Sperm quality, egg size, fecundity and their relationships with fertilization rate of grass (Ctenopharyngodon idella)

Item Type article

Authors Bozkurt, Y.; Öğretmen, F.

Download date 30/09/2021 12:18:46

Link to Item http://hdl.handle.net/1834/37317 Iranian Journal of Sciences 11(4) 755-764 2012 Sperm quality, egg size, fecundity and their relationships with fertilization rate of grass carp (Ctenopharyngodon idella)

*1 2 Bozkurt Y. ; Öğretmen F.

Received: March 2012 Accepted: July 2012

Abstract Gametes were collected by abdominal massage from 15 anesthesized male and female grass carp. In collected sperm samples the following mean spermatological properties were determined: sperm volume 15.64 ± 5.39 mL, sperm motility 81.20 ± 7.61%, motility duration 62.06 ± 19.40s, density 17.88±5.48 x109 mL-1, total density 428.90±302.05 x109, and pH 7.25±0.81. Also egg size (mm) and fecundity (egg/fish) were determined as 1.04±0.028 and 417867 ± 36.274 respectively in female grass carp. The fertilization capacity of sperm was tested with the same egg pool. The insemination dosage was 2x105 spz/egg for each fertilization experiment. Fertilization rate was determined as mean 79.3±2.95% and positively correlated with sperm motility (r=0.932, p<0.01), egg size (r=0.513, p>0.05) and fecundity (r=0.539, p>0.05).

Keywords: Grass carp, Sperm, Motility, Fertilization

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1-Mustafa Kemal Univ., Fac. of Fisheries, Dept. of , Hatay, Turkey. 2-Muğla Univ., Fac. of Science, Dept. of Biology, Muğla, Turkey *Corresponding author’s e-mail: [email protected]

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Introduction Grass carp (Ctenopharyngodon idella), fertilization in grass carp. From this point also known as White Amur, is a vegetarian of view, the objectives of this research was fish living in the Amur River in Asia. designed to determine physical sperm Because of its herbivorous feeding habits, quality parameters and as well as to it has received considerable attention as examine the relationships between biological control of aquatic vegetation. spermatological parameters, egg size, Grass carp can reproduces only its own fecundity and fertilization success in grass original habitat. Therefore, grass carp carp. culture is an important issue to overcome excess aquatic vegetation. So, gamete Materials and methods quality and artificial insemination is very Broodstock management and collection of important to obtain viable larvae for grass gametes carp culture. The experiment was carried out at State The aquaculture industry has been Hydrolic Works (SHW) Fish Reproduction more focused on fish eggs rather than Station, Adana, Turkey, during spawning sperm in spite of both gamete may effect season of grass carp in 2010. The parental fertilization success. However, sperm broodstock were held in sand ponds under quality is very important variable in a natural photoperiod regime and fasted 48 aquaculture broodstock management and it h before sperm collection. The broodstock can influence the fertilization of eggs. were anaesthetized in 100 ppm of MS 222 Parameters to evaluate sperm quality (Argent Labs., Redmond,WA, USA) include sperm volume, spermatozoa before stripping. Water temperature varied density, motility and motility period of between 22-24°C during spawning season. spermatozoa (Billard et al., 1995). Motility Sperm and eggs were collected by is very important parameter since abdominal massage. Sperm was collected Downloaded from jifro.ir at 15:57 +0330 on Wednesday February 14th 2018 spermatozoa must be motile to achieve egg from 15 mature males (TW 4.86±0.44 kg, penetration (Chauvaud et al., 1995). TL 53.9±1.23 cm) by manual abdominal Spermatozoa motility and duration can stripping 12 h after a single injection of 2 vary according to fish species and mg/kg of carp pituitary extract (CPE) at temperature during spermiation period 24°C water temperature. Sperm was (Billard, 1986). In addition, spermatozoa sampled into glass tubes and used in case density and sperm pH are considered other of uncontaminated with water, blood, urine indicators of sperm quality as they can also and faeces. Sperm was stored on ice (4°C) effect the fertilization rate (Lahnsteiner et until used for experiment. Eggs were also al., 1998). Therefore fecundity, egg size collected from 15 mature (4-5 years old) and sperm quality parameters should be females (TW 6.44±0.79 kg, TL 56.7±4.19 considered together to estimate cm) that were stripped gently massaging of fertilization success. the abdomen 10-12 h after a double There is a lack of literature data on injection of 3.5 mg/kg CPE. The first sperm quality and its relationship with injection, 10% (0.35 mg/kg) CPE was

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given 10h before the second (3.15 mg/kg). 30 min of sampling. Semen colour was Only transparent, well rounded and evaluated visually immediately following unwrinkle eggs were used for fertilization. collection. Evaluation of semen Fecundity, egg size and fertilization Sperm was collected in glass tubes and Fecundity was calculated by volumetric sperm volume was registered immediately method and egg size was determined using following collection by abdominal a sensitive micrometer. Fertilization massage. Motility was evaluated using a experiments were performed in triplicate dark-field microscope at x400 using an egg pool at 24°C water magnification and was expressed as temperature. Eggs pooled from each 15 percentage. 0.3% NaCl was used as females were divided to 45 egg batches in activation solution to estimate motility. small dishes. Fertilization took place in For this aim, about 10 µL semen was these dry plastic dishes and 100 g of eggs placed on microscope slide and 100 µL were placed into each dish. Fertilization activation solution was added. Each capacity of sperm from each males were motility determination was performed in tested with the same egg pool. The triplicate for each semen sample. Same insemination dosage was 2x105 spz/egg for person conducted all the sperm motility each fertilization experiment. The observations in order to decrease the fertilization solution (3 g of urea, 4 g of degree of variation among observers. NaCl in 1 L of water) and the dry Duration of spermatozoa motility fertilization technique were used. Semen was estimated using a sensitive were added over the eggs and gently chronometer (1/100) starting mixed before activation with 20 mL simultaneously with the addition of fertilization solution. activation solution into the sample until Following fertilization eggs were 5% of the spermatozoa maintained forward stirred for 10-15 min and then the eggs Downloaded from jifro.ir at 15:57 +0330 on Wednesday February 14th 2018 swimming activity. Spermatozoa density were rinsed with hatchery water two or was determined by the hemacytometric three times to eliminate the stickness of the method. For this aim, sperm was diluted eggs and placed into the incubation trays. (1/1000) with Hayem solution (5 g The fertilization ratio was counted in the

Na2SO4, 1 g NaCl, 0.5 g HgCl2 200 mL 4-cell stage under a stereomicroscope at bicine) and a droplet of the diluted sperm 20-fold magnification. was placed on Thoma’s hemocytometer Statistical analysis slide (depth 0.1 mm) with a coverslip and Motility data were normalized through counted using dark-field microscope. After arcsine transformation. Correlations a few minutes (to allow sperm between spermatological parameters, sedimentation), the number of fecundity, egg size and fertilization rates spermatozoa was counted at x400 were estimated using Pearson’s correlation magnification and expressed as x109 mL-1. test. Results are presented as mean±SE. Semen pH was measured using pH meter Significance was considered at the level of (Model GLP 21, Crison, Barcelona) within α=0.05. Statistical analyses were

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performed with SPSS 10 for Windows spermatozoa motility (r=0.932, p<0.01) statistical software package. and spermatoza density (r=0.826, p<0.01). Results Correlations between fertilization rates and Physical characteristics of semen were sperm quality parameters are shown in found to be rather variable and are Table 2. Mean fecundity of grass carp presented in Table 1. Semen colour was collected from each female was determined as milky white in all samples. 417867±36.274 (ranged from 202154 to The volume of sperm collected for each 715329) and mean egg size was male was 15.64±5.39 mL (ranged from 4 determined as 1.04±0.028 mm (ranged to 23 mL). Mean spermatozoa motility was from 0.9 to 1.2 mm). A negative 81.2±7.61% (ranged from 70 to 95%) and relationship (r=-0.412, p>0.05) was the mean forward motility duration was determined between fecundity and egg 62.06±19.40 s (ranged from 35 to 117s). size. Statistical analysis shows positive The spermatozoa density was 17.88±5.48 correlation between fertilization rates and x109 spz.mL-1 (ranged from 8 to 35 x109 fecundity (r=0.539, p>0.05) and also spz.mL-1). Sperm motility positively between fertilization rates and egg size correlated with spermatozoa density (r=0.513, p>0.05). Relationships between (r=0.742, p<0.01) and movement duration fertilization rates, sperm quality (r=0.855, p<0.01). Mean fertilization rate parameters, fecundity and egg size are was 79.3±2.95% (ranged from 60% to shown in Figure 1 (a-h). 95%) and highly correlated with

Table 1: Sperm quality parameters, fecundity and egg size of grass carp (n=15) Minimum Maximum Mean SEM Volume (mL) 4 23 15.64 5.39 Motility (%) 70 95 81.20 7.61 Movement duration (s) 35 117 62.06 19.40 Density (x109 mL-1) 8 35 17.80 5.48 Downloaded from jifro.ir at 15:57 +0330 on Wednesday February 14th 2018 Total density (x109) 73 1177 428.90 302.05 pH 6 8.5 7.25 0.81 Egg size (mm) 0.9 1.2 1.04 0.03 Fecundity (egg/fish) 202154 715329 417867 36.274

Table 2: Correlations between sperm quality parameters, and fertilization rates in grass carp Sperm Spermatozoa Movement Spermatozoa Total spz Sperm Volume Motility Duration Density Density pH Spermatozoa 0.327 Motility Movement 0.848* 0.855* Duration Spermatozoa 0.908* 0.742* 0.934* Density Total spz. 0.822* 0.912* 0.935* 0.863* Density Sperm pH 0.244 0.317 0.343 0.250 0.290 Fertilization 0.871* 0.932* 0.894* 0.826* 0.857* 0.273 Rate *Correlation is significant at the 0.01 level

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100 100

90 90 80 80 70 70 y = 1,3178x - 28,006 y = 1,5634x + 55,549 2 2 R = 0,8687 60 R = 0,7595 60 Fertilization(%) 50 Fertilization(%) 50 2 5 8 11 14 17 20 23 26 29 60 70 80 90 100 Sperm volume (ml) Spermatozoa motility (%)

Figure 1a: Relationship between fertilization rates Figure 1b: Relationship between fertilization rates and sperm volume and spermatozoa motility

100 100 90 90 80 80 70 y = 0,5037x + 46,895 70 y = 0,0013x + 56,117 R2 = 0,8 2 60 R = 0,6821 60 Fertilization(%) Fertilization(%) 50 50 20 40 60 80 100 120 0 10000 20000 30000 40000 Spz. movement duration (s) Spermatozoa density (x106/ml) Figure 1c: Relationship between fertilization rates Figure 1d: Relationship between fertilization rates and spermatozoa movement duration and spermatozoa density

100 100 90 90 80 80 70 y = 4E-05x + 63.045 70 y = 11.484x - 3.7633 60 R² = 0.7343 60 R² = 0.676 50 50 Fertilization(%) Fertilization(%) 60000 560000 1060000 4 9 14 6 Spz. total density (x10 ) Sperm pH

Figure 1e: Relationship between fertilization rates Figure 1f: Relationship between fertilization rates Downloaded from jifro.ir at 15:57 +0330 on Wednesday February 14th 2018 and total spermatozoa density and sperm pH

100 100 90 80 80 60 y = 4E-05x + 60.723 70 y = 52,273x + 24,636 40 R2 = 0,2631 R² = 0.2892 60 20 Fertilization (%) Fertilization

50 (%) Fertilization 0 0.8 1 1.2 1.4 0 500000 1000000 Egg Size (mm) Fecundity (egg/fish) . Figure 1g: Relationship between fertilization rates Figure 1h: Relationship between fertilization and and egg size fecundity. Figure1: Variables correlated to fertilization rate: a) sperm volume, b) spermatozoa motility, c) spermatozoa movement duration, d) spermatozoa density, e) total spermatozoa density, f) sperm pH, g) egg size, h) fecundity.

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Discussion The relationship between sperm quality in the present study. This value complies parameters, fecundity, egg size and with that of Zhukinskiy and Alekseenko fertilization poorly understood in grass (1983) and Bozkurt et al. (2008) for this carp. For this purpose, the current study species. The sperm motility range of was aimed to evaluate semen quality of samples studied was 70-95% and the grass carp determining the spermatological fertilization rate range was 60-95%. The parameters and fertilization rate in order to motility was well and positively correlated determine the reproductive capability of (r=0.932, p<0.01) with the fertilization this species under captive conditions. yield. The significant positive correlation Sperm volume is one of the features between sperm motility and fertilization reflecting the semen yield and indicates that grass carp eggs can only be spermatozoa density. The mean sperm fertilized with motile spermatozoa. volume of grass carp (15.64±5.39 mL) was Observations on the fertilization similar to results reported by Bozkurt et al. process in teleosts demonstrate that (2008) for grass carp (14.44±1.16 mL) and spermatozoa must swim actively into the Akcay et al. (2004) for mirror carp egg micropylar channel since immotile (13.26±2.51 mL) but different from spermatozoa cannot perform the reported by Bozkurt and Secer (2004) for fertilization process (Iwamatsu et al., mirror carp (8.95±12.95 mL). The 1993). On the other hand, mean motility difference may be due to differences in duration (62.06±19.40 s) was lower than feeding conditions and regime, that of Zhukinskiy and Alekseenko (1983) environmental factors, or spawning time. for this species. This value also different The spermatozoa motility and its from reported by Akcay et al. (2004) for duration have great influence on successful mirror carp and Bozkurt (2006) for scaly fertilization. However, spermatozoa carp. Downloaded from jifro.ir at 15:57 +0330 on Wednesday February 14th 2018 motility varies in vigor and duration not Spermatozoa density may also only among males but also within effect the fertilization rate (Aas et al., individual males depending on ripeness 1991). Findings on spermatozoa density (Tekin et al., 2003; Bozkurt et al., 2011). It 9 -1 has been shown that sperm motility (17.88±5.48 x10 mL ) in the present performances (percentage of motile sperm study corroborates with the results and period of motility) change at spawning reported by Bozkurt et al. (2008) for grass season in some teleost fish (Koldras et al., carp (15.43±0.72) and Akcay et al. (2004) 1996). The observed changes seem to be 9 -1 related to the changes in seminal fluid for mirror carp (17.33±1.22 x10 mL ) but composition as well as hormonal changes not those reported by Emri et al. (1998). in the hypothalamous (Bozkurt et al., The differences may be due to feeding 2009). conditions, age, environmental factors Mean spermatozoa motility of grass carp was determined as 81.2±7.61% (such as day length and temperature) or

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semen dilution ratio. According to Billard and egg size as separate indicators of et al. (1995), semen pH can effect reproductive performance. It is generally spermatozoa motility and maturation. So, accepted that there is an inverse determination of variation in sperm pH can relationship between fecundity and egg provide information on fertilization size that fish produce either more eggs of a capacity of spermatozoa. Mean pH smaller size or fewer eggs of a larger size determined in this study (7.25±0.81) was (Bromage et al., 1992). Similarly, negative generally confirmed by Saad et al. (1988), relationship (r=-0.412, p>0.05) was Lubzens et al. (1997) and Emri et al. determined between fecundity and egg size (1998). in this research. On the other hand, Fertilization procedure was carried fertilization rates positively correlated with out using 2x105 spermatozoa per egg as fecundity (r=0.539, p>0.05) and egg size stated by Munkittrick and Moccia (1984). (0.513, p>0.05). For this reason, it can be Mean fertilization rate (79.3±2.95%) was concluded that fecundity and egg size lower than the findings of Zhukinskiy and should be considered together to evaluate Alekseenko (1983) that reported their fertilization success in grass carp. results in the range of 90.2-97.7%. Dada In conclusion the present research and Ogunduyile (2011) reported positive showed that mature males releasing sperm relationship between sperm volume and with low motility and low density should fertilization rates in African be culled from the broodstock. Reducing (Clarias gariepinus). Also, Linhart et al. the number of male broodstock maintained Downloaded from jifro.ir at 15:57 +0330 on Wednesday February 14th 2018 (2000) indicated good correlation (r=0.53) for spawning can significantly improve between motility and fertilization at hatchery efficiency and minimize feed spermatozoa/egg ratios of around 2x105:1. costs. This investigation provides useful Similarly, higher positive relationship information for efficient gamete (r=0.932, p<0.01) was determined between management and increased fry yields in motility and fertilization ratio at 2x105 aquaculture operations and can help spermatozoa/egg ratio. preperation of gametes for Fecundity and egg size was cryopreservation experiments. On the determined similar to the values reported other hand, further experiments are needed for Ctenopharyngodon idella (Horvath et to increase the viability, survival and al., 1984). Most studies on fish development of larvae used in farming reproduction tend to consider fecundity conditions.

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Acknowledgements Bozkurt, Y., 2006. Relationship between The author would like to thank the staff of body condition and spermatological the State Hydrolic Works (SHW) Fish properties in scaly carp (Cyprinus Reproduction Station for their most efficient assistance during the carpio) semen. Journal of and experimental period. Veterinary Advances, 5(5), 412-414. Bozkurt, Y., Öğretmen, F., Erçin, U. References and Yıldιz, Ü., 2008. Seminal plasma Aas, G.H., Refstie, T. and Gjerde, B., composition and its relationship with 1991. Evaluation of milt quality of physical spermatological parameters of Atlantic . Aquaculture, 95, 125– grass carp (Ctenopharyngodon idella) 132. semen: with emphasis on sperm Akcay, E., Bozkurt, Y., Secer, S. and motility. Aquaculture Research, 39, Tekin, N., 2004. Cryopreservation of 1666-1672. mirror carp semen. Turkish Journal of Bozkurt, Y., Öğretmen, F. and Seçer F. Veterinary and Animal Sciences, 28(5), S., 2009. Effect of different extenders 837-843. and storage periods on motility and Billard, R., 1986. Spermatogenesis and fertilization success of grass carp spermatology of some teleost fish (Ctenopharyngodon idella) sperm species. Reproduction Nutrition during spawning season. Journal of Development, 26, 877-920. Agricultural Sciences, 15 (3), 277-284. Billard, R., Cosson, J., Crim, L.W. and Bozkurt, Y., Öğretmen, F., Kökçü, Ö.

Downloaded from jifro.ir at 15:57 +0330 on Wednesday February 14th 2018 Suquet, M., 1995. Biology of sperm and Erçin, U. 2011. Relationship and artficial reproduction in carp. between seminal plasma composition Aquaculture, 129, 95-112. and sperm quality parameters of the Bozkurt, Y. and Secer, S., 2004. Salmo trutta macrostigma Determination of some spermatological (Dumeril,1858) semen: with emphasis properties of mirror carp (Cyprinus on sperm motility.Czech Journal of carpio L. 1758) semen and evaluation Animal Sciences, 56 (8), 355-364. of short-term preservation using Bromage, N.R., Jones, J., Randall, C., different extenders. In: 3th Nat. Cong. Thrush, M., Davies, B., Springate, J., Reprod. Art. Insem., September 30- Duston, J. and Barker, G., 1992. October 2. Antalya, Turkey. Broodstock management, fecundity, egg quality and the timing of egg

Iranian Journal of Fisheries Sciences, 11(4), 2012 763

production in the rainbow Koldras, M., Loir, M., Maise, G. and Le (Oncorhynchus mykiss). Aquaculture, Gac, F., 1996. Study of the 100, 141-166. composition of seminal fluid and of Chauvaud, L., Cosson, J., Suquet, M. sperm motility along the genital tract, and Billard, R., 1995. Sperm motility during the spawning season, in rainbow in , Scopthalmus maximus: trout (Oncorhynchus mykiss). Aquatic initiation of movement and changes Living Resources, 9, 337–45. with time of swimming characteristics. Lahnsteiner, F., Berger, B., Weismann, Environmental Biology of Fishes, 43, T. and Patzner, R. A., 1998. 341-349. Determination of semen quality of the Dada, A. A. and Ogunduyile, F.D., 2011. , Oncorhynchus mykiss, Effects of velvet bean (Mucuna by sperm motility, seminal plasma pruriens) on sperm quality of African parameters, and spermatozoal catfish Clarias gariepinus (Burchell, metabolism. Aquaculture, 163, 163–81. 1822) Broodstock. Journal of Fisheries Linhart, O., Rodina, M. and Cosson, J., and Aquatic Sciences, 6 (6), 655-661. 2000. Cryopreservation of sperm in Emri, M., Marian, T., Tron, L., Balkay, Cyprinus carpio: Sperm L. and Krasznai, Z., 1998. motility and hatching success of Temperature adaptation changes ion embryos. Cryobiology, 41, 241–250. concentrations in spermatozoa and Lubzens, E., Daube, N., Pekarsky, I., seminal plasma of common carp Magnus, Y., Cohen, A., Yusefovich, Downloaded from jifro.ir at 15:57 +0330 on Wednesday February 14th 2018 without affecting sperm motility. F. and Feigin, P., 1997. Carp Aquaculture, 167, 85-94. (Cyprinus carpio L.) spermatozoa Horvath, L., Tamas, G. and Tolg, I., cryobanks – strategies in research and 1984. Special methods in pond fish application. Aquaculture, 155,13-30. husbandry. Akademiai Kiado. Munkittrick, K.R. and Moccia, R.D., Budapestsh.148 p. 1984. Advances in the cryopreservation Iwamatsu, T., Ishijma, S. and of salmonid semen and suitability for a Nakashima, S., 1993. Movement of production-scale artificial fertilization spermatozoa and changes in micropyles program. Theriogenology, 21,645-659. during fertilization in medaka eggs. Saad, A., Billard, R., Theron, M.C. and Journal of Experimental Zoology, 266, Hollebecq, M.G. 1988. Short-term 57-64.

764 Bozkurt & Ogretmen , State of Mnemiopsis leidyi (Ctenophora: Lobata)…

preservation of carp (Cyprinus carpio) Zhukinskiy, V.N. and Alekseenko, V.R., semen. Aquaculture, 71,133-150. 1983. Semen quality in common carp, Tekin, N., Secer, S., Akcay, E., Bozkurt, Cyprinus carpio,and white amur, Y. and Kayam, S., 2003. The effect of Ctenopharyngodon idella (), age on spermatological properties in in different periods of the spawning rainbow trout (Oncorhynchus mykiss season and as influenced by extraction W., 1792). Turkish Journal of methods. Journal of Icthyology, 23, Veterinary and Animal Sciences, 27, 124-133. 37-44.

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