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BOR - Papers in Press, published online ahead of print April 11, 2007.
Biol Reprod 2007, 10.1095/biolreprod.106.058248
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BIOLOGY OF REPRODUCTION 77, 120–126 (2007)
DOI: 10.1095/biolreprod.106.058248
© 2007 by the Society for the Study of Reproduction, Inc.

Milk Caseins Decrease the Binding of the Major Bovine Seminal Plasma Proteins to Sperm and Prevent Lipid Loss from the Sperm Membrane During Sperm Storage1

Annick Bergeron 3, Yves Brindle 4, Patrick Blondin 5, and Puttaswamy Manjunath 2 3

Department of Medicine,3 University of Montreal and Guy-Bernier Research Center, Maisonneuve-Rosemont Hospital, Montreal, Québec, Canada H1T 2M4 Centre d'Insémination Artificielle du Québec,4 Ste-Hyacinthe, Québec, Canada J2S 7B8 L'Alliance Boviteq Inc.,5 Ste-Hyacinthe, Québec, Canada J2T 5H1

ABSTRACT

Milk is used as a medium for sperm preservation. Caseins, the major proteins of milk, appear to be responsible for the protective effect of milk on sperm. Recently, we have shown that egg yolk, which is also widely used to preserve semen, protects sperm functions by preventing the binding to sperm of the major proteins of bull seminal plasma (BSP proteins), thereby preventing BSP protein-mediated stimulation of lipid loss from the sperm membrane. In the present study, we investigated whether milk caseins protect sperm in the same manner as egg yolk. Bovine ejaculates were diluted with skimmed milk permeate (skimmed milk devoid of caseins) or permeate that was supplemented with caseins and stored at 4°C for 4 h. In the semen diluted with permeate, sperm viability and motility decreased in a time-dependent manner. However, in semen diluted with milk or permeate supplemented with caseins, sperm functions were maintained. In addition, lower amounts of the BSP proteins were associated with sperm in semen diluted with milk or permeate supplemented with caseins, as compared to semen diluted with permeate. No milk proteins were detected in the sperm protein extracts. Furthermore, sperm diluted with milk or permeate supplemented with caseins showed 3-fold lower losses of cholesterol and choline phospholipids than sperm diluted with permeate during storage. Thus, milk caseins decreased the binding of BSP proteins to sperm and reduced sperm lipid loss, while maintaining sperm motility and viability during storage. These results support our view that milk caseins prevent the detrimental effects of BSP proteins on the sperm membrane during sperm preservation.

acrosome reaction, gamete biology, male reproductive tract, sperm motility and transport, sperm preservation

INTRODUCTION

For sperm preservation, heated skimmed milk or whole milk is used as a medium in which semen is diluted directly and stored at 4°C or frozen in the presence of glycerol (reviewed in [1]). Since skimmed milk is almost free of lipids (≤0.1%, mostly triglycerides) and is as efficient as whole milk in protecting sperm during semen storage at 4°C or during cryopreservation [2, 3], lipids do not seem to be the constituent responsible for the protection afforded by milk. Lactose, which is present in cow milk at 4.8% (w/v) [4], can improve the efficiency of extenders used to dilute semen, but is not sufficient by itself to protect sperm during storage at 4°C or cryopreservation [5–7]. The protective constituent of milk is most likely micelles of caseins, which are the major proteins of milk. Caseins ({alpha}, ß and {kappa}) are present in milk at ~27 g/L of total milk proteins and exist as heterogeneous colloidal particles, named casein micelles [4]. Basically, casein micelles are constituted of a hydrophobic core of {alpha} and ß caseins surrounded by {kappa} caseins [8]. It has been shown that casein micelles isolated from milk can protect stallion, goat, ram, and bull sperm during storage at 4–5°C [6, 9–13]. In addition, casein micelles can protect bull sperm during freezing in the presence of glycerol [5]. However, the mechanism by which casein micelles protect sperm during storage has not been explained. Other proteins, such as {alpha}-lactalbumin, ß-lactoglobulin, albumin, and lactoferrin, are soluble in milk (~3.5 g/L) and are collectively called whey proteins [4]. However, it is not clear whether whey proteins have any protective effect on sperm during storage.

Egg yolk is also widely used as a protective agent for sperm preservation. The low-density lipoproteins (LDLs) of egg yolk protect the sperm against damage during storage, cooling, and freezing [14–16]. Bull seminal vesicles secrete a family of acidic proteins designated BSP-A1, BSP-A2, BSP-A3, and BSP-30-kDa (BSP proteins), which constitute approximately 60% of the total bovine seminal plasma proteins. Upon ejaculation, these proteins are added to sperm, bind to sperm membrane choline phospholipids, and cause a continuous loss of cholesterol and phospholipids from the sperm membrane, which is detrimental to sperm storage [17]. Interestingly, egg yolk LDLs can bind BSP proteins [18]. This finding led us to propose a novel mechanism of sperm protection by egg yolk [18, 19]. Upon dilution of semen with extender that contains egg yolk, the LDLs present in egg yolk sequester the BSP proteins present in the seminal plasma and prevent their binding to the sperm membrane, thereby preventing the stimulation of loss of cholesterol and phospholipids. It is known that sperm from species with low levels of cholesterol in their sperm membranes have decreased tolerances to cold shock, as compared to sperm from species with high levels of cholesterol [20, 21]. In addition, the incorporation of cholesterol into bull or stallion sperm membranes increases sperm tolerance to cryopreservation [22–25], while the removal of lipids from the sperm during storage is deleterious for sperm functions [17]. Therefore, the prevention of cholesterol removal from the sperm membrane during storage at 4°C or before freezing is important for sperm protection. Egg yolk and milk have been used in combination to store bull semen [7, 26–28]. However, it is not clear whether these two components have a synergistic protective effect on sperm.

In order to gain insight into the mechanism of sperm protection by milk, we investigated whether casein micelles in milk protect sperm by preventing the BSP proteins from binding to sperm and removing lipids from the sperm membrane during storage.

MATERIALS AND METHODS

Materials

BSA (fraction V), lactoperoxidase, erythrosin B, flavianic acid (naphtol), and delipidized whole rabbit antiserum directed against bovine milk whey proteins were purchased from Sigma (St. Louis, MO). Anti-rabbit IgG (H+L), acrylamide, bisacrylamide, SDS, and other electrophoresis products were obtained from Bio-Rad (Mississauga, ON, Canada). The Low molecular weight (LMW) electrophoresis calibration kit was from Pharmacia Biotech (Baie d'Urfé, QC, Canada). Polyethylene glycol (PEG) was obtained from ICN Biomedicals (Cleveland, OH). Immobilon-P membranes and the enhanced chemiluminescence (ECL) reagent kit were purchased from Mandel Scientific (Boston, MA). The I125 was purchased from Perkin Elmer Life Science (Boston, MA). Goat anti-RGG was from Medicorp Inc. (Montréal, QC, Canada). Tylosin was from Elanco (Guelph, ON, Canada), lincomycin and spectinomycin were from Pharmacia Animal Health (Orangeville, ON, Canada). Rabbit polyclonal antibodies directed against bovine milk caseins (Immunology Consultants Laboratory Inc., Newberg, OR), were a generous gift from Dr. Gary Killian (Pennsylvania State University). All other chemicals used were of analytical grade and obtained from commercial suppliers. Milk permeate was obtained from Natrel (St-Laurent, QC, Canada). Before use, the milk permeate was heated for 10 min at 95°C and centrifuged at 12 000 x g for 10 min at 20°C, to pellet any precipitated materials [6]. This permeate (pH 6.7, 260 mOsm) contained all of the components of milk, with the exception of casein micelles. Skimmed milk was purchased at a local store. Before use, the skimmed milk was heated for 10 min at 95°C and the formed coagulum was removed.

Freshly ejaculated bovine semen collected with an artificial vagina was obtained from the Centre d'Insémination Artificielle du Québec (St-Hyacinthe, QC, Canada). Bulls were handled by qualified technicians according to the Guide for the Care and Use of Agricultural Animals established by the Québec Ministry of Agriculture and Fisheries. Crude bovine seminal plasma (cBSP) proteins were prepared by ethanol precipitation of bovine seminal plasma, followed by dialysis of the precipitates against 50 mM ammonium bicarbonate and lyophilization [29].

Isolation of Casein Micelles

Casein micelles were isolated as described previously [30]. Skimmed milk (heated for 10 min at 95°C with the coagulum removed) was allowed to reach room temperature, and was transferred into 11.5-ml Quick Seal tubes (Mandel Scientific Co., Guelph, ON, Canada) and centrifuged (Sorval ultracentrifuge, T-865 rotor) at 70 000 x g at 20°C for 90 min. The supernatants were discarded and the pelleted casein micelles were resuspended in milk permeate (heated for 10 min at 95°C) with gentle stirring overnight at 4°C. The dispersion was then centrifuged at 5000 x g for 30 min at room temperature, to pellet any undispersed material. The protein concentration was adjusted to 27 g/L with heated milk permeate and used as casein extender.

Preparation of Semen Samples

Single ejaculates from two different bulls were pooled immediately after collection, split into three parts and diluted immediately with heated skimmed milk, heated milk permeate or heated milk permeate that contained caseins (27 g/L), to give a sperm concentration of 40 x 106/ml, the concentration routinely used in the artificial insemination center. Diluted semen was then stored at 4°C for 4 h, as is the procedure in artificial insemination centers, before the addition of glycerol and freezing. This experiment was repeated four times with pools of semen from eight different bulls. After dilution, the final concentration of seminal plasma in the diluted pool of semen was between 2.5% and 3.4% (v/v).

The final pH of the heated skimmed milk and heated milk permeate with or without casein was 6.7, and the osmolality was 260 mOsm. All of the extenders were prepared 1 day before use and contained gentamicin (500 µg/ml), tylosin (100 µg/ml), lincomycin (300 µg/ml), and spectinomycin (600 µg/ml). Extenders were maintained at 37°C prior to semen dilution. After 0, 2, and 4 h of storage at 4°C, samples of the semen were removed; one aliquot was used to assess sperm functions and two aliquots were used for sperm protein and lipid analyses. For the protein and lipid analyses, the semen samples were diluted 1:20 with 50 mM PBS in 15-ml plastic tubes and centrifuged at 1840 x g for 10 min. This washing procedure was repeated five times, to remove the extender and seminal plasma from the sperm. The sperm pellets were resuspended in 900 µl PBS, transferred into 1.5-ml tubes, and centrifuged at 10 000 x g for 10 min. The supernatants were discarded and the sperm pellets were immediately stored at –20°C until used for protein or lipid extraction. As controls, permeate, milk, and permeate supplemented with caseins were subjected to the same washing procedure, to determine if any proteins from the milk were pelleted during centrifugation.

Preparation of Protein Extracts from Sperm Membranes

The proteins were extracted from the sperm membranes in the presence of proteases, as described previously [31], and stored at –20°C until used for sperm protein analysis.

Determination of Protein Concentration

The protein contents of the seminal plasma or sperm extracts were determined by the modified Lowry procedure [32].

Analyses of Proteins by SDS-PAGE and Immunoblotting

Sperm protein samples were reduced, denatured, and separated in 15% polyacrylamide gels. For immunoblotting, the proteins in the gel were transferred to an Immobilon-P membrane, as described by Towbin et al. [33], and immunodetection was performed using polyclonal antibodies directed against each BSP protein [31, 34], polyclonal antibodies against bovine milk caseins, and antiserum directed against bovine milk whey proteins. The intensity of the bands obtained after immunoblotting were quantified by densitometry using the Alphalmager 2200 ver. 5.5 software (Alpha Innotech, San Leandro, CA).

Quantification of BSP Proteins by Radioimmunoassay

Iodination of the BSP proteins was performed by the lactoperoxidase method, as described previously [35]. Radioimmunoassays (RIAs) for each BSP protein were performed as described by Nauc and Manjunath [31]. Briefly, the assay tubes that contained the I125-labeled and unlabeled antigen, the primary antibodies (anti-BSP-A1/-A2, anti-BSP-A3 or anti-BSP-30-kDa protein) and normal rabbit serum (1.5% [v/v]) were incubated at 37°C. After 20 h, 50 µl of 10% goat anti-RGG were added and the assay tubes were incubated for 16 h. Then, 500 µl of 10% polyethylene glycol were added and the antibody-antigen complex was separated by centrifugation (2200 x g for 20 min). The radioactivity associated with the pellet was measured in the 1470 Wallac Wizard gamma counter (Perkin Elmer). The intraassay variation, assessed as the coefficient of variation of triplicate assay samples, was less than 8% for BSP-A1/A2 and less than 10% for both the BSP-A3 and BSP-30-kDa proteins. The interassay variation was within 12% for all three proteins.

Measurements of Sperm Cholesterol and Choline Phospholipids

Sperm lipids were extracted from pellets kept at –20°C using a chloroform:methanol mixture, as described previously [36]. After solvent evaporation under N2, the lipids were resuspended in isopropanol. The amounts of cholesterol and choline phospholipids were determined following the protocols described in the cholesterol determination kit (Boehringer Mannheim, Mannheim, Germany) and the phospholipid B determination kit (Wako Chemicals GmbH, Neuss, Germany), respectively.

Sperm Function Analysis

The sperm motility for each sample was assessed by estimating the percentage of motile sperm in Tris-citrate buffer (200 mM Tris, 73 mM citric acid) on a warm slide using light microscopy. Viability was assessed by staining the sperm according to the protocol of Dott and Foster [37]. Acrosomal integrity was assessed by determining the percentage of acrosome-intact sperm on air-dried sperm smears stained according to the naphtol yellow-erythrosin B staining procedure [38].

Data Analysis

The GLM procedure was used to test the effect of incubation time on different variables, such as sperm functions and RIAs. Sperm cholesterol and phospholipid losses or gains after 4 h of incubation were analyzed for significant differences by ANOVA. Significant differences among treatments were determined using the protected Fisher least-significant difference (LSD) test within each incubation time. A value of P < 0.05 was considered to be statistically significant.

RESULTS

Effect of Casein Micelles on Bull Sperm Functions During Cold Storage

Sperm acrosomes remained intact during the storage of semen in all three extenders (milk, permeate, permeate supplemented with caseins) (Table 1). The percentages of sperm viability and motility did not change significantly during storage at 4°C when the semen was diluted with milk or milk permeate supplemented with caseins. However, after 4 h of storage, the percentage motility and percentage viability were lower in semen diluted with permeate (P < 0.05).


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  		TABLE 1. Effect of milk, caseins, and permeate on sperm functions during 4 h storage at 4°C.

SDS-PAGE Patterns of Sperm Proteins

SDS-PAGE (Fig. 1) was performed to investigate whether modifications occurred in the sperm protein patterns during 4 h of storage of semen at 4°C. There were no differences in the protein patterns of sperm diluted with permeate (Fig. 1, lanes 2–4), milk (lanes 5–7) or permeate supplemented with caseins (lanes 8–10), with the exception of the intensities of the protein bands at 15–16.5 kDa (corresponding to the molecular masses of the BSP-A1/-A2 and BSP-A3 proteins), which were higher in the sperm extract from semen diluted with permeate alone.


Figure 01
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  		FIG. 1. SDS-PAGE patterns of sperm proteins isolated from semen diluted with permeate, milk or permeate supplemented with caseins and stored at 4°C. Aliquots of sperm proteins (15 µg) that correspond to different incubation times (0, 2, and 4 h) in semen diluted with permeate (lanes 2–4), milk (lanes 5–7) or permeate supplemented with caseins (lane 8–10) were denatured, separated on a 15% polyacrylamide gel, and stained with Coomassie blue R-250 dye. Lane 1, low-molecular-weight standard; lane 11, cBSP (5 µg); lane 12, milk proteins (15 µg). The experiment was performed on sperm from four ejaculates; a representative experiment is shown.

Immunoblotting of BSP Proteins in Sperm Extracts

In order to confirm the SDS-PAGE results, sperm protein extracts from each incubation time (0, 2, and 4 h of storage at 4°C) were subjected to immunoblotting using antibodies directed against the BSP-A1/-A2, BSP-A3, and BSP-30-kDa proteins. In semen diluted with milk permeate, the intensities of the bands corresponding to BSP-A1/-A2 and BSP-A3 (Fig. 2, A and B) at each incubation time (0, 2, and 4 h) were 1.4–1.8-times higher than in semen diluted with milk (lanes 4–6) or permeate supplemented with caseins (lanes 7–9). Immunoblotting with antibodies against the BSP-30-kDa protein (Fig. 2C) revealed that the intensity of the band that corresponded to BSP-30-kDa protein was 1.4–1.9-times higher in sperm proteins from semen diluted with milk (lanes 4–6) or permeate supplemented with caseins (lanes 7–9) than from semen diluted with permeate (lanes 1–3). Antibodies directed against the BSP-30-kDa protein also detected a protein band of 14 kDa. The intensity of the 14-kDa immunoreactive band increased in a time-dependent manner when the semen was diluted with permeate (lanes 1–3, corresponding to 0, 2, and 4 h of storage, respectively). Furthermore, the intensity of the 14-kDa band was higher in sperm protein extracts from semen diluted with permeate (lanes 1–3) than in those from semen diluted with either milk (lanes 4–6) or permeate supplemented with caseins (lanes 7–9).


Figure 02
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  		FIG. 2. Immunoblot analysis of BSP proteins associated with sperm during storage at 4°C in permeate, milk or milk permeate supplemented with caseins. Aliquots of sperm protein samples (0.5 µg) that correspond to different incubation times (0, 2, and 4 h) in semen diluted with permeate (lanes 1–3), milk (lanes 4–6) or permeate supplemented with caseins (lanes 7–9) were separated by SDS-PAGE. The separated proteins were transferred to Immobilon-P membranes and probed with purified antibodies directed against the BSP-A1/-A2 (A), BSP-A3 (B), and BSP-30-kDa (C) proteins. In A, B, and C, lane 10 contains cBSP (25, 75, and 125 ng, respectively). The experiment was performed on sperm from four ejaculates; a representative experiment is shown.

Amounts of BSP Proteins in Sperm During Storage of Semen

To confirm the immunoblot results, the concentrations of BSP proteins in sperm protein extracts were assessed by RIAs. At the start of incubation (0 h), a decrease in the quantity of BSP-A1/-A2 proteins associated with sperm was evident in semen diluted with milk (10.3 ± 0.4 ng/106 sperm) or permeate supplemented with caseins (8.3 ± 0.9 ng/106 sperm), as compared to semen diluted with permeate (15.8 ± 3.7 ng/106 sperm) (Fig. 3A). In addition, after 4 h of storage, the quantity of sperm-bound BSP-A3 was slightly higher for sperm diluted with permeate (6.7 ± 0.4 ng/106 sperm) than for sperm diluted with either milk (5.1 ± 0.1 ng/106 sperm) or permeate supplemented with caseins (5.1 ± 0.5 ng/106 sperm) (Fig. 3B). However, after 4 h of incubation, no significant differences were detected between the quantities of sperm-bound BSP-30-kDa protein in semen diluted with permeate, milk or permeate supplemented with caseins (Fig. 3C).


Figure 03
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  		FIG. 3. Amounts of BSP proteins associated with sperm during storage at 4°C in milk, permeate or permeate supplemented with caseins. Each BSP protein was quantified by RIA in sperm protein extracts of semen stored for 4 h with milk (solid squares), permeate (solid circles) or permeate supplemented with caseins (solid triangles). A) BSP-A1/-A2 (extender effect; P < 0.001). B) BSP-A3 (extender effect; P < 0.05). C) BSP-30-kDa protein. The results shown represent the mean ± SEM of four ejaculates performed in triplicate. Different letters within a time-point denote significant differences (P < 0.05).

Analyses of Sperm Cholesterol and Choline Phospholipids

The average concentrations of cholesterol and choline phospholipid associated with sperm at the start of the incubation were 202.7 ± 5.1 and 901.2 ± 49.0 µg/109 sperm, respectively. Immediately after dilution of the semen with milk, permeate or permeate supplemented with caseins (0 h), no differences were observed in the amounts of cholesterol and choline phospholipids associated with the sperm. As shown in Table 2, losses of sperm cholesterol and choline phospholipids were observed during the 4-h storage of semen diluted with permeate, milk or permeate supplemented with caseins. However the losses of cholesterol and choline phospholipids were ~3-fold higher in milk permeate (8.9 ± 1.0% and 12.0 ± 2.6%, respectively) than for semen diluted with milk (4.9 ± 1.7% and 3.6 ± 1.8%, respectively) or permeate supplemented with caseins (3.9 ± 1.1% or 3.5 ± 1.8%, respectively) (P < 0.05).


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  		TABLE 2. Effect of milk, caseins, and permeate on sperm lipid loss after 4 h storage at 4°C.

Immunoblotting of Milk Proteins in Sperm Protein Extracts

In order to determine whether milk proteins bind to sperm during sperm storage, we subjected sperm protein extracts from each incubation time (0, 2, and 4 h) to immunoblotting using antiserum directed against bovine milk whey proteins (Fig. 4A) and antibodies directed against bovine milk caseins (Fig. 4B). Neither whey proteins nor caseins were detected in the sperm protein extracts.


Figure 04
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  		FIG. 4. Immunoblot analysis of milk proteins associated with sperm during storage of semen at 4°C. Aliquots of sperm protein samples (2 µg) that correspond to different incubation times (0, 2, and 4 h) in semen diluted with permeate (lanes 2–4), milk (lanes 5–7) or permeate supplemented with caseins (line 8–10) were separated by SDS-PAGE. The separated proteins were transferred to Immobilon-P membranes and probed with (A) antiserum directed against the whey proteins of bovine milk and (B) purified antibodies directed against bovine milk caseins. Lane 1, protein extracts of undiluted fresh sperm (2 µg); lane 11, TCA-precipitated permeate proteins (100 ng); lane 12, TCA-precipitated milk proteins (500 ng). The experiment was performed on four ejaculates; a representative experiment is shown.

DISCUSSION

The present study shows that casein micelles present in skimmed milk are responsible for sperm protection during storage of bull semen. The protective action of milk on sperm is analogous to the protective action of egg yolk, which is widely used as an extender in the storage of bull semen. As in the case of egg yolk, skimmed milk prevents the binding of BSP proteins to sperm and reduces sperm lipid loss, while maintaining sperm motility and viability during storage. Interestingly, while sperm protection by egg yolk involves the sequestration of BSP proteins by the LDLs present in egg yolk (i.e., BSP protein-lipoprotein interaction), the protection afforded by skimmed milk does not involve the participation of lipids or lipoproteins. It is the casein micelles present in skimmed milk that sequester the BSP proteins. Thus, sperm protection by milk may involve a BSP protein-casein micelle (protein-protein) interaction. The nature of this interaction is under investigation.

Despite the fact that BSP proteins are detrimental for sperm storage because they stimulate continuous lipid removal from the sperm membrane, BSP proteins play a positive role in sperm fertility [19]. In fact, high levels of BSP proteins in seminal plasma are correlated with increased fertility [39, 40]. In addition, BSP proteins are involved in the stimulation of sperm capacitation [41] and in sperm binding to the oviduct [42–44]. Therefore, the severe depletion of BSP proteins would be deleterious for sperm fertility. It appears that egg yolk and milk are efficient protective agents for sperm because they allow the sequestration of excess BSP proteins present in the seminal plasma, which would otherwise be deleterious for the sperm membranes, while retaining some BSP proteins bound to sperm, which should be beneficial for fertility.

In the present study, the prevention of BSP binding to sperm was probably not due to the binding of milk proteins to sperm during storage, since no milk proteins were detected in the sperm protein extracts (Fig. 4). An immunofluorescence study also failed to detect any binding of caseins to stallion sperm during storage [11]. Therefore, the prevention of BSP binding to sperm in milk may be due to an interaction between BSP proteins and casein micelles.

In the present study, milk caseins did not prevent the binding of BSP-30-kDa protein to sperm as determined by RIAs (Fig. 3). Immunodetection with antibodies directed against the BSP-30-kDa protein revealed the presence of a 14-kDa immunoreactive protein in the sperm protein extracts (Fig. 2C). The intensity of the band that corresponded to this 14-kDa protein increased in a time-dependent manner when the semen was diluted with permeate. However, the intensity of this band was higher when the semen was diluted with permeate, as compared to semen diluted with milk or permeate supplemented with caseins. Concomitantly, the intensity of the band that corresponded to the BSP-30-kDa protein was higher in sperm extracts from semen diluted with milk or permeate supplemented with caseins as compared to sperm extracts from semen diluted in permeate (Fig. 2C). The 14-kDa immunoreactive protein may correspond to a proteolytic fragment of the BSP-30-kDa protein. Therefore, it is possible that milk caseins prevent the proteolysis of the BSP-30-kDa protein on the sperm surface, which may be beneficial for sperm fertility after storage.

It is possible that the protective effect of casein micelles on sperm functions observed in the present study is due to the presence of proteins (27 g/L) rather than the casein micelles. However, in the context of sperm storage in milk, as used in artificial insemination centers, it has been clearly established that sperm protection is afforded by casein micelles. In fact, a standard reference extender (containing no whey proteins) [6, 10] or milk permeate (present study) supplemented with milk caseins (concentration found in milk) is as efficient as milk in preserving sperm functions. Therefore, it is most likely that permeate constituents (whey proteins, salt or lactose) are not essential for sperm protection during storage in milk despite the fact that they can be beneficial for sperm storage [6, 10].

When semen was diluted with permeate (absence of caseins), sperm lost ~10% of their lipids after 4 h storage (Table 2). In a previous study, it was shown that when diluted in extender devoid of egg yolk, sperm lost ~15% of their lipids after 4 h storage at 4°C [17]. This difference in the amount of lipid loss could be due to the concentration of seminal plasma in the diluted semen. The removal of lipids from the sperm membrane by seminal plasma (or BSP proteins) is concentration dependent [41]. In the present study, the concentration of seminal plasma in the diluted semen was lower (2.5–3.4%, v/v) as compared to the previous study (4.7–10%, v/v) [17].

It is remarkable that skimmed milk is also used to store semen from other mammals such as ram (reviewed in [45]), goat (reviewed in [46]), stallion (reviewed in [47]), buffalo (reviewed in [48]) and dog [49]. In addition, it has been shown that casein micelles isolated from milk can also protect sperm from stallion, goat and ram during storage [6, 9, 10, 12, 13]. Interestingly, proteins that are deleterious to sperm storage (BSP protein homologs that stimulate lipid loss from the sperm membrane) are also present in the seminal plasma of those species (reviewed in [19]). Therefore, it may be possible that the mechanism of sperm protection by milk caseins could be similar in these species.

In summary, as observed for egg yolk, which is frequently a component of extenders used to store bull semen, caseins from milk decreased the binding of BSP proteins to sperm and minimized lipid loss from the sperm membranes during storage. This may reflect a major mechanism for sperm protection by milk during storage. Interestingly, unlike egg yolk, the sperm-protective effect of milk involves proteins rather than lipids. We hope that the present findings will facilitate the development of novel extenders for the storage of semen from mammals.

ACKNOWLEDGMENTS

We thank Ms. Jasmine Lefebvre for proofreading the manuscript.

FOOTNOTES

1Supported by a grant from the Natural Science and Engineering Council of Canada. Back

Correspondence: 2Puttaswamy Manjunath, Centre de Recherche Guy-Bernier, Hôpital Maisonneuve-Rosemont, 5415 boul. l'Assomption, Montréal, Québec, Canada, H1T 2M4. FAX: 514 252 3430; e-mail: puttaswamy.manjunath{at}umontreal.ca

Received: 21 October 2006.

First decision: 22 December 2006.

Accepted: 6 April 2007.

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