A Study on the Physical and Biochemical Characteristics of Semen of Tor putitora- An Endangered Fish Species in Himalayan Water

The Semen evaluation both physical and biological characteristics provides fundamental knowledge for management of fish breeding (Alavi et al., 2008). For controlled and high seed production of target fish species in the hatchery, it is necessary to have adequate knowledge of physical characteristics and biochemical composition of semen of concerned fish species (Billard et al., 1995). The Seminal plasma in the semen has a unique composition containing substances that support sperm cells and the substances that reflect the functioning of the reproductive system and spermatozoa. These substances are also involved in the process of activation or inhibition of sperm motility (Morisawa, 1985). After the release of spermatozoa from the testes, their viability is also maintained by the ionic environment provided by the seminal plasma (Ciereszko, 2008). The seminal plasma composition has a significant effect on the biological quality of the semen, as expressed by sperm viability and motility (Sood et al., 2020). Slight change in the seminal plasma composition will reduce semen quality and, subsequently, may lead to a decrease in the fertilization rate.
       
There is wide intra- and inter-species variation in the biochemical composition of seminal plasma of fish species (Alavi and Cosson, 2006). Though there are some information on the semen characteristics of some fishes viz. Cyprinids (Billard et al., 1995; Lahnsteiner et al., 1996), salmonids (Scott and Baynes, 1980; Linhart et al., 1991; Billard and Cosson, 1992; Bozkurt et al., 2011) and sturgeons (Gallis et al., 1991). But studies on the biochemical composition of seminal plasma of Indian fish species are limited (Verma et al., 2009). There is no information about the semen quality of Tor putitora except a very preliminary work on physical parameters by Charak et al., (2018). Therefore, physical and biochemical (inorganic and organic) components of semen plasma of golden mahseer (Tor putitora) have been examined to know its composition and prevailing relationships between various semen parameters. This study will help in understanding the functional aspects of seminal plasma for the development of extender during preservation of semen and assessment of semen quality in artificial fertilization.

Procurement of brooders and collection of semen
 
A total of 51 ripe male brooders with average total fish length of 381.57±60.94 mm and average fish weight of 440.78±159.65 g of golden mahseer (Tor putitora) were procures from the River Bhagirathi, Bhilangana and its impoundment (Tehri Dam reservoir) during May 2017–September 2017. Before stripping, the brooders were anaesthetized using 100ppm of MS 222 at the fishing sites. The semen samples were collected in 4.5 ml cryovials by gently pressing the abdomen towards their genital papilla. The initial 1-2 drops of semen were discarded to avoid contamination of semen from body fluid-viz. mucous, blood and urine etc. After the collection of semen, the brooders were released in their natural habitat. The semen samples in cryovials were brought to the laboratory within two hours of sample collection in crushed ice (0 - 4^oC) and processed for physical and biochemical analysis.
 
Semen colour and volume
 
The semen volume was directly measured in cryovials graded in ml. The colour of semen was visually assessed following its collection.
 
pH and osmolality
 
The pH was measured with a digital pH meter (Hanna make) immediately after sampling. The Osmolality was measured using Micro- osmometer (FISKE ASSOCIATES, Norwood, Massachusetts, USA).
 
Sperm motility
 
The motility of sperm was evaluated within two hours of semen collection. A fine drop of semen was taken on a microscopic glass slide and sperm motility was activated by adding activating solution (distilled water) directly under the microscopic observation. The spermatozoa motility duration and percentage of motile spermatozoa were observed under the Phase contrast microscope (x 200 magnification). Only forward-moving sperm were considered as motile, whereas simple pulsating, agitating or rotating on their axis were considered as immotile. The duration of motility (in seconds) was recorded from the moment of activation to the complete termination of activity under the microscopic field.
 
 
Sperm density and spermatocrit value
 
Haemocytometeric method was used for determining the sperm density. Undiluted semen contains a large number of sperm cells which are difficult to count, hence, the semen was diluted 1000 times (10 µl of semen in 990 µl of 0.7% NaCl) as done by Ciereszko and Dabrowski (1993). A drop of the diluted semen was placed on a haemo-cytometer slide (depth 0.1mm) with a cover-slip on it. After allowing the sperm to settle for 2-3 minutes, sperm count was performed in 10 squares at random under phase contrast microscope and mean value for sperm number is calculated. The sperm number per ml of semen is expressed in number ×10⁹. The spermatocrit value defined as the percentage of white congested sperm cells to the total volume of semen. For spermatocrit measurement, microhematocrit capillary tubes (75mm in length, 1mm with an inner diameter and 0.1 ml capacity) were used. The capillaries were filled with semen and their both ends were sealed with haemoseal wax. The volume (length) of semen in microhematocrit capillaries was measured by meter scale in mm and centrifuged for 35 minutes at 7 minutes interval at 7500 rpm (6315× g). Three replicates of semen samples per male individual for each time interval were exposed for spermatocrit approximation and the mean values were recorded.
 
Seminal plasma composition
 
Initially, Seminal plasma was separated by centrifuging the semen samples at 4500 rpm for 10 minutes at room temperature. The supernatant (seminal plasma) was centrifuged twice to avoid any remnants of sperm cells in seminal plasma and stored in Eppendorf vials at -20^oC until further analysis. The ionic and organic composition was measured using atomic absorption spectrometer- Parkin Elmner-Optima 8000 and Parkin Elmner- Analyst 400.
 
Data analysis
 
All the parameters are expressed as arithmetic means, standard error of means and standard deviation. (Mean and SEM). Statistical analysis was made with SPSS 10 for Windows statistical software package.

The colour of the semen of Tor putitora was varied from milky-white to creamy-white. The average semen volume was recorded as 1.09 ± 0.14 ml (range 0.3 to 4.5 ml) per ripe male brooders (total length 381.57 ± 8.53 mm). The mean sperm density was found to be 1.712 ± 0.18 ×10⁹ sperm ml-1 with a range of 1.28-2.23 × 10⁹ sperm ml^-1 and mean spermatocrit as 62.96 ± 2.63 with a range of 35.93 - 98.07 (Table 1). The sperm remain motile for the varying period of 31-59 sec with an average value of 48.16 ± 1.68 sec which were within the normal range of fresh water teleosts (Billard et al., 1995, Rurangwa et al., 2004). A correlation between fish length and physical spermatological parameters of semen was established and graphically represented in (Fig 1-4.) The fish length had a significant positive relationship with semen volume (r= 0.3, P<0.05, n=51). However, it does not show any significant relationship with Spermatocrit. They had negative non-significant relationship (r= -0.16, P>0.05, n=51). Fish length is found negatively correlated with sperm density (r= - 0.208, P>0.05, n=14) and motility duration (r= - 0.205, P>0.05, n=14). Similarly, semen yield also exhibit feebly negative correlation (r= - 0.175, P>0.05, n=51) with that of spermatocrit (Fig 5). There was high significant positive correlation (r= 0.807, P<0.01, n=14) between sperm density and spermatocrit (Fig 6).
 

 

 

 

 

 

 

       
The physical characteristics and biochemical composition of fish semen have been found to vary with the species (Kruger et al., 2006). The present study has reported a slight variation in sperm motility percentage (3.734 ± 0.182) and duration (48.16 ± 1.68 sec) among the individuals of Tor putitora. Tekin et al., (2003) and Piironen (1985) opined that the variation in motility percentage and duration appear not only among male brooders of different fish species but also within individual male brooders depending on their ripeness. Seasonal variation in sperm motility percentage and duration in fish species was also observed by various authors (Benau and Terner, 1980; Akcay et al., 2004). The variation in sperm motility percentage and duration in different fish species may be due to various external factors such as feeding habit, spawning season of male brooders (Bromage and Roberts, 1995; Rurangwa et al., 2004) or internal factors such as age, dilution ratio and ionic composition of the seminal plasma (Bozkurt et al., 2009). Motility duration of sperm is a species-specific trait. The duration of motility is very short (20-25 sec) in trouts (Bromage and Roberts, 1995). In most of the carps, sperm remain motile for 75-110 sec. (Verma et al., 2009). In cold water snow trout species, it is comparatively less up to 59.7±16.5 sec (Agarwal et al., 2009). In Tor khudree, spermatozoa motility lasts for 60sec (Basavaraja and Hegde, 2005). Our finding on sperm motility duration (48.16±1.68 sec) for Tor putitora is very close to the earlier reports on coldwater fish species.
       
Large variation in sperm density has also been noticed among different fish species. Sperm count in six species of carp ranges between 2.6 to 3.5 ×10¹⁰ ml^-1 (Verma et al., 2009). While the average sperm density of Tor putitora (1.712 ± 0.18 × 10⁹ ml^-1) was very close to Tor khudree (4.90 - 7.45  × 10⁹ ml^-1, Basavaraja et al., 1998) but comparatively lower than the Indian major carps (2.6 to 3.5×10¹⁰ ml^-1, Verma et al., 2009). On the other hand, it was higher than the coldwater snow-trouts Schizothorax richardsonii (3.77±0.78 × 10⁸ ml^-1, Agarwal and Raghuvanshi, 2009; Raghuvanshi et al., 2019) and Schizothoraichthys progastus (8.67±0.50 ×10⁸ ml^-1, Agarwal et al., 2013). Sperm density has been reported as high as 5.0-6.5 ×10¹⁰ ml^-1 in Bluefin Tuna (Doi et al., 1982) and lowest figure 2.0 ×10⁶ ml^-1 in Pike (Kolders and Moczarski, 1983). These variations may be due to the size of sperm and species-specific feature. Significant correlation between sperm density and spermatocrit was reported in snow trout (Agarwal and Raghuvanshi, 2009) and other teleost species (Rakitin et al., 1999; Bhatt et al., 2017). The present study also exhibits high positive significant correlation between sperm density and spermatocrit and suggest the use of regression equation for quick and reliable estimation of sperm concentration in Tor putitora.
       
Findings on the inorganic and organic composition of semen are summarized in Table 2. The sodium (102.06± 5.115 mM l^-1), potassium (51.66±3.205 mM l^-1), calcium (1.38±0.304 mg dl^-1) and inorganic-phosphate (4.000±1.102 mg dl^-1) ions predominate in the seminal plasma. The concentration of other inorganic constituents of semen was found as-copper 0.004 ± 0.001 mg l^-1, magnesium 0.860± 0.261 mg l^-1 and zinc 0.062±0.005 mg l^-1.
 

       
Alike, several teleosts (Alavi and Cosson, 2006), sodium and potassium ions predominate in seminal plasma of Tor putitora. Seminal plasma of mahseer has higher Na^2± concentration (102.06 ± 5.115 mM l^-1) than the mirror carp (67.12 mM l^-1, Bozkurt et al., 2009), common carp (75 mM l^-1, Morisawa et al., 1983a), grass carp (98 mM l^-1, Bozkurt et al., 2008), rainbow trout (75 mM l^-1, Morisawa et al., 1983b), rainbow trout (80 mM l^-1, Secer et al., 2004), but lower than the perch (124 mM l^-1, Lahnsteiner et al., 1995), muskellunge (129 mM l^-1, Lin et al., 1996) and catfish (164 mM l^-1, Tan-Fermin et al., 1999). However, concentration of potassium (51.66±3.205 mM l^-1) in Tor putitora is lower than mirror carp (105.1 mM l^-1, Bozkurt et al., 2009) and higher than perch (10.0 mM l^-1, Lahnsteiner et al., 1995), Asian catfish Clarias microcephalus (18.0 mM l^-1, Tan-Fermin et al., 1999) and rainbow trout (46.0 mM l^-1, Secer et al., 2004). The calcium and magnesium ions contribute significantly to the inorganic composition of seminal plasma in fish semen. The average concentration of calcium (1.38 ±0.304 mg l^-l) and magnesium (0.86 ±0.261 mg l^-l) ions in Tor putitora are found close to the brown trout (calcium 1.7±0.2 mM^-l, magnesium 1.0±0.1 mM^-l) reported by Hatef et al., (2007). This variability in ionic composition seems to be a species-specific character.
       
The higher concentration of potassium inhibits sperm motility in salmonids (Morisawa and Suzuki, 1980; Bozkurt et al., 2011) but it increases sperm motility in carp (Billard and Cosson, 1992). Several studies (Cosson et al., 1989; Boitano and Omoto, 1991) indicated that the divalent cations (mainly calcium and magnesium) concentration of seminal plasma increases when sperm motility is initiated. Scott and Bynes, (1980) opined that the ionic concentration of semen has a key role in regulating sperm motility either by contributing the intracellular ionic composition or by their osmolality. Majumder et al., (2020) reported trace minerals like copper and zinc improve the quality and quantity attributes of semen in cattle. According to Stoss (1983), osmolality, pH and ionic composition of semen are the most important factor responsible for inhibition or activation of sperm motility.
       
Seminal plasma osmolality in teleosts varies between 250-450 mOsmol kg^-1. In marine species osmolality is reported as 306 mOsmol kg^-1 in turbot (Suquet et al., 1992), 355.6 mOsmol kg^-1 in ocean pout and 364.6 mmol kg^-1 in sea bream (Chambeyron and Zohar, 1990). Among freshwater species, it is 254.0-334.5 mOsmol kg^-1 in cyprinids (Billard and Cosson, 1992; Lahnsteiner et al., 1994), 300 mOsmol kg^-1 in salmonids (Billard and Cosson, 1992). Our results on osmolality in Tor putitora seminal plasma (256.8±31.940 mOsmol kg^-1) are comparable to the other carp species (269-289 mOsmol kg^-1, Verma et al., 2009). Hypo-osmotic or hyper-osmotic environment triggers the initiation of fish sperm motility. According to Morisawa et al., (1983), sperm of freshwater fishes become motile when diluted in a hypotonic solution. Carp sperm motility also initiated in media of hypo-osmotic pressure i.e. below 150-200 mOsmolkg^-1 (Perchecet_al1995).
       
The results on the organic components (viz. glucose, cholesterol, triglycerides, total protein, creatinine and albumin) of semen of Tor putitora are summarized in Table 2. The Glucose concentration (9.6±0.678 mg dl^-1) in Tor putitora is higher than Atlantic Salmon (0.45±0.15 mMl^-1, Aas et al., 1991), Perca fluviatilis (0.063±0.019 mM l^-1, Lahnsteiner et al., 1995), Indian major carps, grass carp and silver carp (1.2-2.0 mg dl^-1, Verma et al., 2009). The triglyceride (11±0.316 mg dl^-1) is also higher in Tor putitora than the rainbow trout (0.18±0.12 mMl^-1, Lahnsteiner et al., 1998). It is very close to Cyprinus carpio (10.3±1.01 mg dl^-1, Bozkurt et al., 2009) and Labeo rohita (11.7±1.1 mg dl^-1, Vermaet_al2009) but significantly lowers than silver carp and Grass carp (18.0±0.2 mg dl^-1 and 39±2.44 mg dl^-1, Verma et al., 2009). Low level of triglycerides was also found in the seminal plasma of cyprinids (Lahnsteiner et al., 1994). The seminal plasma of Tor putitora has shown higher cholesterol level (21.0±1.049 mg dl^-1) comparative to Indian major carps, grass carp and silver carp (12.7 -17.4 mg dl^-1, Verma et al., 2009). Triglycerides and glucose present in the semen serve as energy source for spermatozoa during immotile storage and during the regeneration phase after motility (Stoss 1983; Lahnsteiner et al., 1993). Lipids and cholesterol in the seminal plasma might have a protective role to spermatozoa and glucose serve as an external cryoprotectant (Maisse, 1996; Secer et al., 2004; Bozkurt et al., 2008; Agarwal, 2011).
       
The low concentration of protein (0.1±0.0 g dl^-1) was found in Tor putitora. It is considerably lower than rainbow trout (1.47±0.84 mg ml^-1, Lahnsteiner et al., 1998) and Caspian brown trout (0.74±0.14 mg ml^-1, Hatef et al., 2007). In general, fish seminal plasma has several times low protein concentration than the mammalian seminal plasma. (Ciereszko and Dabrowski, 1993; Cheema and Babbar 2008). The functions of fish seminal plasma protein are not completely known. It may have some role in sperm motility. Landsteiner et al., (2004) found that seminal plasma protein prolongs the motility rate of rainbow trout spermatozoa. While, Cheema and Babbar (2008) reported that protein had a protective role and has significant correlation with freezability and fertility in cattle bulls. The remarkable concentration of urea (12.6±1.720 mg dl^-1) was also determined in seminal plasma. Urea is considered with protein metabolism as it occurs as a result of digestion of protein. Creatinine level in this study was 1.2±0.255 mg dl^-1. Creatinine kinase is a crucial enzyme for sperm function because it participates in ATP regeneration and acts as a chemical shuttle between creatinine and creatinine phosphate. The activity of creatinine kinase may be a useful marker of damage to Clupea harengus spermatozoa (Grzyb et al., 2003). The uric acid level in T. putitora was 0.50±0.10 mg dl^-1. High concentrations of uric acid have been reported in the seminal plasma of various teleosts (Ciereszko et al., 1999). Uric acid acts as a significant antioxidant, so it has a potential role in the protection of fish spermatozoa against oxidative damage (Alavi et al., 2008).

The results of the present study on physical and biochemical characteristics of semen of endangered golden mahseer Tor putitora provides baseline information about the inorganic and organic constituents of semen that will help in optimizing the selection process for high-quality male spawners for artificial reproduction in hatchery operations. The study will also help in the formulation of extenders and dilutants for semen storage of Tor putitora.

The first author is thankful to the University Grant Commission, New Delhi, for providing CSIR- UGC-Junior Research Fellowship. The authors are grateful to Dr Praveen Kumar, Department of Endocrinology, SGPGI Lucknow for his support in the Biochemical analysis.