Effect of relaxin on motility, acrosome reaction and viability of cryopreserved boar spermatozoa

Abstract

Background and Aims:   Relaxin has an important role in stimulating motility and the acrosome reaction (AR) of fresh boar spermatozoa. The objective of the present study was to determine whether relaxin can improve the motility, AR and viability of cryopreserved boar spermatozoa.

Methods:   Cryopreserved boar spermatozoa were thawed, washed and incubated at 37°C for 4 h in modified Beltsville thawing solution supplemented with 0, 20 or 40 ng/mL relaxin. Sperm motility, AR, viability, and incorporation and oxidation of ¹⁴C‐glucose were evaluated during 0–4 h of incubation.

Results:  The results show that the supplementation of relaxin (especially at 20 ng/mL) in the thawing solution improved sperm motility significantly (P < 0.05) at 1–3 h of incubation. The percentage of acrosome reacted live spermatozoa was improved significantly (P < 0.05) when the spermatozoa were treated with 20 ng/mL relaxin. Viability was not significantly (P > 0.05) improved by supplementation with relaxin. The rates of incorporation and oxidation of ¹⁴C‐glucose were increased in correlation with AR up to 4 h of incubation.

Conclusion:  We conclude that relaxin can improve the sperm motility and AR, and enhance the glucose metabolism of cryopreserved boar spermatozoa. (Reprod Med Biol 2006; 5: 215–220)

INTRODUCTION

DESPITE THE POTENTIAL applications of cryopreserved spermatozoa in the animal breeding industry, in vitro fertilization (IVF) or artificial insemination (AI) techniques with cryopreserved spermatozoa are not widely used in the pig breeding industry. The main reasons are the lower rate of IVF, lower farrowing rate and smaller litter size obtained after using cryopreserved semen compared with fresh semen. ¹ When carrying out IVF techniques and during the evaluation of spermatozoa, sperm washing is a routine stage, during which spermatozoa loses its physiological activity as the result of the lack of essential components naturally present in seminal plasma. ² Thus, the main problems in sperm washing or cryopreservation are the low recovery of sperm activities and a resultant low fertilization rate after AI or IVF.

Relaxin is an important component of seminal plasma that is present in fresh boar semen at 2.35 ng/mL. ³ We observed in a previous study that relaxin has physiological roles in influencing motility and the acrosome reaction (AR) of fresh boar spermatozoa. ⁴ Relaxin also increases the fertilization rate of mouse ⁵ and porcine oocytes ⁶ in vitro and enhances the penetration capability of porcine as well as human spermatozoa. ⁷ However, relaxin is adversely affected when semen is washed and or cryopreserved, ⁸ which might lead to lower sperm activities. ⁹ , ¹⁰ Currently, there is no report on the effect of relaxin on cryopreserved boar spermatozoa. The present study has been undertaken with the objective of determining whether relaxin supplementation can improve the motility, AR and viability of cryopreserved boar spermatozoa.

MATERIALS AND METHODS

Semen processing and cryopreservation

EJACULATES FROM FOUR Duroc boars aged between 2 and 3 years, having proven fertility used for artificial insemination, were collected by the gloved‐hand technique at Nagano Animal Industry Experiment Station, Nagano, Japan. After collection, semen characteristics (total sperm number per ejaculate, subjective sperm motility and normal morphology) were evaluated by microscope, using standard laboratory techniques. ¹¹ Only ejaculates with more than 75% motile spermatozoa and more than 80% normal acrosome were cryopreserved using the straw‐freezing procedure described by Zeng et al. ¹² Briefly, the fresh semen was centrifuged at 700 × g for 5 min to remove the seminal plasma. The sperm sediments were resuspended with modified Hulsenberg VIII diluents (mHM) ¹³ and kept at 25°C for 2 h. After incubation, spermatozoa were washed twice using mHM, centrifuged at 700 × g for 10 min, and then diluted with modified BF5. ¹⁴ The suspensions were cooled slowly from 25 to 5°C over a 3 h interval, and further diluted with the same volume of BF5 diluents containing 4% (v/v) glycerol (final concentration 100 × 10⁶ spermatozoa/mL). After equilibration in glycerol at 5°C, sperm suspensions were packed into 5 mL maxi‐straws and held for 30 min on solid CO₂, then plunged into liquid N₂ for storage.

Preparation of the sperm suspension

Frozen semen samples were thawed in a circulating water bath at 37°C for 10 min with modified Beltsville thawing solution (mBTS) ¹⁴ (1 : 2 [v/v]) and then centrifuged at 600 × g for 5 min to separate the freezing extender. Sperm pellets were resuspended in mBTS to final concentrations of 5 × 10⁶ and 20 × 10⁶ spermatozoa/mL for evaluation of motility and viability, and acrosome status, respectively, where mBTS was supplemented with 0 (control), 20 or 40 ng/mL relaxin. Then, the suspensions were incubated at 37°C for 0–4 h to evaluate the parameters under study.

Progressive motility

After completion of the specified incubation period, sperm motility was determined subjectively on the basis of speed progression and on the type of forward movement of spermatozoa using a phase contrast microscope (Olympus, IMT‐2; Tokyo, Japan) with a thermal stage (37°C).

Viability and acrosome status

To evaluate the viability and acrosome status, spermatozoa were stained after the specified incubation period by applying the triple staining technique as described previously. ¹⁵ The sperm acrosome was stained with Rose Bengal for acrosome status evaluation after the distinction of live from dead spermatozoa using trypan blue. The triple stained slides were examined by light microscopy under oil immersion (×1000). Viability and acrosome status of the spermatozoa was evaluated from randomly selected fields of the triple stained slides until 400 spermatozoa had been examined.

Incorporation and oxidation of ¹⁴C‐glucose

To assess the effect of relaxin on cryopreserved boar spermatozoa by ¹⁴C‐glucose incorporation and oxidation, semen samples were prepared as described above. A 100 µL sample of spermatozoa (5 × 10⁶/mL) from each treatment was incubated with 18.5 KBq/mL of ¹⁴C‐glucose (specific activity 9.25 GBq/mmol; MP Biochemicals, Irvine, CA, USA) and prepared for scintillation counting by applying the method as described in our previous study. ⁴ The incorporation and oxidation of ¹⁴C‐glucose by spermatozoa was determined by a liquid scintillation counter (LS‐6500, Beckman Instruments, Fullerton, CA, USA).

Statistical analysis

The mean values with SEM of sperm motility, viability, acrosome reaction, and ¹⁴C‐glucose incorporation and oxidation of each treatment after the different incubation periods were calculated and statistical significance of the differences among the treatments (0, 20 or 40 ng/mL) were evaluated using the protected Fisher's least significant difference test. The ncss (Number Cruncher Statistical System; NCSS Statistical Software, Kaysville, UT, USA) version 5.01 computer software package was used for all statistical analyses. Each experiment was repeated four times with spermatozoa from four different animals. Differences were considered significant as P < 0.05.

RESULTS

THE EFFECT OF relaxin on progressive motility of cryopreserved boar spermatozoa at 0–4 h of incubation is shown in Figure 1. Compared with the control, 20 ng/mL relaxin improved progressive motility of spermatozoa at 1–3 h, whereas 40 ng/mL improved motility only at 1 h of incubation. Sperm motility decreased over the incubation period at 37°C in all treatments and there was a correlation between treatment and incubation time. Viability declined over the incubation period and showed no significant (P > 0.05) differences among the treatments (Fig. 2).

Figure 1.

Effect of relaxin during 0–4 h of incubation on progressive motility (%) of cryopreserved boar spermatozoa. (○) Control, (▵) addition of relaxin 20 ng/mL of modified Beltsville thawing solution (mBTS) and (▴) addition of relaxin 40 ng/mL of mBTS. Each line with error bar represents the means ± SEM from four replicates. Data points without a common lower case letter indicate differences (P < 0.05) among the treatments.

Figure 2.

Effect of relaxin during 0–4 h of incubation on viability (%) of cryopreserved boar spermatozoa. (○) Control, (▵) addition of relaxin 20 ng/mL of modified Beltsville thawing solution (mBTS) and (▴) addition of relaxin 40 ng/mL of mBTS. Each line with error bar represents the means ± SEM from four replicates. Data points have no significant differences (P > 0.05) among the treatments.

The percentage of acrosome reacted live spermatozoa observed in the treatments is shown in Figure 3. In comparison with the control, 20 ng/mL relaxin enhanced the live spermatozoa to undergo AR at 1–4 h, but 40 ng/mL relaxin enhanced AR only at 1–2 h of incubation. The percentage of acrosome reacted live spermatozoa was increased in accordance with increasing incubation period, and maximum AR occurred at 3–4 h of incubation in all treatments with or without relaxin. The effect of relaxin on the incorporation and oxidation of ¹⁴C‐glucose by spermatozoa is shown in4, 5. A significantly greater (P < 0.05) rate of ¹⁴C‐glucose incorporation and oxidation occurred in the spermatozoa treated with 20 ng/mL relaxin at 1–4 h, but only at 1–2 h when treated with 40 ng/mL. The incorporation and oxidation rate of ¹⁴C‐glucose in the spermatozoa of all treatments increased up to 4 h of incubation.

Figure 3.

Effect of relaxin during 0–4 h of incubation on acrosome reacted live spermatozoa (%) of cryopreserved boar semen. (○) Control, (▵) addition of relaxin 20 ng/mL of modified Beltsville thawing solution (mBTS) and (▴) addition of relaxin 40 ng/mL of mBTS. Each line with error bar represents the means ± SEM from four replicates. Data points without a common lower case letter indicate differences (P < 0.05) among the treatments.

Figure 4.

Effect of relaxin during 0–4 h of incubation on ¹⁴C‐glucose incorporation of cryopreserved boar spermatozoa. (○) Control, (▵) addition of relaxin 20 ng/mL of modified Beltsville thawing solution (mBTS) and (▴) addition of relaxin 40 ng/mL of mBTS. Each line with error bar represents the means ± SEM from four replicates. Data points without a common lower case letter indicate differences (P < 0.05) among the treatments.

Figure 5.

Effect of relaxin during 0–4 h of incubation on ¹⁴C‐glucose oxidation of cryopreserved boar spermatozoa. (○) Control, (▵) addition of relaxin 20 ng/mL of modified Beltsville thawing solution (mBTS) and (▴) addition of relaxin 40 ng/mL of mBTS. Each line with error bar represents the means ± SEM from four replicates. Data points without a common lower case letter indicate differences (P < 0.05) among the treatments.

DISCUSSION

THE QUALITY OF boar semen is markedly affected by cryopreservation. ¹⁶ , ¹⁷ , ¹⁸ , ¹⁹ The traditional sperm quality parameters, motility and viability, were significantly decreased as a result of freeze‐thawing. ¹⁰ In the present study, the effect of relaxin on cryopreserved boar spermatozoa was evaluated up to 4 h of incubation, because survivability of spermatozoa in the female reproductive tract is no longer than this period; ²⁰ therefore, incubation at 37°C for 4 h gives the best indication of the fertilizing ability of cryopreserved boar spermatozoa. ²¹ Concentrations of relaxin of 20 and 40 ng/mL were used on the basis of our previous results. ⁴ Lessing et al. reported that the biological activity of relaxin is adversely affected as a result of freeze‐thawing of spermatozoa. ⁸ They observed that sperm motility was improved when relaxin was added to the semen only at the time of thawing, not before freezing. In the present study, relaxin was added to the post thaw‐washed spermatozoa, not at the prefreezing stage where we observed also that relaxin improved the rate of progressive motility of cryopreserved boar spermatozoa. There was a correlation between relaxin effects and incubation period, where the progressive rate of decreasing motility over the incubation period was comparatively lower in relaxin treated spermatozoa than untreated spermatozoa. Cryopreservation of human spermatozoa in a 10% glycerol solution resulted in a decrease in post‐thaw motility and this loss of motility could be reduced by adding relaxin to the sample after thawing. ⁸ The present result shows that relaxin did not improve the motility when reaching values near the baseline after 4 h of incubation in all treatments. Lessing et al. ² reported that relaxin cannot improve motility of damaged spermatozoa after a certain level from which recovery was no longer possible. Previous studies showed that relaxin increased motility of fresh‐washed boar spermatozoa. ⁴ , ²² Thus, the present result agreed with the results of previous studies, indicating that relaxin can also improve the motility of cryopreserved boar sperm up to a certain limited period of incubation.

All mammalian spermatozoa undergo capacitation after residing in the female genital tract. Spermatozoa also can be capacitated in vitro, during incubation under certain conditions. In vitro studies have shown that AR can be initiated in preincubated spermatozoa either spontaneously or by physiological ionomycine inducers, such as zona pellucida glycoproteins, follicular fluid, bovine serum albumin and non‐physiological calcium ionophore. ²³ The present result clearly indicates that relaxin induced AR in cryopreserved boar spermatozoa.

At the onset of incubation, the acrosome reacting effect of relaxin was very mild, but after 1 h of incubation of spermatozoa in mBTS, the percentage of acrosome reacted live spermatozoa was raised significantly in all treatments. A significantly higher incidence of AR was observed in the spermatozoa incubated with 20 ng/mL compared with control spermatozoa. Similar effects of relaxin were observed in our previous study, where relaxin resulted in an increase of AR in fresh‐washed boar spermatozoa. ⁴ The increase was to a similar level as observed in the present study, but the difference was marginally higher. In the present study, it was shown that relaxin stimulates the rate of incorporation and oxidation of ¹⁴C‐glucose. The present results also indicate that the metabolism of glucose increased with the incubation time, being almost linear at 4 h to provide required energy for inducing AR. Relaxin stimulated the induction of AR and hyperactivity of spermatozoa proportionately with ¹⁴C‐glucose incorporation and oxidation. Results of several studies show the importance of energy supply for motility progression and inducing AR in mammalian spermatozoa. It is well known that either glucose or a combination of glucose and other monosaccharides is the most important exogenous energy source to promote AR in vitro. ²⁴

However, the mechanisms of relaxin activity responsible for these events are still not clearly understood. It might be thought that supplementation of relaxin in the medium might increase intracellular cyclic adenosine monophosphate (cAMP) concentration that in turn could be responsible for improving sperm motility and inducing AR in spermatozoa. Hicks et al. stated that cAMP plays an important role in the glycolytic pathway of spermatozoa and, through its effect on glycolysis, it can influence the energy generation required for sperm motility and inducing AR. ²⁵ Glycolysis for adenosine triphosphate (ATP) production from glucose is required for vigorous motility and hyperactivation of mammalian spermatozoa. ²⁶

Effects of freezing and thawing might be manifested during prolonged incubation with relaxin, stimulating the induction of AR. This postulation might be supported by the fact that after cryopreservation, levels of intracellular Ca²⁺ in spermatozoa are double the values observed before cryopreservation. ²⁷ Relaxin might be thought to stimulate Ca²⁺ influx into the sperm plasma membrane, which is another essential step of mammalian sperm AR. An increased Ca²⁺ influx might also enhance membrane‐bound adenylate cyclase, resulting in increased intracellular concentrations of cAMP, both of which are known to be important regulators of sperm activities. ²⁸ The values of cyclic nucleotides and Ca²⁺ are the most important of the cell signaling events that control the induction of AR in mammalian spermatozoa. ²⁹ , ³⁰ , ³¹ , ³² , ³³ , ³⁴ Elevation of sperm cAMP content by membrane permeant cAMP analogs or phosphodiesterase inhibitors increases the motility of mammalian spermatozoa. ³¹ , ³⁵ , ³⁶ Furthermore, AR of hamster spermatozoa is associated with a progressive increase in cAMP concentrations that precede the onset of motility. ³⁷ A rise in cAMP values during AR is a consequence of an increase in bicarbonate‐stimulated adenylate cyclase activity. ³⁵

The effect of relaxin on intracellular concentration of cAMP and Ca²⁺ influx by mammalian spermatozoa needs to be studied further for a complete understanding of the mechanism of relaxin in improving motility and inducing AR. The results of the present study suggest that relaxin is able to improve progressive motility, AR and enhance the glucose metabolism of cryopreserved boar spermatozoa.