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. 2021 Jul 25;58(3):186–191. doi: 10.2141/jpsa.0200071

Effect of Anserine and Carnosine on Sperm Motility in the Japanese Quail

Prodip Kumar Sarkar 1, Ai Egusa 2, Mei Matsuzaki 3, Tomohiro Sasanami 4,*
PMCID: PMC8371539  PMID: 34447283

Abstract

Sperm motility is considered as one of the most important traits for successful fertilization, but the motility of an ejaculated sperm decreases with time when stored as liquid. It is reported that seminal plasma serves as a nutrient rich medium for sperm and plays an important role in sperm motility and its fertilization ability. Several studies have reported that imidazole dipeptides such as anserine and carnosine affect sperm motility and its fertilization ability in mammals. In this study, we report the presence of anserine and carnosine in the male reproductive tract of the Japanese quail. Abundant levels of anserine (44.46 µM) and carnosine (41.75 µM) were detected in the testicular fluid and seminal plasma respectively using the amino acid analyzer; however, seminal plasma solely contained carnosine. When the ejaculates were incubated with anserine or carnosine, we found that both the dipeptides improve sperm motility parameters such as straight line velocity, curvilinear velocity, average path velocity and amplitude of lateral head displacement after in vitro sperm storage at 15°C. These results indicate that imidazole dipeptides are present in the male reproductive tract and may improve sperm quality during in vitro sperm storage in the liquid states.

Keywords: anserine, carnosine, imidazole dipeptide, male reproductive tract, the Japanese quail

Introduction

In animals, sperm motility is an important trait for successful fertilization (Florman and Ducibella, 2006). In birds, ejaculated sperms are stored in sperm storage tubules (SSTs) of the female reproductive tract before fertilization (Sasanami et al., 2013); motility is considered to be an important factor responsible for sperm uptake into the SSTs (Froman, 2003). Essentially, an avian sperm undergoes an intense selection process in the female reproductive tract; immotile sperms are incapable of fertilizing the eggs (Allen and Grigg, 1957).

The modern poultry industry promises to provide superior quality meat and eggs. To propagate the next generation, artificial insemination (AI) is now widely being used in breeder farms (Łukaszewicz, 2010). Although cryopreservation has long been recognized for germplasm preservation in mammals including livestock and laboratory animals, the technique is not reliable in birds due to low fertilization ability of frozen/thawed sperms (Long, 2006; Long et al., 2014). It is reported that cryopreservation damages sperm plasma membrane (Bakst and Sexton, 1979) and decreases the morphological integrity of the sperm (Blesbois et al., 2005). To minimize fertility loss, sperm may be stored as liquid in birds, but the motility of an ejaculated sperm decreases with time upon storage as liquid in vitro (Kotłowska et al., 2007).

Seminal plasma (SP), a complex fluid added to the sperm in the male reproductive tract (Duncan and Thompson, 2007) serves as a nutrient rich medium (Juyena and Stelletta, 2012) and plays an important role in sperm maintenance, viability and transportation into female reproductive tract (Poiani, 2006). The role of SP proteins, peptides and amino acids has been widely investigated in mammals (Manjunath et al., 2007; Koppers et al., 2011; Crawford et al., 2015; Viana et al., 2018). Several studies have emphasized the important effects of imidazole dipeptides such as anserine and carnosine on sperm motility and its fertilization ability in various species (Siems et al., 2003; Tareq et al., 2008; Bosler et al., 2014). Carnosine is a naturally occurring dipeptide consisting of β-alanine and histidine while anserine is a methylated form of carnosine (Boldyrev et al., 2013). Because both anserine and carnosine are histidine-containing dipeptides, it is thought that they play similar physiological roles (Boldyrev et al., 2013) such as exhibiting buffering capacity at neutral pH (Tanokura, 1983; Sale et al., 2010). Essentially, both these imidazole dipeptides have been reported to maintain muscle buffering capacity and muscle functions (Begum et al., 2005). In domestic birds such as chicken, imidazole dipeptides are reported to be present in the skeletal muscles and are known for their antioxidant capacity (Sato et al., 2008); however, to our knowledge, there are no reports on the presence of these dipeptides in the male reproductive tract of birds. It was therefore interesting to investigate their existence in the male reproductive tract. Here, we report the presence of imidazole dipeptides, anserine and carnosine in the male reproductive tract of the Japanese quail and demonstrate that the dipeptides improve sperm motility even when stored in vitro in the liquid state.

Materials and Methods

Animal Care and Management

Male Japanese quails (Coturnix japonica), 40–60 weeks of age (Quail cosmos, Toyohashi, Japan) were maintained in individual cages under photoperiods of 14h light and 10h dark cycle (lights went on at 05:00). Birds were allowed to eat commercial feed (Toyohashi Feed Mills, Toyohashi, Japan) and drinking water ad libitum. Animal management and all experimental procedures were carried out in accordance with the approved guidelines of the Animal Care Committees of Shizuoka University (Approval number: 2018A-5).

Preparation of Testicular Fluid, Seminal Plasma, Cloacal Gland Secretion and Blood Serum

Three male quails were decapitated and dissected to harvest the testis. Briefly, the testis was cut and fluid was collected from inside the testis using a micropipette. of The fluid (100 µl) from each of the testis was pooled and the mixture was centrifuged at 15,000×g for 10 min at 4°C; the supernatant was collected as testicular fluid. For the preparation of SP, ejaculates from 10 mature male quails were pooled, centrifuged at 5,000×g for 3 min at 4°C, and supernatant was collected. For the preparation of cloacal gland secretion, cloacal foam was obtained from 10 male quails by pressing the gland. The foam was centrifuged at 20,400×g for 5 min at 21°C. Blood was collected from the wing vein of 3 males and centrifuged at 2,000×g for 20 min at 4°C for collection of serum; 100 µl of each sample was pooled. The isolated testicular fluid, SP, cloacal gland secretion and blood serum were stored at −20°C until further use.

Semen Collection and Processing

Hanks balanced salt solution (HBSS) containing 136 mM NaCl, 5.4 mM KCl, 0.8 mM MgSO4, 1.26 mM CaCl2, 4.2 mM NaHCO3 and 5.6 mM glucose was used as sperm diluent. HBSS was incubated at 37°C for 10 min before semen collection. Semen was obtained during mating from mature male quails prior to ejaculation, in accordance with the procedure by Kuroki and Mori (1997) and suspended in 500 µl HBSS. Sperm cells were counted microscopically using a hemocytometer (BX 51, Olympus Optics, Tokyo, Japan) and prepared to reach a final concentration of 1×108 sperm/ml. 100 µl of HBSS containing varying concentrations of anserine (L-Anserine Nitrate, Fujifilm Wako Pure Chemical Co., Osaka, Japan) or carnosine (L-Carnosine, Wako Pure Chemical Corporation) were added to the sperm suspension to achieve a final concentration of 2×107 sperm/ml. Suspensions were incubated at 15°C upto 12 h. The optimum concentration of anserine and carnosine was determined from our preliminary experiments in which the ejaculated spermatozoa were incubated with either 1 µM, 3 µM, 10 µM, 30 µM or 100 µM of the dipepetides. We found that more than 3 µM of anserine and carnosine had no further potentiation effect on sperm motility (data not shown).

Sperm Motility Test Using Computer Assisted Semen Analysis (CASA)

Sperm suspensions were collected from the droplets at varying incubation times: 1 h, 3 h, 6 h, 9 h and 12 h. Sperm motility was analyzed using the CASA system (SMAS, Ver. 3, DITECT Digital Image Technology, Japan). Glass slides were coated with 2% BSA and warmed on a hot plate at 39°C. Sperm suspensions were placed on the warm glass slide, covered with cover glass, and observed microscopically (Nikon ECLIPSE E200, Nikon, Tokyo, Japan). Sperm motility parameters such as straight line velocity (VSL µm/s), curvilinear velocity (VCL µm/s), average path velocity (VAP µm/s), linearity (LIN) (LIN=VSL/VCL), straightness (STR) (STR=VSL/VAP), wobble (WOB) (WOB=VAP/VCL), amplitude of lateral head displacement (ALH µm), beat-cross frequency (BCF Hz) and motility rate (%) were recorded.

Detection of Anserine and Carnosine in the Male Reproductive Tract

The testicular fluid, SP, cloacal gland secretion and blood serum were diluted 2 times with water and mixed with 3% sulfosalicylic acid in a ratio of 1:1. The mixture was centrifuged at 20,400×g for 10 min at 4°C and the supernatant was analyzed using an amino acid analyzer (JCL-500/V2; JEOL, Tokyo, Japan), as previously described (Imanari et al., 2010). In brief, free amino acids were separated on a strong cation exchange column using lithium citrate buffers of different pH for elution. The amino acid derivatives were detected using ninhydrin at 440 nm or 570 nm at a reference wavelength of 690 nm. A solution of amino acid standards (Fujifilm Wako Pure Chemical Co.) was used as an external standard.

Data Analysis

All the data analyses were performed using the statistical package R version 3.3.3 (The R Foundation for Statistical Computing Platform, https://cran.r-project.org/bin/macosx/). Shapiro-Wilk test was performed to confirm the distribution normality. Percentage data were arcsine transformed before analysis and significance was based on the transformed mean value. Data was analyzed for significant differences using one-way analysis of variance (ANOVA). Differences between treatments were analyzed with Tukey's honestly significant difference test. Declaration of significance were based on P<0.05.

Results

Presence of Anserine and Carnosine in the Male Reproductive Tract

Male reproductive tract of the Japanese quail was investigated for the presence of anserine, carnosine and their constituents? β-alanine, 1-methylhistidine, and histidine (Table 1). In the testicular fluid, anserine was detected but carnosine was below detection limits; seminal plasma solely contained carnosine. The constituents histidine, β-alanine, and 1-methylhistidine were present in both the samples. In case of cloacal gland secretion, neither anserine nor carnosine was detected. Blood serum contained both imidazole dipeptides as well as their constituents except for β-alanine.

Table 1. Presence of imidazole dipeptides in the male reproductive tract of the Japanese quail*.

Sources Anserine
(µM)		 Carnosine
(µM)		 β-alanine
(µM)		 Histidine
(µM)		 1-methylhistidine
(µM)
Testicular fluid 44.46 —   54.06 210.00 60.65
Seminal plasma — 41.75 109.67   50.63 52.98
Cloacal gland secretion — —   51.30   18.70   7.48
Blood serum 41.91   2.56 —   75.16 43.88
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*

single analysis was performed

- indicates value under detection limit

Effects of Anserine and Carnosine on Sperm Motility

In the next set of experiments, we incubated the sperm with different concentrations of either anserine or carnosine: 0 µM, 0.1 µM, 0.3 µM, 1 µM or 3µM (Table 2). No significant difference was observed in sperm motility at 0.1 µM concentration of anserine or carnosine compared to controls, but a significant increase in sperm motility parameters was observed at 0.3 µM, 1 µM or 3µM of anserine or carnosine. The most effective dose of anserine and carnosine for sperm motility improvement was found to be 1 µM and 0.3 µM, respectively. Next, the sperms were treated with 1 µM anserine or 0.3 µM carnosine for varying incubation time. Upon storage, a decreasing trend in sperm motility was noted with time and no significant change was observed upto 3 h of incubation; however, 6 h onwards, a significant improvement in motility parameters was observed in comparison to controls (Fig. 1). The beneficial effects of anserine or carnosine on sperm motility were observed even after 9 h of incubation; the sperm motility parameters continued to improve after 12 h of incubation with carnosine.

Table 2. Effect of various concentrations of anserine and carnosine on sperm motility after 6h of sperm storage.

Parameters Control Anserine (µM) Carnosine (µM)
0.1 0.3 1 3 0.1 0.3 1 3
VSL (µm/s) 5.5±2.7a 5.8±2.6ab 10.5±2.4ab 11.6±3.6b 9.7±4.6ab 6.9±2.1a 9.7±3.4a 8.9±1.8a 9.6±4.5a
VCL (µm/s) 38.4±9.9a 44.9±5.9ab 56.1±10.3ab 61.9±7.6b 56.5±12.5ab 54.9±3.7ab 60.1±11.7b 56.8±10.6b 60.3±12.8b
VAP (µm/s) 12.7±3.8a 14.0±2.2ab 22.3±4.5b 23.6±4.4b 21.7±8.5b 17.6±3.1ab 22.5±6.7b 20.1±3.9ab 22.8±8.5b
LIN 0.2±0.1a 0.2±0.1a 0.2±0.1a 0.2±0.1a 0.2±0.1a 0.1±0.02a 0.2±0.03a 0.2±0.1a 0.1±0.03a
STR 0.4±0.1a 0.3±0.1a 0.4±0.1a 0.4±0.1a 0.4±0.1a 0.3±0.04a 0.4±0.04a 0.4±0.1a 0.3±0.03a
WOB 0.4±0.1a 0.3±0.1a 0.4±0.1a 0.4±0.1a 0.4±0.1a 0.3±0.03a 0.4±0.04a 0.4±0.02a 0.4±0.1a
ALH (µm) 1.1±0.2a 1.1±0.2ab 1.6±0.3b 1.7±0.3b 1.5±0.5ab 1.6±0.2ab 1.8±0.4b 1.6±0.3b 1.7±0.4b
BCF (Hz) 8.2±1.0a 7.9±1.3a 8.9±1.1a 8.2±0.7a 8.2±0.9a 7.7±0.5a 7.8±0.4a 8.1±0.9a 7.7±0.6a
Motility rate (%) 59.7±12.3a 58.6±11.3a 68.1±19.4a 74.2±12.9a 71.1±20.6a 64.4±12.7a 72.1±16.9a 74.4±9.6a 77.6±11.7a
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Data (n=5 independent experiments) is expressed as mean±SD. Different superscript letters suggesting significant differences at P<0.05 among treatments within the same row.

Fig. 1.

Fig. 1.

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Time-dependent study of anserine on sperm motility parameters. Sperm samples were incubated with (1 µM) or without anserine. Sperm motility was recorded at varying incubation times— 1 h, 3 h, 6 h, 9 h and 12 h using CASA. The graph shows (a) straight line velocity (VSL); (b) curvilinear velocity (VCL); (c) average path velocity (VAP); (d) linearity (LIN); (e) straightness (STR); (f) wobble (WOB); (g) amplitude of lateral head displacement (ALH); (h) beat-cross frequency (BCF); and (i) motile rate. Data (n=5 independent experiments) is expressed as mean±SD. * indicates statistically significant difference at P<0.05.

Discussion

In this study, we observed that imidazole dipeptides, anserine and carnosine are present in the male reproductive tract of the Japanese quail. Although the levels were far lower than that reported in the human (Mannion et al., 1992) or chicken (Crush, 1970) skeletal muscle, this is the first report demonstrating the presence of imidazole dipeptides in the reproductive system of birds.

Analysis of biological fluid derived from the male reproductive tract revealed that the testicular fluid contains anserine, but not carnosine. In chickens, carnosine is reportedly produced from β-alanine and histidine by carnosine synthase and is further converted to anserine by the action of histamine N-methyltransferase-like protein (Boldyrev et al., 2013). In addition, it is also known that carnosine is sensitive to carnosinase such as CNDP1 and 2 responsible for the degradation of carnosine to β-alanine and histidine (Boldyrev et al., 2013). It is therefore assumed that testicular anserine is a product of carnosine methylation and the remaining carnosine is probably degraded by the action of CNDPs or similar enzymes. To ascertain this, further expression studies and localization experiments of these testicular enzymes is warranted. If the testicular fluid is transported to epididymis and vas deferens when the sperm passes through the male reproductive tract, anserine should also be present in the SP; however, as per our findings, SP contains only carnosine (Table 1). The presence of carnosine in SP suggests that it is not derived from the testis but is newly synthesized and added later in the male reproductive tract; the enzymes responsible for either carnosine methylation or degradation are absent. Similarly, dominant localization of carnosine was reported in the muscles of horses and humans (Harris et al., 1990), though the exact mechanism of its stabilization is unknown. Another possibility is that demethylation of methylhistidine occurs in the latter part of the testis in the reproductive tract and testicular anserine is again converted to carnosine during its passage through the male reproductive tract. Although the enzyme responsible for demethylation of anserine has not been demonstrated in animals yet, it would be interesting to explore a novel demethylase in the male reproductive tract of birds.

In this study, carnosine (0.3 µM) and its derivative—anserine (1 µM) improved sperm motility upon in vitro storage. In physiological condition, avian species may uplift the production of antioxidant enzymes to protect the sperm (Mavi et al., 2020) but the antioxidant defense mechanism may be disturbed in vitro due to oxidative stress (Sikka, 1996). Avian sperm contains high amounts of polyunsaturated fatty acids (Surai et al., 1998). The sperm integrity becomes susceptible with increased reactive oxygen species and lipid peroxidation (Khan, 2011). Several studies revealed the antioxidant properties of anserine and carnosine. The antioxidant activity of histidine and its derivatives is due to their imidazole moiety which decreases the rate of oxidation (Kohen et al., 1988). A histidine containing compound is linked to having antioxidant ability and the peptide linkage between β-alanine, histidine, and 1-methylhistidine is associated with the antioxidant properties (Wu et al., 2003). In addition, the antioxidant activity of carnosine is facilitated by chelation of metal ions, scavenging of reactive oxygen species and peroxyl radicals (Kohen et al., 1988; Boldyrev et al., 2013). The effect of carnosine on sperm motility has been investigated in mammals. For instance, carnosine improves the VSL of sperm in rams (Tan and Han, 1995), prevents oxidative stress in testis of rats (Aydın et al., 2018), and improves the sperm mitochondrial activity in humans (Adami et al., 2020). Carnosine acts as a malondialdehyde scavenger? lower malondialdehyde concentration was observed in stallion semen with higher carnosine content (Rocha et al., 2018). From the above mentioned studies, we believe that anserine or carnosine addition may improve sperm motility by reducing oxidative stress during its storage.

Fig. 2.

Fig. 2.

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Time-dependent study of carnosine on sperm motility parameters. Sperm samples were incubated with (0.3 µM) or without carnosine. Sperm motility was recorded at varying incubation times? 1 h, 3 h, 6 h, 9 h and 12 h using CASA. The graph shows (a) straight line velocity (VSL); (b) curvilinear velocity (VCL); (c) average path velocity (VAP); (d) linearity (LIN); (e) straightness (STR); (f) wobble (WOB); (g) amplitude of lateral head displacement (ALH); (h) beat-cross frequency (BCF); and (i) motile rate. Data (n=5 independent experiments) is expressed as mean±SD. * indicates statistically significant difference at P<0.05.

To conclude, our study indicates that imidazole dipeptides exist in the male reproductive tract and may improve sperm quality during its in vitro storage as liquid. Although we did not examine the membrane integrity of stored sperms, imidazole dipeptides could be potential additives for sperm storage in vitro to minimize membrane damage.

Acknowledgments

This work was supported by a Grant-in-Aid for Scientific Research (B) (General) (17H03902 to TS). All authors approved the final manuscript.

Conflicts of Interest

The authors declare no conflict of interest.

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