Improvement of sperm motility of Oncorhynchus mykiss and Salvelinus fontinalis by L-tryptophan

ABSTRACT

Experiments were designed to test the influence of L-tryptophan supplementation of the extender on the sperm motility parameters and bacterial flora of brook trout (Salvelinus fontinalis) and rainbow trout (Oncorhynchus mykiss). The extender containing 0.3 M glucose in 10% Dimethyl sulfoxide (DMSO) was supplemented with 0, 0.5, 1, 2, and 4 mM L-tryptophan. Sperm samples stored for up to 12 days at 4°C for brook trout were motile while motility was not observed after six days of storage for rainbow trout. Diluted sperm samples were spread-plated onto Plate Count Agar (PCA) (Total Bacteria Count), Rose Bengal Agar (RBC) (Yeast-Mold Count), Violet Red Bile Dextrose (VRBD) (Enterobacteriaceae count) and Mannitol Salt Agar (MSA) (Micrococcus/Staphylococcus count) and counts were performed in duplicate and sampling occurred on day 0, 2, 4, 6, and 12 of cold storage. L-tryptophan supplementation of the extender yielded a positive effect, significantly prolonging sperm motility in S. fontinalis and O. mykiss compared to the control group (p < 0.05). The ideal results were obtained above a concentration of 0.5 mM for both species. Total bacteria count in fresh sperm (undiluted samples) was not detectable and not detected in sperm samples treated with different L-tryptophan concentrations. Yeast-mold, Enterobacteriaceae and Micrococcus/Staphylococcus were not detected from fresh and treated sperm brook trout and rainbow trout. On the basis of the results, L-tryptophan-based extender is useful for maintaining sperm quality in short-term storage. L-tryptophan had a negative effect on the bacterial flora. The results of the current study encourages further studies related to long-term storage and reproduction management.

Abbreviations: DMSO: dimethyl sulfoxide; PCA: plate count agar; RBC: rose Bengal agar; VRBD: violet red bile dextrose; MSA: mannitol salt agar; SCA: sperm class analyzer; CFU: colony-forming unit

KEYWORDS:

• Amino acid
• short-term sperm storage
• sperm quality
• trout

Introduction

Short-term storage is an important method for fish farms and used commonly in aquaculture applications (Shaliutina et al. 2013). In particular, it is needed for large-scale hatchery operations and transporting gametes a short distance may affect fertilization and hatching success (Penaranda et al. 2010; Shaliutina et al. 2013; Kutluyer and Kocabas 2016; Kocabas and Kutluyer 2017a, 2017b, 2017c; Yavas et al. 2017). In this technique, non-diluted sperm or sperm diluted in ionic or multi-component media is preserved at 4°C under different atmosphere composition conditions (O₂ supplementation or presence of CO₂) for lengthy periods of time (Penaranda et al. 2010; Shaliutina et al. 2013). Success of the method depends on several factors such as atmosphere composition, diluents, control of temperature, dilution rate, a range of additives (e.g., antioxidants, antibiotics) and varied seminal plasma composition (Alavi and Cosson 2005; Penaranda et al. 2010; Shaliutina et al. 2013). Recently, different methodological approaches have been applied for developing conservation protocols. Especially, researchers have focused on using different additives in short-term storage of fish spermatozoa.

Assessing bacterial contamination is important for artificial fertilization due to the risk of disease transmission (Ercan and Ekici 2016). Bacterial contamination may be due to inappropriate sperm collection methods (Boonthai et al. 2016). Apart from this, sperm motility and viability may decrease during short-term storage due to anaerobic conditions and associated microbial contamination (Halimi and Norousta 2014). Moreover, increasing load of bacteria can result from oxygen consumption and the production of extracellular enzymes (Jenkins and Tiersch 1997; Halimi and Norousta 2014). For these reasons, reducing contamination of pathogenic and spoilage bacteria is needed to provide high sperm quality and successful fertilization (Boonthai et al. 2016).

Amino acids have a well-known role on multiple biologic and psychological processes (Kutluyer and Kocabas 2016), and an effective primary defense for protection of the sperm membrane structure owing to their antioxidant properties. The aromatic amino acid L-tryptophan is versatile and needed for the biosynthesis of proteins (Zhao et al. 2010). Due to metabolic functions, it has been widely used in research and clinical trials (Richard et al. 2009). Only a single study has been conducted about the in-vitro use of L-tryptophan as an agent on endangered trout S. coruhensis, Anatolian trout S. rizeensis and rainbow trout O. mykiss sperm to date (Kutluyer 2018a).

Salvelinus fontinalis or common name brook trout is a freshwater fish that belongs to the family Salmonidae and lives in lakes and ponds throughout Eastern North America and around the world. Because of its economic value and recreational importance, S. fontinalis has been over harvested, leading to a decline of this trout population. Furthermore, its spawning sites have diminished, habitat fragmented, and atmospheric changes are also factors that have led to the declining population (Kocabaş et al. 2013). Also, increased human activity, the pollution of natural habitats and the river bottom have negatively influenced their reproduction. To date, few studies have explored short-term storage of brook trout (S. fontinalis) (Köse and Şahin 2015) although studies have been conducted about cryopreservation of brook trout (Lahnsteiner 2000; Lahnsteiner et al. 2011; Nynca et al. 2015; Judycka et al. 2016, 2018). Rainbow trout Oncorhynchus mykiss is commercially Salmonid fish species and most studies to date have focused on short-term sperm storage of rainbow trout (Pérez-Cerezales et al. 2009; Lahnsteiner et al. 2011; Pourkazemi et al. 2013; Sarosiek et al. 2013; Şahin et al. 2013a, 2013b; Aguilar-Juárez et al. 2014; Ubilla et al. 2014; Risopatrón et al. 2018). As far as the authors of this work are aware, there is no available information concerning brook trout (S. fontinalis) and rainbow trout (O. mykiss) spermatozoa to understand how the supplementation of L-tryptophan influences sperm motility after short-term storage. Thus, we hypothesized that addition to L-tryptophan will affect spermatozoa motility and storage duration of S. fontinalis and O. mykiss. Therefore, the current study tested the effects of increasing concentration of L-tryptophan on sperm motility and bacterial flora of brook trout S. fontinalis and rainbow trout O. mykiss.

Results and discussion

Mean values for measured sperm quality parameters in S. fontinalis and O. mykiss are given in Table 1. Current data are in contrast with those reported by previous studies. The reason of differences in present results can be explained with ecology and spawning behavior of brood stock, weight and age of the male, sampling term, and method and stage of spawning (Piironen and Hyvarinen 1983; Suquet et al. 1994, 1998; Tekin et al. 2003; Kocabas and Kutluyer 2017b).

Table 1. Summary of standard sperm quality parameters (mean ± SD) assessed for S. fontinalis and O. mykiss (n = 6).

	Salvelinus fontinalis		Oncorhynchus mykiss
Parameters	Mean	Range	Mean	Range
Color	White		White
Volume (mL)	4.15 ± 1.26	1–6	8.50 ± 4.24	4–15
pH	7.28 ± 0.15	7.25–7.59	7.26 ± 0.10	7.13–7.35
Spermatocrit (%)	58.20 ± 2.35	55.00–60.00	31.64 ± 4.38	27.63–37.04
Sperm density (×10^9)	8.34 ± 0.34	8.15–8.65	7.26 ± 0.18	7.17–7.38

The percent and survival of motile fresh spermatozoa in S. fontinalis and O. mykiss were 89.50 ± 6.14% and 80.50 ± 19.54 s, and 98.00 ± 1.73% and 47.67 ± 4.51 s, respectively. Two-way ANOVA indicated a significant treatment effect for the percent and survival of motile sperm cells in O. mykiss (F_value = 6.113 and p = 0.000; F_value: 14.103 and p = 0.000, respectively) and S. fontinalis (F_value = 12.847 and p = 0.000; F_value: 19.324 and p = 0.000, respectively) while the progressive motility and duration of motility showed significant decline in O. mykiss (F_value = 36.713 and p = 0.000; F_value: 49.093 and p = 0.000, respectively) and S. fontinalis (F_value = 56.781 and p = 0.000; F_value: 36.713 and p = 0.000, respectively) with storage duration. After 6 and 11 days of storage, no motile sperms were shown in any samples of O. mykiss and S. fontinalis, respectively (Tables 2–5), while in non-supplemented groups, no motile spermatozoa were recorded in O. mykiss and S. fontinalis after 2 and 3 days of storage. Sperm motility and duration gradually decreased over the course of the experiment. Significantly lower values were observed in S. fontinalis and O. mykiss after 2 and 3 days, respectively. In O. mykiss, no motile spermatozoa were recorded after 6 days of storage at concentration of 0.5 mM L-tryptophan. In S. fontinalis, there were no motile spermatozoa after 11 days of storage at a concentration of 0.5 mM and 7 days of storage in other concentrations of L-tryptophan from 1 to 4 mM. For both species, the best results were obtained at concentration of 0.5 mM compared to control groups (non-supplemented). The decline in sperm motility rates and durations was significant for O. mykiss and S. fontinalis (p < 0.05). Thus far, a great deal of past research has focused on short-term sperm preservation of rainbow trout (Pérez-Cerezales et al. 2009; Lahnsteiner et al. 2011; Pourkazemi et al. 2013; Sarosiek et al. 2013; Şahin et al. 2013a, 2013b; Aguilar-Juárez et al. 2014; Ubilla et al. 2014; Risopatrón et al. 2018) although few studies have conducted short-term storage and cryopreservation of brook trout (S. fontinalis) (Lahnsteiner 2000; Lahnsteiner et al. 2011; Nynca et al. 2015; Köse and Şahin 2015; Judycka et al. 2016, 2018). This report is apparently the first to examine the effect of sperm storage medium supplemented with L-tryptophan on fish sperm although a single complementary study has been reported about using L-tryptophan in different activation medium for S. coruhensis, O. mykiss and S. rizeensis spermatozoa by Kutluyer (2018a). Kutluyer (2018a) demonstrated that best concentrations of L-tryptophan for S. coruhensis, O. mykiss and S. rizeensis were 2, 0.5, and 5 mM, respectively. Generally, the present study indicated that the addition of L-tryptophan in short-term storage media caused the positive effect on storage duration. The diluents tested for two species provided beneficial conditions for spermatozoa during preservation. However, the sperm motility and storage duration were decreased slightly at higher concentration of L-tryptophan (1 mM above). The results of the current study clearly reveal that the best concentration of L-tryptophan was 0.5 mM for S. fontinalis and O. mykiss sperm. The results of the present study are in line with the report presented by Kutluyer et al. (2018). Production of free oxygen radicals increases during short-term storage due to anaerobic conditions (Penaranda et al. 2010). In oxidative stress conditions, deteriorated sperm function as reactive oxygen species (ROS) was generated (Sikka 1996; Kowalowka et al. 2007; Penaranda et al. 2010). L-tryptophan is one of the aromatic amino acids and important in a number of other metabolic functions and protein synthesis (Richard et al. 2009; Zhao et al. 2010). L-tryptophan acts by transferring an electron to free radicals resulted in the compound becomes stable and protect the plasma membrane injuries. The sperm plasma membrane is able to modulate electrolyte movement at the cellular level appropriately (Muthmainnah et al. 2018) in metabolic processes that produce ATP, a sperm energy source (Gonçalves et al. 2018; Muthmainnah et al. 2018).

Table 2. Effect of extenders supplemented with L-tryptophan at different concentrations on mean (±SD) percentages of progressive motility in S. fontinalis sperm after short-term storage.

Storage day (days)	Fresh sperm	Control	0.5 mM	1 mM	2 mM	4 mM
0	89.50 ± 6.14^a,x	90.00 ± 5.00^a,x	90.00 ± 5.00^a,x	87.50 ± 6.45^a,x	85.00 ± 9.13^a,x	83.75 ± 2.50^a,x
1	60.00 ± 0.01^a,y	66.67 ± 5.77^b,y	78.00 ± 9.17^c,y	80.25 ± 0.50^c,x	83.33 ± 2.89^c,x	82.50 ± 2.89^c,x
2	40.00 ± 0.01^a,z	57.20 ± 8.17^b,y	77.00 ± 5.48^c,y	76.00 ± 8.94^c,x	75.00 ± 4.08^c,x	72.50 ± 5.00^c,x
3	0.00 ± 0.00^a,t	15.00 ± 7.07^b,z	59.17 ± 9.17^d,yz	50.80 ± 11.34^d,y	22.50 ± 25.98^c,y	15.00 ± 5.77^b,y
5	0.00 ± 0.00^a,t	13.33 ± 5.77^b,z	58.75 ± 7.50^c,yz	17.00 ± 4.47^b,z	15.00 ± 0.01^b,y	12.50 ± 6.45^b,y
6	0.00 ± 0.00^a,t	0.00 ± 0.00^a,t	54.33 ± 9.43^c,yz	10.00 ± 0.01^b,z	6.67 ± 2.89^ab,yz	4.83 ± 2.50^ab,yz
7	0.00 ± 0.00^a,t	0.00 ± 0.00^a,t	50.83 ± 14.29^b,yz	7.00 ± 0.01^a,z	5.00 ± 0.01^a,yz	3.00 ± 0.01^a,yz
8	0.00 ± 0.00^a,t	0.00 ± 0.00^a,t	44.17 ± 3.76^b,z	0.00 ± 0.00^a,t	0.00 ± 0.00^a,z	0.00 ± 0.00^a,z
9	0.00 ± 0.00^a,t	0.00 ± 0.00^a,t	20.00 ± 0.01^b,t	0.00 ± 0.00^a,t	0.00 ± 0.00^a,z	0.00 ± 0.00^a,z
10	0.00 ± 0.00^a,t	0.00 ± 0.00^a,t	15.00 ± 0.01^b,t	0.00 ± 0.00^a,t	0.00 ± 0.00^a,z	0.00 ± 0.00^a,z
11	0.00 ± 0.00^a,t	0.00 ± 0.00^a,t	8.33 ± 1.53^b,t	0.00 ± 0.00^a,t	0.00 ± 0.00^a,z	0.00 ± 0.00^a,z
12	0.00 ± 0.00^a,t	0.00 ± 0.00^a,t	0.00 ± 0.00^a,v	0.00 ± 0.00^a,t	0.00 ± 0.00^a,z	0.00 ± 0.00^a,z

^a,b,cDifferent superscripts within the same rows demonstrate significant differences (p < 0.05).

^x,y,z,t,vDifferent superscripts within the same columns demonstrate significant differences (p < 0.05).

Table 3. Effect of extenders supplemented with L-tryptophan at different concentrations on mean (±SD) duration of motility in S. fontinalis sperm after short-term storage.

Storage duration (day)	Fresh sperm	Control	0.5 mM	1 mM	2 mM	4 mM
0	80.50 ± 19.54^a,x	67.33 ± 2.52^a,x	86.00 ± 24.09^a,x	71.50 ± 5.69^a,x	76.25 ± 9.46^a,x	67.50 ± 15.00^a,x
1	40.50 ± 14.06^a,y	61.33 ± 2.31^b,x	81.67 ± 18.93^c,x	64.50 ± 4.20^bc,xy	71.00 ± 9.64^bc,x	64.00 ± 4.90^bc,x
2	20.00 ± 0.01^a,z	58.00 ± 2.74^bc,x	75.33 ± 8.59^d,x	61.00 ± 9.62^c,xy	60.00 ± 0.01^bc,y	51.25 ± 2.50^b,y
3	0.00 ± 0.00^a,t	22.75 ± 7.63^b,y	57.17 ± 11.72^c,y	50.80 ± 8.32^d,y	41.25 ± 10.31^e,y	38.75 ± 2.50^f,z
5	0.00 ± 0.00^a,t	20.00 ± 0.01^b,y	56.67 ± 10.33^d,y	38.00 ± 2.74^c,yz	28.33 ± 2.89^z	27.50 ± 2.89^b,z
6	0.00 ± 0.00^a,t	0.00 ± 0.00^a,z	51.67 ± 4.08^d,y	30.00 ± 0.01^c,yz	26.67 ± 2.89^bc,z	25.00 ± 5.77^b,z
7	0.00 ± 0.00^a,t	0.00 ± 0.00^a,z	45.83 ± 5.85^b,yz	20.00 ± 0.01^c,z	18.00 ± 0.01^c,t	15.00 ± 0.01^c,t
8	0.00 ± 0.00^a,t	0.00 ± 0.00^a,z	40.83 ± 3.76^b,z	0.00 ± 0.00^a,t	0.00 ± 0.00^a,v	0.00 ± 0.00^a,v
9	0.00 ± 0.00^a,t	0.00 ± 0.00^a,z	36.67 ± 2.89^b,z	0.00 ± 0.00^a,t	0.00 ± 0.00^a,v	0.00 ± 0.00^a,v
10	0.00 ± 0.00^a,t	0.00 ± 0.00^a,z	20.00 ± 0.01^b,t	0.00 ± 0.00^a,t	0.00 ± 0.00^a,v	0.00 ± 0.00^a,v
11	0.00 ± 0.00^a,t	0.00 ± 0.00^a,z	12.50 ± 3.54^b,t	0.00 ± 0.00^a,t	0.00 ± 0.00^a,v	0.00 ± 0.00^a,v
12	0.00 ± 0.00^a,t	0.00 ± 0.00^a,z	0.00 ± 0.00^a,v	0.00 ± 0.00^a,t	0.00 ± 0.00^a,v	0.00 ± 0.00^a,v

^a,b,c,d,eDifferent superscripts within the same rows demonstrate significant differences (p < 0.05).

^x,y,z,t,vDifferent superscripts within the same columns demonstrate significant differences (p < 0.05).

Table 4. Effect of extenders supplemented with L-tryptophan at different concentrations on mean (±SD) percentages of progressive motility in O. mykiss sperm after short-term storage.

Storage duration (day)	Fresh sperm	Control	0.5 mM	1 mM	2 mM	4 mM
0	98.00 ± 1.73^a,x	98.00 ± 0.05^a,x	98.00 ± 0.01^a,x	90.00 ± 5.00^ab,x	86.67 ± 5.77^b,x	76.67 ± 5.75^b,x
1	90.00 ± 0.01^a,x	91.67 ± 0.05^a,x	90.00 ± 2.89^a,x	84.67 ± 0.58^ab,x	76.67 ± 5.75^b,xy	66.67 ± 5.70^c,xy
2	0.00 ± 0.00^a,y	82.50 ± 4.47^b,xy	88.00 ± 2.88^b,xy	83.00 ± 1.00^b,x	71.25 ± 2.50^c,xy	53.33 ± 5.71^d,xy
3	0.00 ± 0.00^a,y	72.50 ± 4.46^b,y	78.00 ± 2.85^bc,y	82.00 ± 2.74^c,x	61.25 ± 2.52^d,y	43.33 ± 5.73^e,y
4	0.00 ± 0.00^a,y	37.00 ± 2.50^b,z	38.75 ± 2.80^b,z	37.50 ± 5.00^b,y	26.67 ± 2.89^bc,z	18.33 ± 2.85^c,z
5	0.00 ± 0.00^a,y	26.67 ± 0.01^b,zt	30.00 ± 7.64^b,z	25.00 ± 4.08^b,z	13.33 ± 2.85^c,t	11.67 ± 2.89^c,zt
6	0.00 ± 0.00^a,y	13.33 ± 0.05^b,t	15.26 ± 1.00^c,t	13.00 ± 0.05^bc,t	0.00 ± 0.00^a,v	0.00 ± 0.00^a,t
7	0.00 ± 0.00^a,y	0.00 ± 0.00^a,v	0.00 ± 0.00^a,v	0.00 ± 0.00^a,v	0.00 ± 0.00^a,v	0.00 ± 0.00^a,t

^a,b,c,dDifferent superscripts within the same rows demonstrate significant differences (p < 0.05).

^x,y,z,t,vDifferent superscripts within the same columns demonstrate significant differences (p < 0.05).

Table 5. Effect of extenders supplemented with L-tryptophan at different concentrations on mean (±SD) duration of motility in O. mykiss sperm after short-term storage.

Storage duration (day)	Fresh sperm	Control	0.5 mM	1 mM	2 mM	4 mM
0	47.67 ± 4.51^a,x	113.33 ± 15.28^b,x	108.00 ± 7.21^b,x	76.67 ± 11.55^c,x	73.33 ± 11.54^c,x	70.00 ± 10.00^c,x
1	23.67 ± 0.58^a,y	56.67 ± 7.64^b,y	54.00 ± 3.61^b,y	42.33 ± 2.52^c,y	36.67 ± 5.77^d,y	35.00 ± 5.00^d,y
2	0.00 ± 0.00^a,z	42.50 ± 5.00^b,yz	43.90 ± 5.48^b,z	42.33 ± 0.58^b,y	35.00 ± 4.08^bc,y	28.33 ± 2.89^c,yz
3	0.00 ± 0.00^a,z	40.00 ± 0.50^b,yz	43.75 ± 4.47^b,z	40.75 ± 16.64^b,y	32.67 ± 4.08^bc,y	25.33 ± 2.89^c,yz
4	0.00 ± 0.00^a,z	34.75 ± 10.00^b,z	28.00 ± 2.50^c,t	40.60 ± 4.79^d,y	25.00 ± 2.52^c,yz	18.33 ± 5.03^e,z
5	0.00 ± 0.00^a,z	18.00 ± 1.00^b,t	25.00 ± 5.77^c,t	24.75 ± 0.50^c,z	15.00 ± 0.01^bd,z	12.67 ± 2.62^d,z
6	0.00 ± 0.00^a,z	10.00 ± 0.01^b,t	14.00 ± 0.02^bc,tv	12.00 ± 0.05^c,t	0.00 ± 0.00^a,t	0.00 ± 0.00^a,t
7	0.00 ± 0.00^a,z	0.00 ± 0.00^a,v	0.00 ± 0.00^a,v	0.00 ± 0.00^a,v	0.00 ± 0.00^a,t	0.00 ± 0.00^a,t

^a,b,c,dDifferent superscripts within the same rows demonstrate significant differences (p < 0.05).

^x,y,z,t,vDifferent superscripts within the same columns demonstrate significant differences (p < 0.05).

Former studies reported higher motility and viability in stored sperm and extended storage time with O₂ supplementation (Benic et al. 2000; Christensen and Tiersch 1996; Jenkins-Keeran et al. 2001; Marques and Pereira-Godinho 2004; Bobe and Labbe 2009; Ciereszko et al. 2011; Donaldson et al. 2011). Although, in this study, diluted sperm samples that were kept under anaerobic conditions in covered containers that were mixed during daily measurement of sperm motility with the addition of L-tryptophan had an extended storage period. Additional experiments with O₂ supplementation could be realized for improvement of sperm storage time in addition to L-tryptophan supplementation.

Total bacteria count in fresh sperm (undiluted samples) was not detectable and not detected in sperm samples treated with different L-tryptophan concentrations. Yeast-mold, Enterobacteriaceae and Micrococcus/Staphylococcus were not detected from fresh and treated sperm (day 0, 2, 4, 6 and 12) brook trout (S. fontinalis) and rainbow trout (O. mykiss). Although there is no study in fish sperm about the effect of L-tryptophan on bacterial flora or count after short-term storage, the total bacteria count has been evaluated in fresh sperm of rainbow trout. Kubilay et al. (2009) reported the total bacteria count in milt of rainbow trout as 2.0 × 10³ cfu/mL due to the inadequate hygienic condition in water. Ercan and Ekici (2016) determined that high bacterial colony (>500) in fresh semen samples. Sperm motility and longevity may reduce with microbial contamination in anaerobic conditions (Niksirat et al. 2011). At this point, cell rupture and death can occur (Keogh et al. 2017). Sperm collection method, non-sterile solutions, and process of storage can all contribute to microbial contamination (Niksirat et al. 2011; Keogh et al. 2017).

In conclusion, sperm movement and storage time were better in diluents with L-tryptophan compared to control (without L-tryptophan). Based on the economic consideration, the reasonable concentration of L-tryptophan was 0.5 mM for S. fontinalis and O. mykiss. Further studies are needed to determine the effects of L-tryptophan on the fertility capacity of the sperm, energy metabolism and quantification of ATP levels. Nevertheless, the study reported here will likely provide benefits for practical aquaculture applications, increasing storage period, improving fertilizing capacity, standardization of methods and prolong the sperm motility.

Material and methods

Broodfish handling and collection of semen

Mature males of S. fontinalis (n: 6, 40.12 ± 3.15 cm, 1060.28 ± 198.26 g, mean ± SD; more than 2 years of age) and O. mykiss (n: 6, 35.23 ± 4.75 cm, 599.12 ± 3.15 g, mean ± SD; more than 2 years of age) were used in this study. Broodfish were provided from the facility in Faculty of Marine Sciences, Karadeniz Technical University (Trabzon, Turkey) and Isina trout farm (Rize, Turkey) (January, 2018). This study was conducted according to procedures approved by the Animal Experiments Local Committee of Karadeniz Technical University, Turkey. For sperm collection, stripping of the male was gently realized abdominally after anesthetized using 2-phenoxyethanol (0.6 mL L⁻¹). For wiping the genital pore, a tissue paper was used to avoid water, blood, urine, or feces for preventing sperm activation and contamination in sperm collection. Sperm was collected into a 50 mL vial and kept on ice transport purposes until sperm analysis commenced in less than 30 min after collection.

Experimental design

To investigate the effect of short-term (liquid) storage of brook trout and rainbow trout spermatozoa, semen samples were divided into 15 mL covered tubes and diluted 1:3 (sperm:extender) in a 0.3 M glucose, 10% Dimethyl sulfoxide (DMSO) and 10% egg yolk extender (Tekin et al. 2003), and kept under anaerobic conditions at 4°C. Standard extender was supplemented with L-tryptophan (one per experimental group): Control (0), (a) 0.5 mM, (b) 1 mM, (c) 2 mM, and (d) 4 mM. Sperm was activated with NaCl (52 mM) after short-term storage.

Evaluation of spermatozoa motility

Macroscopic (sperm color and volume) and microscopic analyses (sperm concentration and progressive motility) of fresh semen were performed to determine the quantity and quality of sperm samples. Progressive motility was assessed using a Nikon E50i microscope (Nikon CI, Tokyo, Japan) with a magnification of 200 ×, captured by a Basler A312fc digital camera (Microptic S.L., Barcelona, Spain). Motility analysis from the video sequences was performed using the Sperm Class Analyser (SCA) with the software (Zeiss Axio Scope with AxioVision, Carl Zeiss MicroImaging GmbH, Jena, Germany). The motility rate was determined as actively moved sperm. Time from activation initiation to sperm stop move was described as duration of forward motility. Pooled fresh sperm was used, and sperm with normal pH, volume and motility >80% was selected for the experiment. Spermatozoa density was evaluated using a Burker cell hemocytometer (Brand GMBH + CO KG, Blaubrand®, Germany). Spermatocrit was assessed according to Rurangwa et al. (2004) and expressed as percent (the ratio of white packed material volume/the total volume of semen × 100).

Evaluation of bacterial flora

The stored sperm samples were serially diluted (10-fold dilution) in physiological saline (NaCl: 0.85%) and the spread plating method was used for determination of bacteria group. Aliquots of 0.1 mL of each dilution were spread-plated onto Plate Count Agar (PCA) (total bacteria count), Rose Bengal Agar (RBC) (yeast-mold count), Violet Red Bile Dextrose (VRBD) (Enterobacteriaceae count) and Mannitol Salt Agar (MSA) (Micrococcus/Staphylococcus count). For total bacteria count and Enterobacteriaceae count, media were incubated at 37°C for 48 h (ICMSF 1992; Harrigan 1998). For yeast-mold count, media were incubated at 25°C for 5 days (Mislivec et al. 1992). For Micrococcus/Staphylococcus count, media were incubated at 37°C for 36–48 h (Tamer et al. 1989). All colonies were counted as CFU mL⁻¹. Under the detectable level was <1.0 × 10² cfu.mL⁻¹. Counts were performed in duplicate and sampling occurred on day 0, 2, 4, 6 and 12 of cold storage.

Statistical analysis

Results are presented as mean ± standard deviation (SD). Two-way ANOVA with Duncan test was used for comparison of data. Significance level for all analyses was accepted as p < 0.05. The software SPSS version 14.0 was performed for statistical analyses.

Notes on contributors

Broodfish handling and collection of semen, and evaluation of spermatozoa motility: MK; Evaluation of spermatozoa motility, analyzed the data, and wrote the manuscript: FK; Evaluation of bacterial flora: ÖE; Evaluation of spermatozoa motility: ÖA; Broodfish handling and collection of semen: NB.

Disclosure statement

No potential conflict of interest was reported by the authors.