Effect of fatty acids bound to bovine serum albumin‐V on acrosome reaction and utilization of glucose in boar spermatozoa

Abstract

Aim:  The present study has been designed with the objective of determining if fatty acids bound to bovine serum albumin‐V (BSA‐V) can improve motility, viability, and increase acrosome reaction (AR) and utilization of glucose in boar spermatozoa.

Methods:  Boar spermatozoa were washed, swum‐up and incubated at 37°C for 6 h in TALP medium supplemented with fatty acids bound to bovine serum albumin fraction V (BSA‐V), fatty acid free BSA (BSA‐FAF), polyvinyl alcohol + main fatty acids bound to BSA‐V (PVA + FA) and PVA. Sperm motility, viability, AR, and the incorporation and oxidation of ¹⁴C‐glucose were evaluated during 6 h of incubation.

Results:  The results show that the BSA‐V was superior to BSA‐FAF and PVA in improving motility and AR. Viability was significantly increased (P < 0.05) by only BSA‐V compared with PVA. When the main fatty acids compound of BSA‐V were added to PVA, the sperm motility, viability and AR became almost the same as with BSA‐V. The rate of incorporation and oxidation of ¹⁴C‐glucose were significantly increased (P < 0.05) by BSA‐V compared with BSA‐FAF and PVA. Fatty acids bound to BSA‐V are important for improvement of sperm functions.

Conclusions:  The present study postulates that fatty acids bound to BSA‐V are important to acrosome reaction and the utilization of glucose in boar spermatozoa.

INTRODUCTION

CULTURE MEDIA HAVE a great impact on the results obtained from reproductive techniques such as in vitro fertilization (IVF) in domestic animals. Sperm motility, viability and acrosome reaction (AR) depend strongly on the media used during the process of in vitro manipulation. Improvements in the culture systems that are most commonly used have relied on macromolecules, such as bovine serum albumin (BSA), or complex biological fluids, such as serum in the culture medium, for the provision of components essential for development. However, these macromolecules are inconsistent in their composition and their ability to support development in vitro.

Most sperm incubation media contain BSA for supporting sperm motility, viability and AR. ¹ Bovine serum albumin maintains the osmolality of media, and a variety of molecules, such as fatty acids, lipids, steroids, amino acids and growth factor bound to BSA, are released from it and stimulate sperm functions. BSA preparations can contain up to 2–3 mol of fatty acids/mol of protein, ² which could represent the introduction of as much as 15 µg of fatty acid/mg of BSA. ³ Concentration of bovine serum albumin fraction V (BSA‐V) used in culture media usually ranges from about 1–30 mg/mL or more, which may carry a large amount of fatty acids. Spermatozoa can use these fatty acids as a source of energy. ⁴ , ⁵ BSA serves as a highly efficient fatty acids delivery system, making a distinct contribution in inducing acrosome reaction of guinea pig spermatozoa. ⁶ Therefore, the beneficial effects of BSA to culture medium on sperm motility, viability and AR are presumably at least partly due to those of the fatty acids.

However, it is not clear what role the fatty acids bound to BSA play in inducing AR of boar spermatozoa. Thus, the present study has been undertaken to investigate the effect of the fatty acids bound to BSA on boar sperm motility, viability, AR and utilization of glucose.

MATERIALS AND METHODS

Chemicals

RADIOACTIVE GLUCOSE WAS purchased from American Radiolabeled Chemicals Inc (St Louis, MO, USA). Fatty acids bound to BSA (BSA‐V), fatty acid free BSA (BSA‐FAF) and polyvinyl alcohol (PVA) came from Sigma Chemical Company (St Louis, MO, USA). The purity of BSA was verified by sodium dodecyl sulfate‐agarose gel electrophoresis (SDS‐AGE). The purity of BSA‐V was ≥98% (SDS‐AGE) and BSA‐FAF was ≥99% (SDS‐AGE). All other chemicals were analytical grade and purchased from Nacalai Tesque (Kyoto, Japan).

Culture medium

Boar spermatozoa were prepared in a basic TALP medium. ⁷ According to the experimental design, the basic medium was supplemented with BSA‐V and BSA‐FAF as 0.3 g/100 mL and PVA as 0.1 g/100 mL concentration. To find out the reason for the excellent performance of BSA‐V, fatty acids of BSA‐V were mixed together then added with PVA and compared with BSA‐V for sperm functions. Fatty acids mixture (FA) was prepared on the basis of the fatty acids binding to BSA ⁸ and the percentage of fatty acids (myristic 0.030 mg, palmitic 0.066 mg, palmitoleic 0.028 mg, stearic 0.069 mg, oleic 0.111 mg, linoleic 0.025 mg, arachidonic 0.156 mg, docosahexaenoic 0.051 mg and lignoceric 0.096 mg/100 mL) present in BSA‐V. ⁹ A total of 0.64 mg fatty acids mixture was added per 100 mL medium.

Semen collection and preparation

Semen from Duroc boars aged between 2 and 3 years was collected by the gloved‐hand technique at Nagano Animal Industry Experiment Station, Nagano, Japan. The semen was diluted according to the method of Johnson et al., ¹⁰ with the Modena extender giving a sperm concentration of 1 × 10⁸/mL at room temperature. The vial of semen was carried to the laboratory within 30 min using a cork box to keep it at 21°C temperature. The upper most 10 mL of the semen was removed from the vial and incubated for 10 min in a water bath at 37°C temperature for anabiosis. Then, 3 ml of the anabiosed sperm sample was washed twice with TALP medium by centrifugation at 900 × g for 5 min. The sperm pellet was suspended in TALP medium and pre‐incubated at 37°C, 95% humidified air and 5% CO₂ for 1 h to swim‐up. A 50 µL of sperm aliquots obtained from swim‐up separation was resuspended to the previously prepared drops with the specified medium to give a final concentration of spermatozoa 5 × 10⁶/mL. It was then incubated at 37°C, 95% humidified air and 5% CO₂ for 0–6 h to evaluate the sperm motility. To evaluate the acrosome status, swim‐up spermatozoa were resuspended separately at a concentration of 20 × 10⁶/mL and incubated at 37°C for 0–6 h in the same conditions.

Progressive motility

After completion of the specified incubation periods, three subsamples of each sample were placed on warm glass slides for motility and movement quality evaluations. Slides were warmed at 37°C by using a stage warmer set (MPF‐10‐SZX, Kitazato supply, Tokyo, Japan). The slides were then examined under a light microscope (×400). Sperm motility was assessed by determining the percentage of spermatozoa that showed the following four categories of movement: (a) rapid progressive motility; (b) slow or sluggish progressive motility; (c) non‐progressive motility; and (d) immotility. It was then expressed as a progressive motility percentage. The percentage of spermatozoa with progressive motility was determined subjectively by scoring 400 individual spermatozoa in each sample.

Acrosome reaction and viability

To evaluate the AR and viability, spermatozoa were stained by applying the triple staining technique. ¹¹ Namely; dead spermatozoa were stained with trypan blue at the post‐acrosomal region, whereas live spermatozoa were not stained with this dye but were stained with Bismark brown. The acrosome was stained by Rose Bengal. The triple stained slides were examined by light microscopy under oil immersion (×1000). Viability and acrosome reaction of the spermatozoa were evaluated from randomly selected fields of the triple stained slides until 400 spermatozoa had been examined. Therefore, based on the staining patterns, the spermatozoa might be categorized according to the following characteristics: (a) dead and acrosome unreacted (Fig. 1a); (b) live and acrosome unreacted (Fig. 1b); (c) dead and acrosome reacted (Fig. 1c); and (d) live and acrosome reacted or lost (Fig. 1d).

Figure 1.

Triple stained boar spermatozoa with different acrosome status. (a) Dead spermatozoa which had not undergone acrosome reaction (AR). (b) Live spermatozoa which had not undergone AR. (c) Dead spermatozoa which had undergone AR. (d) Live spermatozoa which had undergone AR.

Incorporation and oxidation of ¹⁴C‐glucose

To assess the metabolic activity of spermatozoa treated with fatty acids bound to BSA‐V by ¹⁴C‐glucose incorporation and oxidation, semen samples were prepared as above for motility evaluation. A 100 µL sample of spermatozoa (5 × 10⁶/mL) from each treatment was incubated with 18.5 kBq/mL (specific activity 370 MBq/mmol) of ¹⁴C‐glucose and prepared for scintillation counting according to the method described by Miah et al. ¹² The incorporation and oxidation of ¹⁴C‐glucose by spermatozoa was determined by a liquid scintillation counter (LS‐6500, Beckman Instruments, Fullerton, CA, USA). The value of incorporation and oxidation was expressed directly by counts per minute (cpm).

Statistical analysis

For these experiments, data were subjected to the protected Fisher's least significant difference test. The ncss (Number Crunchier Statistical System; NCSS Statistical Software, Kaysville, UT, USA) Version 5.01 computer software package was used for all statistical analysis. Each experiment was repeated four times and differences were considered significant for P < 0.05.

RESULTS

EFFECTS OF THE BSA‐V, BSA‐FAF and PVA on progressive motility, viability and AR in boar spermatozoa during 0–6 h of incubation are shown in Figure 2. BSA‐V performed better for progressive motility and AR. The percentage of progressive motility and AR were observed to be significantly higher (P < 0.05) in BSA‐V treated spermatozoa compared with BSA‐FAF from 1 to 4 h of incubation, and compared with PVA from almost all incubation periods. Compared with PVA, viability was only significantly improved (P < 0.05) by BSA‐V, not by BSA‐FAF during 1–4 h of incubation.

Figure 2.

Effect of bovine serum albumin fraction V (BSA‐V), fatty acid free BSA (BSA‐FAF) and polyvinyl alcohol (PVA) on the percentage of (a) progressive motility, (b) viability and (c) acrosome reacted live spermatozoa during 0–6 h of incubation. (▴) BSA‐V; (▪) BSA‐FAF; (◆) PVA. This observation started after 1 h pre‐incubation. Each line with an error bar represents the mean ± SEM from four replicates. Data points without a common lower case letter indicate differences (P < 0.05) among the treatments.

Effects of BSA‐V, PVA + FA and PVA on motility, viability and AR of boar spermatozoa are compared in Figure 3. Compared with PVA, there was no significant difference between BSA‐V and PVA + FA treated spermatozoa with regard to improving progressive motility, viability and increasing AR.

Figure 3.

Effect of bovine serum albumin fraction V (BSA‐V), polyvinyl alcohol + main fatty acids bound to BSA‐V (PVA + FA) and PVA on the percentage of (a) progressive motility, (b) viability and (c) acrosome reacted live spermatozoa at 2 and 3 h of incubation. This observation started after 1 h pre‐incubation. Each line with an error bar represents the mean ± SEM from four replicates. Data points without a common lower case letter indicate differences (P < 0.05) among the treatments.

The rates of incorporation and oxidation of ¹⁴C‐glucose by spermatozoa is shown in Figure 4. The incorporation rate of ¹⁴C‐glucose was higher (P < 0.05) in the BSA‐V treated spermatozoa than in the BSA‐FAF and PVA from 1 to 4 h of incubation. The trend of ¹⁴C‐glucose oxidation was also the same as incorporation; where performance of BSA‐V was greater (P < 0.05) than BSA‐FAF and PVA from 1 to 4 h incubation periods. The incorporation and oxidation rate of ¹⁴C‐glucose in the spermatozoa treated by BSA‐V, BSA‐FAF and PVA increased up to 3 h of incubation and then decreased up to 6 h of the incubation period.

Figure 4.

Effect of bovine serum albumin fraction V (BSA‐V), fatty acid free BSA (BSA‐FAF) and polyvinyl alcohol (PVA) on (a) incorporation and (b) oxidation of ¹⁴C‐glucose in pre‐incubated boar spermatozoa for 0–6 h. (▴) BSA‐V; (▪) BSA‐FAF; (◆) PVA. This observation started after 1 h pre‐incubation. Each line with an error bar represents the mean ± SEM from four replicates. Data points without a common lower case letter indicate differences (P < 0.05) among the treatments.

DISCUSSION

IN THE PRESENT study, motility was increased by fatty acids bound to BSA‐V compared with fatty acid free BSA or PVA. This study showed that fatty acids play an important role in improving sperm motility. Motility of rabbit ¹ and hamster ¹³ spermatozoa was improved by fatty acids bound to BSA; boosting of motility might have been the result of supplying sufficient energy from fatty acids bound to BSA. The viability was also improved by fatty acids bound to BSA‐V. Lipids bound to BSA improved boar sperm viability during cooling and after cryopreservation. ¹⁴ In the present study, BSA‐V increased sperm motility, viability and acrosome reaction more than the BSA‐FAF and PVA. Sperm functions were also stimulated by PVA but not greatly in a phenomenon observed by Bavister. ¹⁵ From the first experiment it appears that fatty acids of BSA‐V significantly improved sperm functions, but this BSA‐V might have contained some contaminants. To avoid the risk of this inconvenience, main fatty acid compounds of BSA‐V were added to PVA and observed for sperm functions; it was found that motility, viability and AR were again improved by PVA + FA almost as much as with BSA‐V. The main fatty acids of BSA‐V were oleic, linoleic, palmitic and arachidonic acid, ⁹ which have important roles in improving sperm motility, viability and inducing AR.

During mammalian fertilization, spermatozoa undergo physiological and functional modifications that are known as capacitation. ¹⁶ After capacitation, spermatozoa undergo an exocytotic process called AR; ¹⁷ multiple fusions occur between the plasma membrane and the outer acrosomal membrane. It has been known for some years that free or esterified fatty acids added to media can induce fusion of ordinary cells, but the effect of fatty acids on sperm cell fusion is still a matter of controversy. In the present study, at the onset of incubation, the improvement of AR by fatty acids bound to BSA‐V was very mild but it increased significantly up to 3 h of incubation. Compared with PVA, 1.6‐fold AR of live spermatozoa was increased by fatty acids bound to BSA‐V and 1.2‐fold by fatty acids free BSA at 3 h of incubation. As spermatozoa gradually dies, from 3 h upward there was a decline in AR. In the present study, fatty acids bound to BSA‐V triggered higher induction of AR compared with fatty acids free BSA and PVA in all incubation periods. When fatty acids were added to PVA, AR was almost the same as BSA‐V, whereas PVA without fatty acid failed in both experiments. Thus, the results of the second experiment showed that the additional effect of BSA‐V on inducing AR is a result of the presence of fatty acids in BSA‐V. Stewart ¹⁸ found that capacitation in golden hamster spermatozoa was increased more in the presence of fatty acids bound to BSA than the fatty acid free BSA. Creutz ¹⁹ reported that exogenous unsaturated fatty acids promoted the fusion of chromaffin granules, which are associated with AR. Peter ²⁰ and Meizel ²¹ suggested that a variety of molecules, such as fatty acids and amines, are associated with BSA which possibly act on the sperm plasma membrane and trigger AR. In contrast, oleic, arachidonic and docosahexanoic acid triggered AR in hamster spermatozoa. ²² High amounts of fatty acids in mammalian sperm plasma membrane play a major role in membrane fusions and increasing AR. ²³ The findings of these studies are consistent with the results of the present study, showing that fatty acids of BSA‐V provided an adequate environment for boar sperm AR. Thus, the present study is the first report on the effect of fatty acids bound to BSA‐V on AR in boar spermatozoa.

The present study shows that fatty acids bound to BSA‐V stimulate the rate of incorporation and oxidation of ¹⁴C‐glucose in boar spermatozoa in almost all incubation periods. Although, under normal physiological circumstances, oxidation of glucose has an inverse correlation with oxidation of fatty acid. Wolfe ²⁴ stated that oxidation of glucose increases oxidation of fatty acids. The trend of incorporation and oxidation of glucose being the same as AR is supported by the findings of Miah et al. ¹² and suggests that incorporation and oxidation of glucose increased up to 3 h for more ATP for AR at that time. Tsujii et al. ²⁵ also reported that fructose stimulated induction of the AR in boar spermatozoa. Mayes ²⁶ reported that fatty acids which are bound to BSA have metabolic significance in sperm cells and most of the long chain fatty acids derived from these fatty acids by chain elongation. The function of fatty acid oxidation is, of course, to generate metabolic energy. ²⁷ Octanoate metabolism indicates that inside the mitochondria, there is a stimulatory effect of glucose on β‐oxidation of fatty acids. ²⁴ Each round of β‐oxidation produces one NADH, one FADH₂, and one acetyl‐CoA. Oxidation of acetyl‐CoA through the citric acid cycle generates additional FADH₂ and NADH, which are reorganized through oxidative phosphorylation to form ATP. Complete oxidation of a fatty acid molecule is, therefore, a highly exergonic process which yields numerous ATP. ²⁷ However, ATP is necessary for sperm motility. Boar sperm has a complex system to optimize its ATP levels through a sophisticated regulation of its energy fluxes. ²⁸ It is noteworthy that the control of boar sperm pyruvate kinase and LDH is greatly dependent on the intracellular ATP/ADP ratio. ²⁹ , ³⁰ This seems to show that one of the most important mechanisms by which boar sperm can simultaneously control separate metabolic pathways is through the regulation of its intracellular ATP levels and ATP/ADP ratio, which directly modulates the consumption of separate energy substrates. ²⁸ Obtaining energy substrates could allow boar sperm to increase their survival rate and metabolic strategy that might play an important role in improving sperm functions. The relevance of such metabolic activity in spermatozoa is not yet firmly established. In contrast, Neill ³¹ reported that metabolism of fatty acid is correlated with bovine sperm motility, but we speculate that such metabolism is not only associated with motility but also with acrosome reaction.

However, intracellular signaling cascades induced by fatty acids bound to BSA‐V in the stimulation of AR are still not clearly understood. We suggest that fatty acids present in BSA‐V supplemented the sperm incubation medium thought to increase intracellular Ca²⁺ and cAMP concentration, which are responsible for inducing AR. In another experiment, Tsai ³² found that high‐lipid BSA induces more Ca²⁺ influx in human osteoblast‐like cells than the low lipid BSA. Dominguez ³³ stated that arachidonic acid induces acrosome reaction in human spermatozoa by increasing Ca²⁺ influx. The increase in protein tyrosine phosphorylation during AR is regulated by increasing intracellular cAMP and Ca²⁺ influx. ³⁴ Thus, the effect of individual fatty acids of BSA‐V on sperm functions and intracellular concentration of cAMP, Ca²⁺ influx and protein tyrosine phosphorylation by fatty acids in boar spermatozoa needs to be studied further for a complete understanding of the mechanism by which fatty acids bound to BSA improve motility and induce AR. Fatty acid content of BSA‐V can enhance sperm motility and AR. Fatty acids bound to BSA‐V are able to access the spermatozoa easily, be absorbed by them and are highly activated for sperm metabolism. In conclusion, fatty acids bound to BSA‐V are more capable than fatty acid free BSA to improve AR and enhance the utilization of glucose in boar spermatozoa.