75 GLUCOSE CONCENTRATION OF FREEZING EXTENDER MODULATES THE TYROSINE PHOSPHORYLATION PATTERN OF FROZEN-THAWED BOAR SPERMATOZOA

2009 ◽  
Vol 21 (1) ◽  
pp. 138
Author(s):  
J. E. Rodríguez-Gil ◽  
M. Hernández ◽  
M. M. Rivera ◽  
L. Ramió-Lluch ◽  
J. Ballester ◽  
...  
Keyword(s):  
Protein Phosphorylation ◽  
Tyrosine Phosphorylation ◽  
Glucose Concentration ◽  
Egg Yolk ◽  
Freezing And Thawing ◽  
Precise Control ◽  
Boar Sperm ◽  
Phosphorylation Pattern ◽  
Thawing Process ◽  
Boar Spermatozoa

The optimization of freezing extenders is an essential issue for enhancing boar sperm cryosurvival. The aim of the present study was to disclose the role of glucose concentration of freezing extender on the metabolic activity of frozen–thawed spermatozoa. To achieve it, pooled sperm-rich ejaculate fractions from 5 mature and fertile boars (3 ejaculates per boar) were collected using the gloved-hand method. After centrifugation (2400g for 3 min), the sperm pellet was split into 7 aliquots. The aliquots were diluted to a final concentration of 1 × 109 sperm mL–1, in a Tris-citric extender supplemented with 20% egg-yolk, 3% glycerol, and 0, 0.05, 2, 4, 10, 55, or 185 mm glucose. All the extenders were adjusted to a pH of 6.8 and 310 mOsm kg–1 to avoid osmolarity effects. Extended semen samples were dispensed into 0.5-mL straws, and frozen in a programmable cell freezer at 20°C min–1. Thawing was carried out in a water bath at 37°C for 20 s. Afterward, an analysis of protein phosphorylation in tyrosine residues was carried out through bi-dimensional electrophoresis followed by a Western blot analysis. This analysis indicated that sperm samples frozen in extenders without glucose showed specific changes in the tyrosine phosphorylation pattern compared with fresh sperm. Furthermore, the addition of glucose in increasing concentrations to the freezing extender was accompanied by a concentration-dependent decrease in the overall tyrosine phosphorylation pattern, especially in proteins with a molecular weight ranging from 150 to 200 kDa and an acidic isoelectric point (pI). The maximal decrease was observed in spermatozoa frozen in the extender containing 185 mm glucose, in which an additional decrease in the tyrosine phosphorylation of proteins ranging from 60 to 80 kDa, and a basic pI was also observed. These results suggest that glucose is a modulator in the resistance of boar sperm to support freezing and thawing process, because the precise protein phosphorylation pattern of spermatozoa is directly linked to their functional status. In this way, a precise control of the glucose concentration of the freezing extender would be required to improve boar sperm cryoresistance. Supported by CICYT (AGL2005-00760 and AGL2004-04756-C02-02/GAN), Madrid and GERM (04543/07), Murcia, Spain.

scholarly journals Cryopreservation of boar semen

2020 ◽  
Vol 65 (No. 4) ◽  
pp. 115-123
Author(s):  
Marija Jovičić ◽  
Eva Chmelíková ◽  
Markéta Sedmíková
Keyword(s):  
Animal Species ◽  
Semen Quality ◽  
Egg Yolk ◽  
High Rate ◽  
Freezing And Thawing ◽  
Long Term Storage ◽  
Boar Semen ◽  
Boar Spermatozoa ◽  
Term Storage

Sperm cryopreservation is the best technology for long-term storage of the semen. However, the damage of boar spermatozoa by cryopreservation is more severe than in other animal species and a standardized freezing protocol for efficient cryopreservation has not been established yet. Semen quality and freezability vary greatly between breeds as well as between individual boars and even the season. Boar spermatozoa are sensitive to low temperatures; they sustain damage and a high rate of mortality and freezing/thawing the boar semen may strongly impair the sperm function and decrease the semen quality. The freezability of boar semen can be influenced by a cryopreservation procedure, and also by using various additives to freezing and thawing extenders such as antioxidants. In order to obtain acceptable results after thawing the boar semen, it is necessary to combine an optimal amount of additives (glycerol, egg yolk, sugars, antioxidants), cooling and warming velocities.

37 DOUBLE FREEZING AND THAWING OF NGUNI BULL SEMEN

2016 ◽  
Vol 28 (2) ◽  
pp. 148
Author(s):  
M. L. Mphaphathi ◽  
M. M. Seshoka ◽  
T. R. Netshirovha ◽  
Z. C. Raphalalani ◽  
T. C. Chokoe ◽  
...  
Keyword(s):  
Sperm Motility ◽  
Egg Yolk ◽  
Freezing And Thawing ◽  
Sperm Analysis ◽  
Treatment Groups ◽  
Bull Semen ◽  
Before And After ◽  
Repeated Freezing ◽  
Thawing Process ◽  
Marked Decline

Indigenous bulls semen are important for conservation programs. The objectives of this study were to evaluate the effects of repeated freezing and thawing on sperm motility characteristics. Semen was collected from 4 Nguni bulls by means of electro ejaculator and stored in a thermo flask (37°C). Sperm total motility, progressive and nonprogressive motility, and velocity were assessed using computer-aided sperm analysis before and after freezing. Semen was then diluted with egg yolk citrate extender (fraction A), then followed by 12% of glycerol + egg yolk citrate extender (fraction B, Seshoka et al. 2012). Diluted semen samples were equilibrated for 4 h at 5°C. After the equilibration period, samples were loaded into 0.25-mL straws and transferred into a controlled rate programmable freezer. After the target temperature of –130°C was reached, semen straws were stored in a LN tank (–196°C). After 3 months of storage, straws were thawed at 15°C (first and second freezing and thawing followed the same process) for 5 min and further evaluated post-thawed at 0 and 15 min during incubation at 15°C. Treatment means were separated using Fisher’s protected t-test least. No significant differences were recorded between the raw semen total sperm motility percentage (93.2%) and first frozen-thawed at 0 min (82.6%), with the total sperm motility rate recovery of 88.6%. In addition, there was a marked decline recorded in sperm total motility during the first frozen-thawed at 15 min (77.6%), second frozen-thawed at 0 min (31.3%), and second frozen-thawed at 15 min (30.1%; P < 0.05). The sperm curvilinear velocity and average path velocity was reduced following first frozen-thawed (P < 0.05) but remained constant and stable between the treatment groups (P > 0.05). In conclusion, the freezing-thawing process did not reduce the Nguni bull total sperm motility during the first freezing and thawing process, compared with raw semen. However, a drastic decline was recorded during the second freezing-thawing processes, compared with raw semen.

scholarly journals 79 EFFECT OF PRE-FREEZE ADDITION OF PLATELET-ACTIVATING FACTOR AND PLATELET-ACTIVATING FACTOR:ACETYLHYDROLASE ON THE POST-THAW INTEGRITY OF FROZEN - THAWED BOAR SPERM

2005 ◽  
Vol 17 (2) ◽  
pp. 189
Author(s):  
R. Bathgate ◽  
B.M. Eriksson ◽  
W.M.C. Maxwell ◽  
G. Evans
Keyword(s):  
Platelet Activating Factor ◽  
Egg Yolk ◽  
Large White ◽  
Boar Sperm ◽  
Thawing Process ◽  
Potential Benefits ◽  
Boar Semen ◽  
Acrosome Integrity ◽  
And Control

The use of frozen-thawed boar sperm is not widespread, owing to reduced fertility rates and high cost per dose (Eriksson et al. 2004 Proc. Aust. Assoc. Pig Vet., 61–69). Improvements in post-thaw sperm survival are required for commercialization. Platelet-activating factor (PAF) is a phospholipid involved in regulating sperm function. PAF:acetylhydrolase (PAF:AH) regulates PAF activity by conversion to its inactive isoform. Both occur naturally in boar semen (Kordan et al. 2003 Pol. J. Vet. Sci. 6, 55–60). Removal of PAF and PAF:AH along with seminal plasma during the cryopreservation process may inhibit the ability of sperm to withstand the freeze-thawing process. The aim of this study was to assess the effect of PAF and PAF:AH, added to boar semen pre-freeze, on the post-thaw motility and acrosome integrity of sperm. The sperm rich fraction was collected from a mature Large White × Landrace boar, diluted with Androhep (1:2, semen:Androhep; Minitube, Verona, WI, USA), cooled to 17°C over 2 h, and then centrifuged (10 min, 800g). The sperm pellet was resuspended in cooling extender (11% (w/v) lactose solution with 20% (v/v) egg yolk; control), cooling extender plus 100 ng/mL PAF (PAF), or cooling extender plus 0.4% (v/v) PAF:AH (Pafase; ICOS Corporation, Seattle, Washington, USA), and cooled to 5°C over 2.5 h. Sperm were further diluted with cooling extender plus 9% (v/v) glycerol and 1.5% (v/v) Equex STM (freezing extender), loaded into 0.5-mL straws, and frozen. Straws were thawed (20 s, 42°C) and the motility and acrosome integrity (FITC-PNA; Mortimer etal. 1990 Hum. Reprod. 5, 99–103) assessed at 0, 3, and 6 h post-thaw after incubation at 37°C. Data from three replicates were analyzed by ANOVA and a Tukey test applied where significant differences were found. Post-thaw motility (0 and 3 h) was higher for PAF (60.0 ± 0.0% and 25.0 ± 2.9%) than for control (41.7 ± 1.7% and 10.0 ± 2.9%; P < 0.05), but was similar for Pafase (41.7 ± 1.7% and 16.7 ± 1.7%; P > 0.05). By 6 h post-thaw, motility was similar for PAF (1.7 ± 1.7%), Pafase (6.7 ± 6.8%), and control (1.7 ± 1.7%, all respectively; P > 0.05). Acrosome integrity was higher at 0, 3 and 6 h post-thaw for Pafase (55.7 ± 3.2%, 45.7 ± 3.7% and 23.0 ± 3.1%) than for control (42.7 ± 1.5%, 25.7 ± 5.7% and 12.3 ± 2.7%) and PAF (33.0 ± 3.7%, 26.3 ± 2.2% and 11.7 ± 0.3%, all respectively; P < 0.05), but was similar between control and PAF (P > 0.05). Supplementation of cooling extender with 100 ng/mL PAF increased initial post-thaw motility, but this benefit was lost after 6 h post-thaw. Pafase in the cooling extender improved the proportion of intact acrosomes, even after 6 h post-thaw. In vitro studies investigating the interaction between Pafase-treated frozen-thawed sperm and oviducal epithelial cells would be of interest to further establish the potential benefits of pre-freeze addition of Pafase on the fertilizing potential of frozen-thawed boar sperm.

119 OXIDATIVE STRESS DURING THE CRYOPRESERVATION OF BOAR SPERMATOZOA

2007 ◽  
Vol 19 (1) ◽  
pp. 177 ◽  
Author(s):  
M. Hernandez ◽  
J. M. Vazquez ◽  
E. A. Martinez ◽  
J. Roca
Keyword(s):  
Oxidative Stress ◽  
Egg Yolk ◽  
Ros Production ◽  
Butyl Hydroperoxide ◽  
Tert Butyl ◽  
Tert Butyl Hydroperoxide ◽  
Intracellular Ros ◽  
Boar Sperm ◽  
Ros Formation ◽  
Boar Spermatozoa

The cryopreservation procedure causes dramatic changes in boar sperm survival but it is yet unclear where and how the process affects spermatozoa. Cryopreservation damage appears partly associated with oxidative stress and reactive oxygen species (ROS) generation. The present study evaluates the effect that various steps of a conventional cycle of cryopreservation have on the intracellular production of ROS by boar spermatozoa (spz). Sperm-rich fractions collected from 2 mature boars (3 ejaculates per boar), cooled to 17�C, and kept for 16 h were cryopreserved following a standard freeze–thaw process with 0.5-mL plastic straws. The production of ROS was recorded in 5 steps of the cryopreservation process. These steps were as follows: step (1) after collection, when the fresh semen was extended (1:1, v/v) in Beltsville Thawing Solution (BTS, 205 mM glucose, 20.39 mM NaCl, 5.4 mM KCl, 15.01 mM NaHCO3, and 3.35 mM EDTA); step (2) after cooling and storage for 16 h at 17�C; step (3) after centrifugation (2400g for 3 min) and re-extension of the pellet with lactose-egg yolk extender; step (4) at 5�C, after the addition of lactose-egg yolk-glycerol-Equex Stem Paste to 1 � 109 spz mL; and step (5) immediately after thawing at 37�C for 20 s. For the ROS measurement, all samples were re-extended in BTS (3 � 106 spz mL-1) and incubated without (basal ROS level) or with ROS inducers (1 mM tert-butyl hydroperoxide) for 120 min at 37�C and 5% CO2. Cells were simultaneously stained with 22,72-dichlorodihydrofluorescein diacetate (1 �M) to estimate the production of ROS, and propidium iodide (12 �M) to exclude dead sperm from the analysis. Samples were evaluated at 30 min and 120 min by flow cytometry (Coulter Epics XL; Coulter Corporation, Miami, FL, USA); further analyses of the parameters were done by FCSExpress software (DeNovo Software, Thornhill, Ontario, Canada). ROS production was expressed as the mean of the green intensity fluorescence units of the viable sperm population. Data from 3 replicates were analyzed as a split plot design using a mixed model ANOVA including cryopreservation step, boar, and incubation time as fixed effects and replicate as random effect. Results indicated that the basal ROS formation remained relatively low and constant (P = 0.95) through the cryopreservation process, without differences between boars (P = 0.559), although with a significant increase after 120 min of incubation (P &lt; 0.001). However, the exposure to tert-butyl hydroperoxide significantly increased the intracellular ROS formation in all of the steps (P &lt; 0.001), showing significant differences between them, and being especially raised at steps 3 and 4. In conclusion, the present study confirms that the basal intracellular ROS production during cryopreservation of boar sperm is low. Nevertheless, the susceptibility of those spermatozoa to external stresses vary through the cryopreservation process, especially after centrifugation and later extension at 17�C and after the slow cooling at 5�C. This work was supported by CICYT (AGF2005-00706), Madrid, Spain

73 SUPPLEMENTATION OF FROZEN BOAR SEMEN WITH β-MERCAPTOETHANOL INCREASES THE INCIDENCE OF INTACT CELLS AFTER THAWING

2008 ◽  
Vol 20 (1) ◽  
pp. 117
Author(s):  
H. Funahashi ◽  
S. Yamaguchi ◽  
W. Fujii ◽  
T. Murakami
Keyword(s):  
Liquid Nitrogen ◽  
Dna Fragmentation ◽  
Egg Yolk ◽  
Molecular Probes ◽  
Sperm Cells ◽  
Freezing And Thawing ◽  
Intact Cells ◽  
Boar Semen ◽  
Post Hoc ◽  
Boar Spermatozoa

During the process of freezing and thawing of boar spermatozoa, a large number of the cells appear to be injured by some stresses such as osmotic forces and oxidation, causing reduced viability and penetrability. β-Mercaptoethanol (bME), a strong reducing agent, may ease oxidative stress and rescue sperm cells from those injuries. The aim of this study was to determine the effect of the presence of bME during freezing and thawing of boar spermatozoa on the viability and acrosome status of the sperm cells. Semen samples were collected from 3 boars; only samples with a high motility (more than 80%) were used for this experiment. Each sample was diluted 1:1 with modified Modena solution and kept overnight at 15�C. After centrifugation at 800g for 10 min, the diluent supernatant was removed; spermatozoa were re-suspended at 2 � 109 cells mL–1 in the first diluent (8.8% trehalose solution containing 20% egg yolk and antibiotic) supplemented with 0, 25, or 50 µm bME, and then cooled to 5�C over 2–3 h. At 5�C, semen samples were further diluted 1:1 with the second diluent (same as the first diluent + 5% glycerin + 1.48% Orvus ES Paste (Equex STM; Minitube, Verona, WI, USA)) supplemented with 0, 25, and 50 µm bME, respectively. After packaging the semen into 0.5-mL straws, it was frozen by keeping the straws 4 cm above the surface of liquid nitrogen for 15 min and then storing them in liquid nitrogen until use. After thawing at 37�C for 30 s, semen samples were re-suspended in 10 mL of BTS solution containing 1.15 mm caffeine and 4 mm Ca chloride, and incubated at 37�C under 5% CO2 in air for 90 min. Viability, DNA fragmentation, and acrosome status of spermatozoa were assessed by flow cytometry after staining with SYBR�14/PI (Molecular Probes, Inc., Eugene, OR, USA), acridine orange, and PNA/PI, respectively. Statistical analyses of data from at least 3 replicated trials were carried out by ANOVA and Fisher's protected least-squares difference (PLSD) post-hoc test. Just after thawing, no differences in viability (45.6–51.1%; P = 0.67), DNA fragmentation (0.7–0.9%; P = 0.76), and acrosome status (intact acrosome: 79.2–83.0%; P = 0.26) of the spermatozoa were observed when sperm cells were frozen and thawed in 0, 25, and 50 µm bME. After culture for 90 min, however, the incidence of spermatozoa with an intact acrosome was significantly higher (P < 0.05) when the semen was frozen and thawed in the presence of 50 µm bME (70.9%), compared with 0 (61.7%) and 25 µm bME (61.0%). Chlortetracycline (CTC) analyses were peformed to confirm that the incidence of intact spermatozoa was higher (P < 0.01) in 50 µm bME (67.6%) than that of non-supplementation controls (51.4%). These results demonstrate that supplementation of semen with 50 µm bME during freezing and thawing processes reduces acrosome damage of boar spermatozoa.

244 PROTEIN TYROSINE PHOSPHORYLATION PATTERN OF SPERM BOUND TO THE PORCINE OVIDUCT DURING THE PERIOVULATION STAGE

2013 ◽  
Vol 25 (1) ◽  
pp. 270
Author(s):  
V. Luño ◽  
R. López-Úbeda ◽  
L. Lefièvre ◽  
C. Matás
Keyword(s):  
Tyrosine Phosphorylation ◽  
Protein Tyrosine Phosphorylation ◽  
Fixed Tissue ◽  
Protein Tyrosine ◽  
Boar Sperm ◽  
Phosphorylation Pattern ◽  
Paraffin Block ◽  
Low Levels ◽  
High Level ◽  
One Way Anova

The interaction of spermatozoa and oviductal epithelial cells (OEC) is a controlled process that regulates sperm capacitation and the acquisition of fertilizing ability until the time of ovulation. A crucial signalling event involved in capacitation is protein tyrosine phosphorylation. In previous studies, we have demonstrated changes in the pattern of protein tyrosine phosphorylation in boar sperm after the co-culture with OEC. The aim of this study was to characterise the pattern of protein tyrosine phosphorylation in boar sperm bound or unbound to the oviduct of the sow during the periovulation stage. Eight crossbred multiparous sows were inseminated with 3 × 109 sperm. The animals were anesthetized and laparotomies were performed at 36 h after insemination. Ovaries and oviducts were exposed through a midventral incision for collection. Each oviduct was divided into four parts: the ampulla, ampullary-isthmic junction, isthmus, and utero-tubal junction. All segments of the oviduct were flushed to recover spermatozoa, which were subsequently fixed. Tissue obtained from each of the oviduct segments were fixed and embedded in a paraffin block. Sections were mounted on poly-l-lysine-coated slides and deparaffinized. Flushed sperm and oviductal sections were analysed by indirect immunofluorescence using monoclonal antiphosphotyrosine antibodies. Three different sperm subpopulations were determined according to the distribution of protein tyrosine phosphorylation observed: nonphosphorylated spermatozoa (pattern 1), subequatorial segment or subequatorial segment and flagellum phosphorylation (pattern 2), and subequatorial segment and head or flagellum phosphorylation, or both (pattern 3). Data were analysed with SPSS (IBM, Armonk, NY, USA) using one-way ANOVA. After flushing, most sperm were recovered from the utero-tubal junction segment of the oviduct, and sperm exhibited a higher proportion of pattern 2 (81.62%). Unbound sperm showed a high level of protein tyrosine phosphorylation in the subequatorial segment, head and flagellum in the isthmus (32.34%), ampullary-isthmic junction (37.70%), or ampulla region (35.11%; P < 0.05). Very few sperm were attached to OEC, and sperm oviduct binding was mainly found in the isthmus region. The most common tyrosine phosphorylation distribution observed in sperm attached to OEC was pattern 1 (84.21%), although labelling to the subequatorial segment was also observed. Our results showed that only sperm that did not display tyrosine phosphorylation on the sperm acrosome region (head) were found bound to OEC. In conclusion, distinct protein tyrosine phosphorylation patterns were found on sperm bound to OEC. This interaction could be used as a tool for selecting a population of sperm containing low levels of tyrosine phosphorylation.

129 IMPROVEMENT OF BOAR SPERM CRYOSURVIVAL BY THE OPTIMIZATION OF CRYOPRESERVATION CONDITIONS

2007 ◽  
Vol 19 (1) ◽  
pp. 182
Author(s):  
J. Roca ◽  
M. Hernandez ◽  
T. Cremades ◽  
J. M. Vazquez ◽  
E. A. Martinez
Keyword(s):  
Propidium Iodide ◽  
Sperm Quality ◽  
Membrane Integrity ◽  
Egg Yolk ◽  
Limiting Factors ◽  
Commercial Application ◽  
Glycerol Concentration ◽  
Freezing And Thawing ◽  
Warming Rate ◽  
Boar Spermatozoa

One of the most important limiting factors for the efficient commercial application of frozen–thawed semen on pig artificial insemination programs is the significant and consistent inter-boar variability in sperm cryosurvival. The objective of the present study was to evaluate the effectiveness of different cryopreservation conditions (CCs) for freezing and thawing boar spermatozoa, and to determine their suitability for individual boars, with particular reference to those that showed an intrinsic poor sperm cryosurvival. Using a split-ejaculate technique, single ejaculates from 53 boars were suspended in lactose-egg yolk extender containing 2 or 3% final glycerol concentration, packaged in 0.5-mL straws, cooled at rates of 10, 40, or 60�C min-1 using a programmable cell freezer, and stored in liquid nitrogen; the frozen samples were warmed at ≈1200�C min-1 (37�C water bath for 20 s) or ≈1800�C min-1 (70�C for 8 s). The cryopreservation condition including 2% glycerol, 40�C min-1 of cooling, and ≈1200�C min-1 of warming was considered as the control. Frozen–thawed sperm were evaluated at 30 and 150 min post-thawing for sperm motility (CASA system), plasma membrane integrity (SYBR-14 and propidium iodide), and acrosome membrane integrity (FITC-peanut agglutinin and propidium iodide). Data were analyzed using 2 different ANOVA mixed models. Whereas cooling rate had no influence (P ≥ 0.05), glycerol concentration and warming rate, both independently, affected (P ≤ 0.05) all post-thawing sperm assessments. No interaction (P ≥ 0.05) among effects was detected for any of the sperm parameters assessed. Evaluating the combined effect of glycerol concentration and warming rate, the highest post-thaw sperm quality was achieved from the semen samples frozen with 3% of glycerol and thawed at ≈1800�C min-1. Significant differences (P ≤ 0.05) among ejaculates (boars) to support the different CCs were shown in all post-thaw sperm assessments. Three different (P ≤ 0.05) ejaculate (boar) populations, defined by PATN analysis (PATN software package, CSIRO, Canberra, Australia), were identified according to post-thaw sperm assessments in semen samples frozen and thawed using control CC (populations so-called 'good', 'moderate', and 'bad' sperm freezers). Different (P ≤ 0.05) susceptibility in the tolerance of spermatozoa to support the different CCs was found among the ejaculate populations. Whereas spermatozoa from ejaculates considered as 'good' freezers are relatively unaffected (P ≥ 0.05), those from 'moderate' and, mainly, 'bad' freezers are very sensitive (P ≤ 0.05) to the modifications in the CCs. In conclusion, slight modifications in the CCs — glycerol concentration and warming rate for thawing — can improve the sperm cryosurvival of some ejaculates (boars), the improvement being particularly larger in those ejaculates (boars) classified as ' bad' sperm freezers. This work was supported by CICYT (AGF2005-00760), Madrid, Spain.

79 CHOLESTEROL QUANTIFICATION DURING CRYOPRESERVATION IN BOAR SPERM SAMPLES ENRICHED IN OR DEPRIVED OF CHOLESTEROL

2009 ◽  
Vol 21 (1) ◽  
pp. 140
Author(s):  
C. Tomás ◽  
M. Hernández ◽  
E. Mocé ◽  
E. Martínez ◽  
J. M. Vá ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Mixed Model ◽  
Control Sample ◽  
Egg Yolk ◽  
Colorimetric Test ◽  
Mixed Model Anova ◽  
Boar Sperm ◽  
Boar Spermatozoa ◽  
Plasma Membrane Cholesterol ◽  
Mammalian Spermatozoa

Sperm membranes suffer significant lipid changes during cryopreservation similar to initial steps in capacitation, in which a reduction in plasma membrane cholesterol (pmCHO) is observed. Methyl-β-cyclodextrin (MBCD) or cyclodextrin pre-loaded with cholesterol (CLC; Purdy PH and Graham JK 2004 Cryobiology 48, 36–45) have been used to decrease or increase the pmCHO, respectively, in different mammalian spermatozoa. In this study, pmCHO levels were assessed during the cryopreservation process in boar sperm samples deprived of (D) or enriched in (E) CHO. Single sperm-rich fractions from 14 boars were divided in 4 aliquots and frozen in 0.5-mL straws after dilution in a lactose-egg yolk extender with a final concentration of 20% egg yolk and 3% glycerol (Control sample, C) and supplemented with MBCD (1 mg/120 × 106 cells; D sample) or CLC at 1 (E-1 sample) or 3 (E-3 sample) mg/120 × 106 cells. The pmCHO level was quantified at 17°C in pre-diluted sperm samples (basal pmCHO) and at 3 steps of the cryopreservation process: after cooling at 5°C (step 1), after the addition of 3% glycerol (step 2), and immediately after thawing (step 3). The pmCHO was quantified by an enzymatic colorimetric test (Spinreact®, Sant Esteve de Bas, Spain) at 520 nm, following the protocol described by Moore AI et al. (2005 Cryobiology 51, 241–249). Data (least squares means ± SEM) were expressed as micrograms of CHO/106 sperm and analyzed as a mixed-model ANOVA. Because there were significant differences (P ≤ 0.05) between ejaculates, this effect was included as random. The level of pmCHO in C, D, and E-1 samples followed the same trend without significant differences (P ≤ 0.05) among them. That level increased (P ≤ 0.05) at step 1 (0.55 ± 0.17; 0.52 ± 0.17; and 0.66 ± 0.17 for C, D, and E-1, respectively), compared with the basal level (0.24 ± 0.02), and decreased (P ≤ 0.05) to the basal level at steps 2 (0.23 ± 0.07; 0.23 ± 0.07; and 0.30 ± 0.07 for C, D, and E-1, respectively), and 3 (0.07 ± 0.02; 0.08 ± 0.02; and 0.13 ± 0.02 for C, D, and E-1, respectively). Although the pattern in E-3 was similar to other treatments, the level of pmCHO was greater (P ≤ 0.05) than those at step 1 (0.75 ± 0.17), 2 (0.51 ± 0.07), and 3 (0.16 ± 0.02). In conclusion, the pre-freezing treatment of sperm samples with methyl-β-cyclodextrin, to reduce the CHO, did not modify the CHO level of plasma membrane of boar spermatozoa. However, treatment with cyclodextrin pre-loaded with CHO at 3 mg/120 × 106 cells increased significantly the CHO level of plasma membrane, which was evident throughout the cryopreservation process. Supported by AGL2006-07769/GAN, AGL2005-00760/CICYT, Madrid, and GERM (04543/07), Murcia, Spain.

245 EFFECT OF INCUBATION IN OVIDUCTAL EPITHELIAL CELLS AND CO-CULTURE WITH OOCYTES IN THE PROTEIN TYROSINE PHOSPHORYLATION PATTERN OF BOAR SPERM

2013 ◽  
Vol 25 (1) ◽  
pp. 270
Author(s):  
R. López-Úbeda ◽  
C. Matás
Keyword(s):  
Epithelial Cells ◽  
Tyrosine Phosphorylation ◽  
Petri Dish ◽  
Culture Conditions ◽  
Protein Tyrosine Phosphorylation ◽  
Surface Distribution ◽  
Protein Tyrosine ◽  
Boar Sperm ◽  
Phosphorylation Pattern ◽  
High Level

The oviductal epithelial cells (OEC) play an important role in the process of sperm capacitation and the acquisition of fertilizing capacity. One of the most important changes that can be observed in spermatozoa is the increase in protein tyrosine phosphorylation. In previous studies, we have demonstrated changes in the pattern of protein tyrosine phosphorylation in boar sperm after the co-culture with OEC. The aim of this study was to characterise the pattern of protein tyrosine phosphorylation in boar sperm after incubation in oviductal cells in the presence of oocytes. Epithelial cells were stripped from the oviducts of cycling sows and cultivated for 7 days on a petri dish. Sperm cells treated by a discontinuous gradient of isotonic Percoll were added to the OEC and incubated for 30 min. After 30 min, the spermatozoa bound and unbound were separated and co-cultured with oocytes for 18 h. Five different samples were analysed by indirect immunofluorescence to determine the localization of proteins phosphorylated in tyrosine residues (Tardif et al., 2001 Biol. Reprod. 65, 784–792): ejaculated control, sperm unbound OEC after 30 min, sperm unbound after 30 min with oocytes, sperm bound after 30 min with oocytes, and sperm bound after 30 min and detached after culture with oocytes. Five patterns were determined according to their surface distribution: nonphosphorylated spermatozoa (pattern 1), head, tail, or both phosphorylated (pattern 2), equatorial segment phosphorylated (pattern 3), equatorial segment and head, tail, or both phosphorylated (pattern 4), and phosphorylation of the tail alone and in combination with other areas of the sperm (pattern 5). Sperm without any treatment showed lower tyrosine phosphorylation levels (87.04%; P < 0.05). Unbound sperm showed a high level of protein tyrosine phosphorylation in the subequatorial segment and head, tail, or both phosphorylated (47.71%; P < 0.05), and this pattern was associated with a high level of capacitation. The same pattern was reduced when unbound sperm were co-cultured with oocytes for 18 h (25.67%; P < 0.05), and was even lower in sperm bound with oocytes. The study of phosphorylation of the tail (pattern 5) showed no significant differences between different samples. We concluded that sperm-bound OEC have lower levels of phosphorylation, indicating a selective function in the sperm-OEC interaction. In addition, phosphorylation of the tails appears to be related more to the hyperactivation of sperm than to the different culture conditions.

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