Bull Sperm Membranes Research Articles

Time-resolved Fluorescence and Generalized Polarization: Innovative tools to assess bull sperm membrane dynamics during slow freezing

During slow freezing, spermatozoa undergo membrane alterations that compromise their ability of fertilizing. These alterations are cause either by cold shock or by the use of cryoprotectants known to be cytotoxic. However, little is known about the membrane changes that occurred during freezing. Here, we combined Generalized Polarization (GP), Time-resolved Fluorescence and laurdan fluorescence properties to investigate the changes in membrane fluidity and dynamics during slow freezing of bull sperm. We successfully demonstrated that laurdan may be distributed in three different local environments that correspond to different membrane lipid composition. These environments wont behave the same way when the cells will be subjected to either a chemical treatment (adding the cryoprotectants) or a physical treatment (freezing).

Electrophoretic profile of bull sperm membrane proteins as a tool for selection of breeding bull

Proteins present in the seminal plasma and sperm influence sperm function and fertilization. The present study was carried out to characterize the electrophoretic profile of bull sperm membrane proteins and to correlate with bull fertility. Semen samples were collected from 20 breeding bulls (10 Jersey and 10 Jersey crossbred) and the sperm membrane proteins were extracted by Triton X. Heparin binding proteins were eluted with heparin column, and the molecular weight of the proteins was assessed by discontinuous sodium dodecyl sulphate polyacrylamide gel elecrophoresis (SDS- PAGE). To assess the field fertility of the bulls, 50 frozen semen straws/bull were used for insemination. Results indicated that only 50 % of the bulls screened had 28– 30 kDa heparin binding proteins in their sperm and these bulls had 12 % higher conception than the bulls lack the protein. So, it can be concluded that the bulls positive for of 28– 30 kDa heparin binding proteins in sperm membrane protein had higher chance of fertility and screening for its presence can be used as a tool in addition to routine criteria for selecting bulls.

Lipid Bilayer Composition Affects Transmembrane Protein Orientation and Function

Sperm membranes change in structure and composition upon ejaculation to undergo capacitation, a molecular transformation which enables spermatozoa to undergo the acrosome reaction and be capable of fertilization. Changes to the membrane environment including lipid composition, specifically lipid microdomains, may be responsible for enabling capacitation. To study the effect of lipid environment on proteins, liposomes were created using lipids extracted from bull sperm membranes, with or without a protein (Na+ K+-ATPase or α-amylase). Protein incorporation, function, and orientation were determined. Fluorescence resonance energy transfer (FRET) confirmed protein inclusion in the lipid bilayer, and protein function was confirmed using a colourometric assay of phosphate production from ATP cleavage. In the native lipid liposomes, ATPase was oriented with the β subunit facing the outer leaflet, while changing the lipid composition to 50% native lipids and 50% exogenous lipids significantly altered this orientation of Na+ K+-ATPase within the membranes.

Arrangement of PMCA4 in bovine sperm membrane fractions

The plasma membrane Ca(2+) -ATPase (PMCA) is the main restorer of Ca(2+) balance in sperm. Particularly, PMCA isoform 4 has an essential function in sperm fertility by its participation in gaining sperm hypermotility. PMCA activity is influenced by its lipid environment. Sperm membranes exhibit lipid raft microdomains or detergent-resistant membrane domains, enriched in sphingolipids and cholesterol, forming functional specialized areas. Lipid and protein composition of lipid rafts alters during the capacitation process, which is characterized by a cholesterol efflux. In this study, the localization of PMCA4 in lipid membrane fractions of the sperm plasma membrane was investigated. We identified PMCA4 in both the detergent-resistant membrane (DRM) and in the detergent-soluble (DS) fraction of caput and cauda sperm, respectively. Capacitation did not influence PMCA4 localization. In immunocytochemical studies PMCA4 was co-localized with the lipid raft/DRM marker caveolin in the mid piece of caput and cauda sperm. Functional studies with seminal vesicle major protein PDC-109 showed that the Ca(2+) -ATPase activity in DS fractions of cauda sperm and capacitated cauda sperm was stronger enhanced than in the DRMs. In both fractions the effect was statistically significant. In contrast, in lipid overlay experiments PDC-109 interacted stronger with the lipids extracted from DRMs than with lipids extracted from DS. Our results indicate a possible functional compartmentalization of PMCA in bull sperm membranes and point to a presumptive, yet unknown interaction partner of Ca(2+) -ATPase and PDC-109, mediating the PDC-109 action from DRMs to the DS fraction of sperm plasma membrane.

SPAM1 Isoforms from Two Tissue Origins Are Differentially Localized Within Ejaculated Bull Sperm Membranes and Have Different Roles During Fertilization1

The deduced amino acid sequence of bull sperm, SPAM1, suggests that it possesses a transmembrane domain between the hyaluronidase and the putative zona pellucida (ZP) binding domains. The objective of this study was to determine the orientation and localization of SPAM1 in order to understand how it could fulfill these two roles. We report that two isoforms of SPAM1 are present on ejaculated bull spermatozoa: one localized on the anterior portion of the sperm head, and the other on the postacrosomal portion of the head. The first isoform is expressed intracellularly, while the second one is detected at the sperm surface with its hyaluronidase domain facing the extracellular environment. Two-dimensional electrophoresis revealed that the two isoforms have different masses (80 and 70 kDa, respectively), and LC/MS/MS analyses confirmed our previously published deduced amino acid sequence of bovine SPAM1. In addition, this approach showed that the 70-kDa isoform differs from the 80-kDa isoform in its C terminus. Our results suggest that the shorter SPAM1 form originates from the epididymis, while the longer one is produced during spermatogenesis. These results clearly demonstrate that ejaculated bull sperm possess two forms of SPAM1: one (epididymal) expressed at the surface, and one (testicular) that interacts with the ZP after the acrosome exocytosis.

Delivering cholesterol or cholestanol to bull sperm membranes improves cryosurvival

This study compared the effect of adding other cholesterol conjugates, which should incorporate into and increase sperm membrane fluidity at low temperatures thereby increasing cryosurvival. Ejaculates from each of four bulls were diluted to 120 million cells/ml in a Tris diluent and used in two experiments. Each experiment contained four treatments: No additive (control); 1.5 mg CLC/120 million sperm (positive control); and 1.5 mg cyclodextrin pre-loaded with cholestanol or desmosterol/120 million sperm. In the first experiment, fresh sperm were treated with cyclodextrins that were pre-loaded with cholesteryl conjugates and incubated for 15 min at 22 °C to allow incorporation of the conjugates. The percentages of motile sperm after exposure to solutions ranging from 0 to 1200 mOsm were used to evaluate the osmotic tolerance of the cells. The ability of treated sperm to bind to the bovine zona pellucida (ZP) and chicken egg perivitelline membrane (CEPM) was evaluated to determine if altering the sperm membrane affected the ability of sperm to bind to oocytes. In the final experiment, the cryosurvival rates of control and treated sperm were determined. Control and treated sperm were cryopreserved in a Tris diluent containing 10% egg yolk (EY) and 8% glycerol (final concentrations). Samples were thawed to determine the motility and ability of sperm to bind to the ZP and to CEPM using a CASA and epifluorescence microscope, respectively. Fresh sperm treated with CLC resulted in more binding to the ZP compared to all other treatments ( P < 0.05). No differences were observed between ZP and CEPM binding ( P > 0.05). The percentages of motile sperm were greater for fresh samples treated with cholesterol, cholestanol or desmosterol loaded cyclodextrin than control cells ( P < 0.05) when the sperm were exposed to anisosmotic conditions, and then returned to isosmolality. After cryopreservation the percentages of motile sperm and number of sperm binding to each CEPM were similar for sperm treated with CLC and cholestanol compared to sperm treated with desmosterol ( P > 0.05). All treatments provided greater motility and binding efficiency than control sperm ( P < 0.05). Therefore, adding cholesterol or cholestanol to bull sperm membranes improved cell cryosurvival.

Effect of cholesterol or cholesteryl conjugates on the cryosurvival of bull sperm

Different cholesterol conjugates-loaded-cyclodextrin was added to bull sperm to improve sperm quality after freezing. Ejaculates from four bulls were diluted to 120 million cells/ml in Tris (T) diluent and then sub-divided into 10 treatments as follow: no additive (control); 1.5 mg CLC (positive control); 0.75 mg or 1.5 mg of cyclodextrin pre-loaded with cholesterol conjugated to heptanoate, palmitate, pelargonate or stearate, and incubated for 15 min at 22 °C. The samples were then diluted 1:1 with 20% egg yolk (EY) in T diluent and cooled to 5 °C over a 2 h. Upon reaching 5 °C, the samples were diluted 1:1 with T diluent containing 10% EY + 16% glycerol, and allowed to equilibrate for 15 min, and packaged into 0.5 ml straws and frozen in static liquid nitrogen vapor for 20 min before being plunged into liquid nitrogen for storage. Straws were thawed and the sperm motility, viability and number sperm binding to perivitelline membrane were determined. The ability of bull sperm to bind to the oocyte membranes was conducted using the chicken egg perivitelline membrane (CEPM) as described by Barbato et al. [G.F. Barbato, P.G. Cramer, R.H. Hammerstedt, A practical in vitro sperm-egg binding assay that detects subfertile males. Biol. Reprod. 58 (1998) 686–699] and modified by Amorim et al. [E. Amorim, J.K. Graham, B. Spizziri, M. Meyers, L. Amorim, C. Torres, The effect of adding cholesteryl-heptanoate, -palmitate, -pelargonate, or -stearate loaded cyclodextrin on bull sperm cryosurvival, in: Proceeding 40th Annual Meeting of the Society for the Study of Reproduction (SSR), July, San Antonio, TX, EUA, 2007], where these authors did not observe difference between bovine zona pellucide and CEPM. Higher percentages of motile and viable sperm were maintained after thawing from bull sperm treated with CLC and pelargonate compared to all other treatments ( P < 0.05). Control samples and sperm treated with heptanoate, palmitate, or stearate loaded cyclodextrin exhibited similar motility percentages and viable sperm after freezing ( P > 0.05). The percentage of motile sperm and number sperm binding to chicken egg perivitelline membrane was higher for CLC and pelargonate compared to control ( P < 0.05). Therefore, adding either cholesterol or pelargonate to bull sperm membranes improved cell cryosurvival, whereas treatments with cyclodextrins pre-loaded with other cholesterol-like molecules did not.

Oxidative stress alters membrane sulfhydryl status, lipid and phospholipid contents of crossbred cattle bull spermatozoa

Ferrous ascorbate (FeAA: FeSO 4 + ascorbic acid) has been used in the past by different investigators to induce oxidative stress. The optimum dose of FeAA for inducing oxidative stress by affecting thiols [total thiols (TSH), glutathione reduced (GSH), glutathione oxidized (GSSG), redox ratio (GSH/GSSG)], total lipids and phospholipids has been ascertained in the local crossbred cattle bull spermatozoa. The fractions of spermatozoa suspended in 2.9% sodium citrate were subjected to three doses of FeAA (100 μM:500 μM, 150 μM:750 μM, 200 μM:1000 μM; FeSO 4:ascorbic acid), and were assessed for various parameters. On increasing the concentration of FeAA, a gradual decrease in TSH, GSH, GSH/GSSG, lipid and phospholipid levels, but increase in GSSG content were observed. It is concluded that thiol groups play an important role in antioxidation and detoxification of ROS as well as maintaining intracellular redox status. Thiol groups, thus, serve as defense mechanisms of sperm cells to fight against oxidative stress. In addition, all doses of FeAA cause leakage of lipids and phospholipids from the bull sperm membranes.

258 THE EFFECT OF A PLANT PROTEIN COMPONENT OF MEDIA USED FOR BULL SPERM SEXING ON SPERM MEMBRANE STATUS

The aim of the work was to examine the effect of modified TALP medium (TALP/Pp, Animal Pharma B.V., Hengelo, The Netherlands)—used in the sperm sexing procedure—on bull sperm membrane status. The TALP was modified by replacement of bovine serum albumin (BSA) with a mixture of several plant proteins and soya lecithin (Pp). The Pp component was prepared using a high pressure homogenization process. The TALP/Pp had the same pH and osmotic pressure as the original TALP medium (TALP/BSA). The work was divided into 2 parts: (1) Nine ejaculates collected from 2 bulls (Holstein and Polish Red) were used. Immediately after collection, each ejaculate was split into 2 parts and diluted (1:2) with TALP/BSA or TALP/Pp. The sperm membrane status was examined after 3 days of storage at 15�C. (2) Fifteen ejaculates collected from 5 bulls (Holstein, Polish Red, and Simmental) were used. Each ejaculate was split into 2 parts: the first part was diluted with TALP/BSA, stained, incubated, and sexed according to the XY Inc. bull semen sexing procedure; the second part was diluted, stained, incubated, and collected after sexing into TALP/Pp with no egg yolk addition. In both groups no red food due was used to identify and exclude the dead spermatozoa from the sorted fractions. The sperm sexing procedure was performed with an SX MoFlo high-speed sorter at a speed of 3000–4000 cells/s. After collecting about 10 million spermatozoa, both fractions, X andY, were mixed, centrifuged at 700g for 15 min to concentrate the spermatozoa (20 million mL–1), and the sperm membranes examined. For sperm membrane examination, 'live/dead' samples were stained with SYBR-14/propidium iodide fluorochromes and analyzed by flow cytometry. The data from 20 000 spermatozoa were collected for each sample. The percentage of membrane-intact ('live') spermatozoa was taken for statistical analysis. The mean percentage of live spermatozoa stored for 3 days in TALP/BSA v. TALP/Pp was 25.7% (SD = 7.48) v. 28.58% (SD = 7.04), respectively (P &lt; 0.01). The mean percentage of live spermatozoa in samples of sexed semen was 33.57% (SD = 18.97) for TALP/BSA and 38.51% (SD = 20.22) for TALP/Pp (P &lt; 0.01). It can be concluded that Pp should be considered as a replacement for BSA in the TALP medium used for bull sperm sexing because (1) it results in significantly higher numbers of live spermatozoa after storage and/or sexing; (2) it eliminates a possible source of transmissible diseases (such as bovine spongiform encephalopathy); and (3) it decreases the total cost of the basic media used for the bull sperm sexing procedure.

67 DELIVERING CHOLESTANOL OR DESMOSTEROL TO BULL SPERM MEMBRANES IMPROVES CRYOSURVIVAL

Altering the lipid composition of sperm plasma membranes affects sperm cryosurvival. Cryopreservation induces many stresses on the spermatozoa, including destabilization of the plasma membrane, which results in the loss of sperm motility and function. Treating bull spermatozoa with cholesterolloaded cyclodextrin (CLC) prior to cryopreservation increases sperm cryosurvival rates. This study compared the effect of adding other sterols, which should incorporate into the membrane and increase membrane fluidity at low temperatures, thereby increasing cryosurvival. Ejaculates from four bulls were divided into two experiments (E). In E1, ejaculates were extended with Tris, and then subdivided into four treatments: No additive (control), 1.5 mg CLC/120 million sperm (positive control), and 1.5 mg/120 million sperm in cyclodextrin pre-loaded with either cholestanol or desmosterol. Spermatozoa were incubated for 15 min at 22�C after which both the ability of fresh spermatozoa to bind to the zona pellucida (ZP) and chicken egg perivitelline membrane (EPM) and their osmotic tolerance were evaluated. In E2, sperm were diluted to 120 million cells mL–1 in a Tris diluent and treated as described for E1. Then, samples were diluted 1:1 (v:v) in Tris with 20% Egg Yolk (EY) and cooled to 5�C. After dilution 1:1 (v:v) with Tris containing 10% EY and 16% glycerol, samples were allowed to equilibrate for 15 min, and then were packaged into 0.5-mL straws, frozen in static liquid nitrogen vapor for 20 min, and plunged into liquid nitrogen for storage. Straws were thawed and the motility and zona-binding ability were determined using a Hamilton Thorne Motility Analyzer (Hamilton Thorne Biosciences, Beverly, MA, USA) and epifluorescence microscopy, respectively. Treatment differences for sperm motility, osmotic tolerance, and zona binding were determined using analysis of variance. Treating spermatozoa with CLC resulted in more fresh bull spermatozoa binding to the EPM and ZP compared to cholestanolor desmosterol-loaded cyclodextrin-treated spermatozoa or control cells (P &lt; 0.05). No differences were observed between EPM and ZP binding (P &gt; 0.05). The percentages of total and progressively motile spermatozoa were higher for fresh samples treated with cholesterol-, cholestanol-, or desmosterol-loaded cyclodextrin than for control cells (P &lt; 0.05) when spermatozoa were exposed to anismotic conditions, and then returned to isosmolality. After cryopreservation, the percentages of motile spermatozoa and number of spermatozoa binding to ZP were similar for spermatozoa treated with CLC (56% and 115 sperm/ZP) and cholestanol (53% and 108 sperm/ZP) compared to spermatozoa treated with desmosterol (42% and 86 sperm/ZP; P &lt; 0.05). All treatments provided higher motility and binding efficiency than control spermatozoa (32% and 62 sperm/ZP; P &lt; 0.05). Therefore, adding cholesterol or cholestanol to bull sperm membranes improved cell cryosurvival. Studies to determine if cholestanol affects sperm capacitation need to be conducted.

Fertility-associated proteins in Nelore bull sperm membranes

The present study was undertaken to evaluate the protein composition of the sperm membranes (SM) of Nelore bulls, assessing protein markers associated with bull fertility, and whether these markers can be used for predicting bull fertility. Samples were obtained of 20 Nelore bulls, with fertility ranked and divided into three groups (greater, normal and least). To rank the bull's fertility weighted classification was used (according to the number of pregnant cows, number of AI cows and number of herds, considering three different breeding seasons), using the PROC GENMOD as a statistical model, with 99% significance. A total of 7897 Nelore cows, randomly distributed among 28 different farms, were considered in the statistical analyses. The bulls were divided into three fertility groups (pregnancy rates): greater (% F > 80), normal (79 < % F > 71) and least (<68% F) with 3, 13 and 4 bulls, respectively. Two-dimensional gel electrophoresis (2DE) of sperm membranes indicated in 27 spots (SM40, SM53, SM69, SM93, SM102, SM111, SM137, SM138, SM189, SM196, SM201, SM202, SM204, SM225, SM236, SM237, SM239, SM241, SM246, SM247, SM275, SM283, SM342, SM346, SM355, SM372, SM391) was prevalent in the higher fertility group, and just one spot (SM244) was prevalent in the lower fertility group. Spots SM244 and SM239 had their identification defined by PMF/MALDI-MS, as BSP-A3 and aSFP, respectively. Both these proteins showed a great potential for predicting bull's fertility. The amount of aSFP was 8.5 times greater in the sperm membrane protein profile of the higher fertility groups of Nelore bulls. Besides that, the BSP-A3 was 2.5 times greater in the lower fertility group. For the other spots potentially associated with fertility not yet identified, additional tests will be necessary, but it is clear that the 2D electrophoresis of the sperm membrane can be used for a new approach to predict Nelore bull fertility.

The fluidity of Chinese hamster ovary cell and bull sperm membranes after cholesterol addition

Cell plasma membrane fluidity is affected by membrane lipid and protein composition as well as temperature. Altering the cholesterol content of a membrane can change membrane fluidity at different temperatures and this may affect cell survival during cryopreservation. In these experiments, we examined the effect that adding cholesterol to the membranes of Chinese hamster ovary cells (CHO) and bull sperm had on cell plasma membrane fluidity and cell survival when cells were cooled to 5 °C or were cryopreserved. Cells were treated with 0, 1.5 or 5.0 mg cholesterol-loaded cyclodextrin (CLC), stained with N-((4-(6-phenyl-1,3,5-hexatrienyl)phenyl)propyl)trimethylammonium- p-toluenesulfonate (TMAP-DPH) to evaluate membrane fluidity and with propidium iodide to evaluate cell viability, prior to analysis by flow cytometry at 23, 5 °C, and after cryopreservation. CHO cells exhibited a single cell population with all cells having similar membrane fluidity. Membrane fluidity did not change when temperature had been reduced and then returned to 23 °C ( P < 0.05), however, adding cholesterol to the cells induced membranes to become more rigid ( P < 0.05). Bull sperm samples consisted of two cell subpopulations, one having relatively higher membrane fluidity than the other, regardless of cholesterol treatment or temperature. In addition, cells possessing the highest membrane fluidity did not survive cooling or cryopreservation efficiently. CLC treatment did not significantly alter membrane fluidity after temperature changes, but did maintain higher percentages of spermatozoa surviving cooling to 5 °C and cryopreservation ( P < 0.05). In conclusion, adding cholesterol to cell resulted in detectable membrane fluidity changes in CHO cells and increased survival of bull sperm after cooling to 5 °C and after cryopreservation.

Effect of adding cholesterol to bull sperm membranes on sperm capacitation, the acrosome reaction, and fertility.

When cholesterol is added to sperm membranes before cryopreservation, higher percentages of motile and viable cells are recovered after thawing. However, because one of the first steps in sperm capacitation is cholesterol efflux from the sperm plasma membrane, adding cholesterol to enhance cryosurvival may retard sperm capacitation. These studies evaluated the ability of sperm treated with cholesterol-loaded cyclodextrins (CLC) to capacitate, acrosome react, and fertilize oocytes. Control (non-CLC-treated) and CLC-treated sperm were treated with heparin, dilauroylphosphatidylcholine (PC12), or calcium ionophore A23187 (A23187) to capacitate and induce the acrosome reaction. Sperm capacitation, assessed by an increase in intracellular calcium level, and acrosome-reacted sperm were measured using flow cytometry. Fresh CLC-treated sperm cells underwent capacitation and/or the acrosome reaction at rates different from control samples, and the differences detected were dependent on the method used to induce sperm capacitation and the acrosome reaction. After cryopreservation, however, CLC-treated and control sperm underwent capacitation and the acrosome reaction at similar rates regardless of the method used to induce capacitation and the acrosome reaction. The primary concern for CLC-treated sperm, however, is whether this treatment would affect in vitro or in vivo fertility. Adding either control or CLC-treated cryopreserved sperm to bovine oocytes in vitro resulted in similar oocyte cleavage rates and blastocyst formation rates. In addition, when inseminated into heifers, pregnancy rates for control and CLC-treated sperm were also similar. Therefore, treating bull sperm with CLC permits greater numbers of sperm to survive cryopreservation while preserving the fertilizing potential of each individual sperm.

Determination of bull sperm membrane permeability to water and cryoprotectants using a concentration-dependent self-quenching fluorophore.

The objective of this study was to determine the membrane permeability characteristics of bovine spermatozoa. These included the hydraulic conductivity (Lp), the permeability coefficients (Ps) of four common cryoprotective agents (CPAs) and the associated reflection coefficients (sigma). Stopped-flow fluorometry was applied in order to capture rapid cell volume changes under anisosmotic conditions in the absence or presence of permeant solutes (CPAs). This technique utilizes a concentration-dependent self-quenching entrapped fluorophore. The resulting cell volume changes were used in three-parameter fitting calculations to compute Lp in the absence of permeant solutes and Ps and Lp in the presence of permeating solutes (CPAs) at 22 degrees C. The hydraulic conductivity in the absence of permeating solutes was estimated to be 0.68+/-0.05 microm/min/atm (mean+/-SEM). Hydraulic conductivity (Lp) in the presence of CPAs was 0.91+/-0.27 (mean+/-SEM), 0.29+/-0.04, 0.42+/-0.05, and 0.39+/-0.03 microm/min/atm in the presence of dimethylsulfoxide (Me(2)SO), glycerol, propylene glycol (PG), and ethylene glycol (EG), respectively. The values for Ps were estimated to be 1.72+/-0.36, 1.75+/-0.03, 2.47+/-0.24, and 1.49+/-0.33 x 10(-3)cm/min for Me(2)SO, glycerol, PG, and EG, respectively. The data were used to simulate volume excursions during addition and removal of CPA, to predict the different effects of the four CPAs.

Lipid dynamics in the plasma membrane of ram and bull spermatozoa after washing and exposure to macromolecules BSA and PVP.

Seminal plasma proteins and macromolecules in the external medium have a major influence on the functionality of sperm plasma membranes. In this investigation we have examined their effects on lipid diffusion in the surface membrane of ram and bull spermatozoa as measured by fluorescence recovery after photobleaching (FRAP). Results show that progressive removal of seminal plasma from ram spermatozoa by repeated centrifugation and resuspension in media +/- 4% bovine serum albumin (BSA) or 0.4% polyvinlypyrrolidone (PVP) causes a reduction in lipid diffusion in all regions of the membrane. By contrast, bull sperm membranes respond with an increase in diffusion in all regions. Repeated washing of bull spermatozoa whose membranes were previously immobile (i.e., showed no recovery after FRAP) restored lipid diffusion suggesting an inhibitory effect of seminal plasma proteins. Further analysis by atomic force microscopy revealed a close association between BSA and the plasma membrane. It is concluded that diffusion of lipids in the plasma membrane of ejaculated ram and bull spermatozoa is influenced by seminal plasma proteins and the composition of the suspending medium. Mol. Reprod. Dev. 59:306-313, 2001.

Effects of Various Cryoprotective Agents and Membrane-Stabilizing Compounds on Bull Sperm Membrane Integrity after Cooling and Freezing

In this study attempts were made to improve the survival rates of bull spermatozoa after freezing/thawing and to clarify the importance of certain agents to the cryopreservation of spermatozoa. For that purpose the standard freezing extender was modified by the addition of different concentrations of various cryoprotectants and membrane-stabilizing agents: glycerol, 1.2-propanediol, polyvinylpyrrolidone, sucrose, egg yolk, lipid vesicles, and bovine serum albumin (BSA). Sperm membrane impermeability toward H33258 was employed as the parameter for sperm integrity during cooling and after freezing/thawing. Exclusion of glycerol from the extender did not significantly affect sperm integrity. Replacing 6% glycerol by 6% 1.2-propanediol resulted in reduced sperm survival, whereas replacement of glycerol by 62.5 m M sucrose slightly improved survival rates. Addition of 5 or 10% polyvinylpyrrolidone (either or not in combination with 0.5 M sucrose) significantly reduced sperm integrity. Excluding egg yolk from the extender caused a serious decrease of sperm survival after both cooling and freezing. The cryoprotection offered by egg yolk could not be mimicked by dioleoylphosphalidylcholine (DOPC) vesicles or DOPC/phosphatidic acid/cholesterol vesicles in concentrations up to 29 or 9 m M, respectively. However, the freezing extender containing 6.5 m M DOPC vesicles in combination with 6% BSA yielded results which did not significantly differ from those obtained with the standard extender; higher vesicle concentrations combined with BSA might produce even better results. Further research on the cryopreservation of bovine spermatozoa should focus on membrane stabilization since the membrane-stabilizing compounds yield more promising results than the ice-preventing agents.