquots and each was diluted with freezing extender supplemented with 0% (control) or 10% of SP (1–4). Data from eight replicates were analyzed as a split plot design using a PROMIXED model. The addition of SP to freezing extender had a significant effect (P<0.05) on post-thaw sperm survival compared to control. Moreover, there were significant differences (P<0.05) between SP donors. PSM, PMI and AMI were significantly (P<0.05) higher in SP1 (56.71±4.30; 57.16±4.01 and 57.22±4.01, respectively) and SP2 (59.48±4.30; 60.17±4.01 and 60.05±4.01, respectively) compared to control (50.39±4.30; 49.98±4.01 and 49.54±4.01, respectively). There were no differences (P>0.05) between SP3, SP4 and control. These results indicate that the addition of SP from particular boars (good freezers) to freezing extender may improve post-thaw sperm survival. Individual differences in the SP composition should explain the above results. Supported by INIA (RZ01-019) and MCYT (AGL2001-0471).

scholarly journals 309 SEX SORTED BOAR SPERMATOZOA: TIME COURSE AND PROFILE OF THEIR IN VITRO PENETRATION ABILITY AFTER STORAGE IN THE PRESENCE OF HOMOLOGOUS SEMINAL PLASMA

2005 ◽  
Vol 17 (2) ◽  
pp. 305
Author(s):  
I. Parrilla ◽  
J.M. Vazquez ◽  
M.A. Gil ◽  
I. Caballero ◽  
C. Almiñana ◽  
...  
Keyword(s):  
Seminal Plasma ◽  
Time Course ◽  
Egg Yolk ◽  
Vitro Fertilization ◽  
Penetration Ability ◽  
And Control ◽  
Pig Oocyte ◽  
Boar Spermatozoa ◽  
Penetration Time

Addition of seminal plasma (SP) to the collection medium has been shown to be beneficial for motility and viability of sex-sorted and stored spermatozoa. However, SP could not only delay but also decrease the in vitro fertilization rates of IVM pig oocytes. In the present study, the time-course of IVM pig oocyte penetration of sex sorted boar spermatozoa stored in the presence or absence of SP was evaluated. Spermatozoa were sex-sorted following the Beltsville sperm sexing technology (Johnson and Welch 1999 Theriogenology 52, 1323–1342) and collected in TEST-egg yolk buffer (2%) with (10%) or without (control) SP. Sex-sorted spermatozoa were stored at 20°C during 0, 2, 5, and 10 h after sorting. Oocytes were matured in vitro in NCSU23 (Peters and Wells 1663 J. Reprod. Fertil. 48, 61–73) for 44 h in 5% CO2 in air at 39°C. The in vitro penetration time-course was determined by co-incubating the sex-sorted and stored spermatozoa with IVM oocytes during 3, 6, and 18 h in modified TRIS-buffered medium (mTBM) (Abeydeera and Day 1997 Theriogenology 48, 537–544) at 39°C in an atmosphere of 5% CO2 in air. Penetration rates and number of spermatozoa per oocyte were assessed after fixation and staining of the oocytes. Statistical analyses were conducted by ANOVA. Presence of SP did not delay the onset of the oocyte penetration. Moreover, at 3 h of co-incubation, SP increased (P < 0.05) both penetration rates and mean number of spermatozoa per oocyte in sorted and stored boar spermatozoa when compared with control (45 vs. 20, 50 vs. 32, 38 vs. 23, 15 vs. 8, at 0, 2, 5, and 10 h of storage with SP and control, respectively). High penetration rates were reached after 6 h of co-incubation (82 vs. 51, 96 vs. 76, 83 vs. 48, 31 vs. 24, at 0, 2, 5, and 10 h of storage with SP and control, respectively) in sorted and stored samples, with no further increase at 18 h (70 vs. 63, 92 vs. 79, 87 vs. 53, 55 vs. 40, at 0, 2, 5, and 10 h of storage with SP and control, respectively). Spermatozoa stored 2 h in the presence of SP showed the best penetration rate and highest mean number of spermatozoa per oocyte. The mean number of spermatozoa per oocyte increased as the co-incubation time increased (ranging from 2.1 to 5.8 for sorted spermatozoa stored 2 h in the presence of SP at 3 h and 18 h of co-incubation, respectively). In conclusion, the presence of SP during the storage of sex-sorted spermatozoa improves their in vitro fertilizing ability without affecting the onset of the oocyte penetration time. This work was supported by DGICYT (AGL 2001-0471), Fundación Seneca (PB174/FS/02) and CTIC (2103SIU0040).

138 EFFECT OF SEMINAL PLASMA ON CHILLING AND FREEZING OF CANINE SPERMATOZOA

2007 ◽  
Vol 19 (1) ◽  
pp. 187
Author(s):  
I. Yu ◽  
Y. J. Kim ◽  
I. S. Kim ◽  
S. P. Leibo ◽  
N. Songsasen
Keyword(s):  
Pisum Sativum ◽  
Seminal Plasma ◽  
Membrane Integrity ◽  
Egg Yolk ◽  
Beneficial Effects ◽  
Progressive Motility ◽  
Healthy Dogs ◽  
Sperm Survival ◽  
Acrosome Integrity ◽  
Group 1

Seminal plasma (SP) is usually removed from semen that is to be cryopreserved. However, some reports indicate that SP has beneficial effects on spermatozoa during chilling and freezing (Barrios et al. 2000 Biol. Reprod. 63, 1531–1537; Moore et al. 2005 Theriogenology 63, 2372–2381; Vadnais et al. 2005 Anim. Reprod. Sci. 87, 121–132). The purpose of this study was to determine the effect on sperm survival of adding SP to the extender before cooling and freezing canine spermatozoa. In replicate experiments, ejaculates obtained from 4 healthy dogs (3–4 years old) of various breeds were pooled and centrifuged at 300g for 10 min at 25�C; the supernatant of seminal plasma was decanted. Spermatozoa were suspended in egg yolk-Tris (EYT) buffer. The study comprised 2 experiments: Exp. 1: Sperm were suspended in EYT extender containing 0%, 20%, 50%, 80%, or 100% SP, and were slowly cooled to 4�C for 2 h or held at 25�C as controls. Exp. 2: Sperm samples, each of which contained 1 � 108 sperm mL-1, were assigned to 5 groups to be frozen. In group 1, sperm in EYT + 20% SP were cooled to 4�C, diluted to contain final concentrations of 5% glycerol + 10% SP in EYT, and then frozen. In the 4 other groups, sperm in EYT alone were first cooled slowly to 4�C, then diluted to contain 5% glycerol plus 0%, 20%, 40%, or 50% SP in EYT, and then frozen. Spermatozoa were frozen at 25�C min in plastic straws that were suspended above liquid nitrogen and thawed in water at 38�C for 30 s. Sperm survival was assayed by determining progressive motility and integrity of plasma and acrosome membranes. Progressive motility was determined by microscopic examination at 400� magnification. Membrane integrity was assessed by use of a double fluorescent dye, and acrosome integrity by staining sperm with Pisum sativum agglutinin. The results of the first experiment showed that 20%, 50%, 80%, or 100% SP did not improve motility, membrane integrity, or acrosome integrity of spermatozoa chilled to 4�C compared to those chilled without SP (P &gt; 0.05). Survival of spermatozoa suspended in EYT + 20% SP and maintained at 25�C was significantly higher than for those that were chilled (P &lt; 0.05). The results of the second experiment showed that spermatozoa suspended in EYT + 20% SP and then diluted at 4�C to contain 5% glycerol + 10% SP exhibited the highest progressive motility and membrane integrity after being frozen and thawed (P &lt; 0.05). In summary, although seminal plasma did not affect spermatozoa that were only chilled, addition of seminal plasma did significantly improve survival of canine spermatozoa that were frozen and thawed.

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

scholarly journals Conditioned Medium from Canine Amniotic Membrane-Derived Mesenchymal Stem Cells Improved Dog Sperm Post-Thaw Quality-Related Parameters

Animals ◽  
2020 ◽  
Vol 10 (10) ◽  
pp. 1899
Author(s):  
Feriel Yasmine Mahiddine ◽  
Jin Wook Kim ◽  
Ahmad Yar Qamar ◽  
Jeong Chan Ra ◽  
Soo Hyun Kim ◽  
...  
Keyword(s):  
Kinetic Parameters ◽  
Conditioned Medium ◽  
Amniotic Membrane ◽  
Membrane Integrity ◽  
Mitochondrial Activity ◽  
Flow Cytometry Analysis ◽  
Sperm Cryopreservation ◽  
Control Groups ◽  
Freezing Medium ◽  
And Control

This study investigated the effects of conditioned medium (CM) from canine amniotic membrane-derived MSCs (cAMSCs) on dog sperm cryopreservation. For this purpose, flow cytometry analysis was performed to characterize cAMSCs. The CM prepared from cAMSCs was subjected to proteomic analysis for the identification of proteins present in the medium. Sperm samples were treated with freezing medium supplemented with 0%, 5%, 10%, and 15% of the CM, and kinetic parameters were evaluated after 4–6 h of chilling at 4 °C to select the best concentration before proceeding to cryopreservation. Quality-related parameters of frozen–thawed sperm were investigated, including motility; kinetic parameters; viability; integrity of the plasma membrane, chromatin, and acrosome; and mitochondrial activity. The results showed that 10% of the CM significantly enhanced motility, viability, mitochondrial activity, and membrane integrity (p < 0.05); however, the analysis of chromatin and acrosome integrity showed no significant differences between the treatment and control groups. Therefore, we concluded that the addition of 10% CM derived from cAMSC in the freezing medium protected dog sperm during the cryopreservation process.

scholarly journals Kinetic characterization of the changes in protein tyrosine phosphorylation of membranes, cytosolic Ca2+ concentration and viability in boar sperm populations selected by binding to oviductal epithelial cells

Reproduction ◽  
2001 ◽  
pp. 469-480 ◽  
Author(s):  
AM Petrunkina ◽  
J Friedrich ◽  
W Drommer ◽  
G Bicker ◽  
D Waberski ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Tyrosine Phosphorylation ◽  
Membrane Integrity ◽  
Free Swimming ◽  
Sperm Binding ◽  
Preferential Binding ◽  
Functional Modulation ◽  
And Control ◽  
Boar Spermatozoa

On reaching the oviduct, spermatozoa are retained in the isthmic region of the oviduct until ovulation occurs. The essential steps of capacitation are co-ordinated in this region. In this study, a primary cell culture system of oviductal epithelial cells was established to investigate sperm binding to oviductal epithelium and modulation of sperm function during incubation under capacitating conditions in co-culture with oviductal epithelial cells. Epithelial cells were stripped from the oviducts of sows and cultivated for 5-7 days on Lab-Tek Chamber slides on Matrigel. The preparations on chamber slides and suspensions of control spermatozoa were incubated for 3 h in Tyrode's albumin lactate pyruvate (TALP) medium. At 3, 30, 60, 90 and 180 min the free-swimming spermatozoa were collected by washing, and membrane integrity, tyrosine phosphorylation patterns and [Ca(2+)](i) of bound, unbound and control spermatozoa were assessed with fluorescent probes (propidium iodide, Cy-3 and fluo-3-AM). The cells bound to oviductal epithelial cells showed reduced cytosolic Ca(2+) concentration, reduced and almost absent tyrosine phosphorylation of membrane proteins and higher viability at the time of the first sampling. Increases in Ca(2+) concentration and cell death occurred much more slowly during incubation in cells bound to oviductal epithelial cells compared with free-swimming spermatozoa, and no changes in tyrosine phosphorylation were observed. The preferential binding of viable, low-Ca(2+) cells with suppressed tyrosine phosphorylation and slower functional modulation of boar spermatozoa attached to oviductal epithelial cells might represent a mechanism for selecting functionally competent spermatozoa and prolonging their lifespan by delaying capacitation in the oviductal reservoir.

scholarly journals Development of Sperm Cryopreservation Protocols for Sharks and Rays: New Tools for Elasmobranch Conservation

2021 ◽  
Vol 8 ◽  
Author(s):  
Pablo García-Salinas ◽  
Victor Gallego ◽  
Juan F. Asturiano
Keyword(s):  
Sperm Quality ◽  
Membrane Integrity ◽  
Egg Yolk ◽  
Ex Situ ◽  
Conservation Strategies ◽  
Sperm Cryopreservation ◽  
In Captivity ◽  
Reproductive Techniques ◽  
Elasmobranch Species ◽  
Reproductive Cycles

Elasmobranchs are one of the most endangered vertebrate groups on the planet, but despite this situation the use of reproductive techniques in elasmobranch conservation strategies has been scarce. Among these techniques, sperm preservation is a potential tool for ex situ conservation and aquaria sustainability. However, there are no widespread preservation protocols for elasmobranch sperm, and shark sperm cryopreservation has never been achieved before. Here we present the establishment of successful cryopreservation protocols for elasmobranch sperm, tested in several species. We have formulated a sperm extender that can be used for different elasmobranch species, capable of maintaining sperm motility for several weeks. Additionally, we achieved the cryopreservation of sperm by previously diluting it in our extender and supplementing it with different combinations of cryoprotectants. The effects of methanol and dimethyl sulfoxide as permeating cryoprotectants were evaluated, as well egg yolk as a non-permeating cryoprotectant. Sperm quality was assessed by studying the motility and membrane integrity post-thawing, demonstrating its effectiveness in the 10 species tested, including two which are considered Critically Endangered. This is the first time that shark sperm cryopreservation has been reported, broadening our knowledge of the reproductive techniques that can be applied to elasmobranchs and laying the foundations for the first cryobanks for shark and ray sperm. Outcomes from this study will be useful for ex situ conservation efforts developed by public aquaria. A regular supply of frozen sperm will reduce the problems that result from the transport of specimens, inbreeding or lack of synchronized reproductive cycles in captivity.

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

2007 ◽  
Vol 19 (1) ◽  
pp. 182 ◽  
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.

113 EFFECT OF SPERM COATING ON THE QUALITY OF BOVINE FROZEN - THAWED SPERMATOZOA

2006 ◽  
Vol 18 (2) ◽  
pp. 164
Author(s):  
M. Thys ◽  
A. Van Soom ◽  
J. Dewulf ◽  
T. Rijsselaere ◽  
A. de Kruif
Keyword(s):  
Seminal Plasma ◽  
Sperm Quality ◽  
Univariate Analysis ◽  
Membrane Integrity ◽  
Egg Yolk ◽  
Freezing And Thawing ◽  
Bovine Sperm ◽  
Sperm Characteristics ◽  
Group 2 ◽  
Group 1

The substantial decrease of sperm quality after cryopreservation remains an important issue in the artificial insemination industry. Sperm coating with Triladyl® (Minitübe, Tiefenbach, Germany) during ejaculation can preserve sperm characteristics and oocyte penetrating capacity of fresh bovine spermatozoa stored in egg yolk diluent for up to 6 days (De Pauw et al. 2003 Theriogenology 59, 1109–1122). Since collecting semen in a tube containing egg yolk-Tris extender (sperm coating) limits the period of contact between spermatozoa and seminal plasma, the present experiment was conducted to assess if this slightly adjusted method of sperm collection could also have a significant effect on bovine sperm quality after cryopreservation. Semen of five young Holstein Friesian bulls was collected by means of an artificial vagina connected to an empty tube (Group 1; five ejaculates per bull) or a tube containing 4 mL of an egg yolk-Tris extender (Groups 2 and 3; each five ejaculates per bull). The semen samples of Group 1 were conventionally diluted in straws (60 × 106 sperm/mL), frozen, and stored in liquid nitrogen. The samples of Group 3 were centrifuged, and after removing diluent and seminal plasma, the sperm pellet was conventionally diluted and processed. The samples of Group 2 were processed without removal of the supernatant. After thawing each ejaculate was analyzed for average path velocity (VAP), beat cross frequency (BCF), and progressive motility (PROG) using CASA (Minitübe, Tiefenbach, Germany). Furthermore, the membrane integrity of each sample was evaluated using fluorescent SYBR®–14/PI staining (BD Biosciences, Erembodegem, Belgium). All parameters were compared among the three groups of sperm using univariate analysis of variance (SPSS 12.0; SPSS, Inc., Chicago, IL, USA). No significant differences could be observed among the three groups for all of the evaluated sperm characteristics (Table 1). A significant effect of the bull could be determined for all analyzed parameters (P ≤ 0.02), except for the percentage of moribund cells. Nevertheless, the group-bull interaction was never statistically significant. Coating bovine sperm with an egg yolk-Tris extender during ejaculation cannot prevent the substantial deterioration of the spermatozoa that occurs during freezing and thawing since this method of sperm collection does not significantly influence the motility parameters or the membrane integrity after thawing. Table 1. VAP, BCF, PROG, and percentage of membrane-intact, dead, and moribund spermatozoa for the three groups of sperm This research was supported by IWT (no. IWT/020727).

scholarly journals The Re-Addition of Seminal Plasma after Thawing Does Not Improve Buck Sperm Quality Parameters

Animals ◽  
2021 ◽  
Vol 11 (12) ◽  
pp. 3452
Author(s):  
Uchechi Linda Ohaneje ◽  
Uchebuchi Ike Osuagwuh ◽  
Manuel Alvarez-Rodríguez ◽  
Iván Yánez-Ortiz ◽  
Abigail Tabarez ◽  
...  
Keyword(s):  
Seminal Plasma ◽  
Sperm Quality ◽  
Membrane Integrity ◽  
Egg Yolk ◽  
Quality Parameters ◽  
Mitochondrial Activity ◽  
Dna Integrity ◽  
Vitro Fertilization

In order to achieve a higher post-thaw buck sperm quality, an approach in the thawing protocol of cryopreserved sperm doses under in vitro capacitation conditions mimicking the in vivo female environment was studied. Therefore, functional and kinetic characteristics of buck thawed sperm from males of different ages, the season of collection, and melatonin implanted males in the non-breeding season were assessed after 3 h of incubation in an in vitro fertilization (IVF) media with 20% of buck seminal plasma (SP). Previously, fresh ejaculates were collected via artificial vagina from eight males of the Cabra Blanca de Rasquera breed during two consecutive years in breeding and non-breeding periods. Prior to semen collection in non-breeding seasons, males were split into two groups: one group was implanted with melatonin, while the other was not. In each group, semen samples were pooled, centrifuged, and diluted in an extender containing 15% powdered egg yolk and 5% glycerol before freezing. After thawing, sperm were washed and incubated in three different media: (a) control media (modified phosphate-buffered saline (PBS), (b) IVF commercial media, and (c) IVF media + 20% SP. Sperm motility was evaluated by CASA, while plasma and acrosome membrane integrity, mitochondria activity, and DNA fragmentation were analysed by flow cytometer at 0 h and after 3 h incubation. A significant reduction in motility, mitochondrial activity, plasma, and acrosome membrane integrity were observed after incubation in the presence of SP, although similar to that observed in IVF media alone. DNA integrity was not affected under in vitro capacitation conditions, regardless of SP addition. In conclusion, the addition of SP failed to improve post-thaw buck sperm quality under in vitro conditions irrespective of male age, the season of collection, and melatonin implant.

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

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