Introduction

Most fishes are reproduced by external fertilization; success is influenced by several factors including sperm motility (Lahnsteiner et al. 1998), breeding season (Methven and Crim 1991), sperm competition within males (Stockley et al. 1997), health condition (Rakitin et al. 1999), and gamete interaction time (Rakitin et al. 1999; Turner and Montgomerie 2002). Despite the recent work about influence of the mentioned factors on fertilization success, relatively few information is available on the effect of male age (Casselman and Montgomerie 2004). Age-related decrease in sperm quality has been documented in Atlantic salmon Salmo salar (Kazakov 1981), rainbow trout Oncorhynchus mykiss (Schmidt-Baulain and Holtz 1991; Chechun et al. 1994), mature turbot Scophthalmus maximus (3–6 years old) (Suquet et al. 1998), and striped bass Morone saxatilis (Vuthiphandchai and Zohar 1999). It has been reported that swimming velocity of spermatozoa is strongly affected by the aging of mature male bluegill Lepomis macrochirus (Casselman and Montgomerie 2004) and guppy Poecilia reticulata (Gasparini et al. 2010). An age-related increase in sperm production, carotenoid pigmentation, and black spots on their body were documented in mature male guppies P. reticulata (Evans et al. 2002). In contrast, aging of male zebrafish Danio rerio is associated with a change in sperm content but not in sperm motility traits or fertility (Kanuga et al. 2011). Sperm motility and spermatocrit have been commonly used as reliable factors to evaluate the semen quality in teleosts (Harrell et al. 1990; Suquet et al. 1992; Rana 1995). Information about the sperm quality of different age groups of broodstock is of great importance for optimizing successful artificial propagation.

The effect of age on the reproductive performance in females has been reported in rainbow trout (Pitman 1979), striped bass (Monteleone and Houde 1990; Zastrow et al. 1989; Vuthiphandchai and Zohar 1999), bullhead Cottus gobio (Abdoli et al. 2005), common carp Cyprinus carpio (Mordenti et al. 2003; Aliniya et al. 2013), and African catfish Clarias gariepinus (Jokthan 2013). Egg size and fecundity have been considered as key factors used to evaluate the reproductive performance of females. Egg quality can be defined as the ability of the egg to be fertilized and produce normal embryo with high survival rate (Brooks et al. 1997; Bobe and Labbé 2010). Egg size has been shown to be positively correlated with fertilizing capacity (Buckley et al. 1991), while absolute fecundity increases with female body size (Bagenal 1978; Kamler 1992). However, the effect of female age on fecundity is variable (Wootton 1979) and should be considered within the context of the species biology such as longevity and age of sexual maturity.

The bighead carp Hypophthalmichthys nobilis is one of the important Asian carps. It is native to the large rivers and associated flood plain lakes of eastern Asia. Their range extends from southern China to the Amur River system, which forms the northern border of China and the southern border of Russia. It has been widely distributed in Europe and America (Kolar et al. 2005). Adults usually have a mottled silver-gray coloration, a large and scaleless head, and a large mouth. This species has been ranked as the fifth-highest production (7.5%) of all cultured freshwater fish worldwide. In spite of its significant commercial value and the high proportion of propagation in hatcheries, there is no information available on the age-related reproductive performance of bighead carp. Therefore, the objectives of this study on bighead carp were (i) to determine the age-related changes in male and female characteristics and (ii) to examine the effects of age on the male and female reproductive fitness in terms of fertilization, hatching, and larvae survival rate.

Material and methods

Broodstock husbandry and gamete collection

For this study, eight mature males (4 of them 3 years old and others 4 years old) and eight females (4 of them 4 years old and others 5 years old) were selected. The average weight and length for 3 and 4-year-old males were 5200 ± 80.5 g, 75.0 ± 0.8 cm, and 9300 ± 80.7 g, 93.0 ± 0.8 cm, respectively. The average weight and length for 4 and 5-year-old females were 11,400 ± 89.5 g, 90.5 ± 0.5 cm, and 15,005 ± 67.7 g, 95.0 ± 1.0 cm, respectively. To induce spermiation, a single dose of intramuscular injection of carp pituitary extract (CPE) at 0.5 mg kg−1 body weight was used. Milt was collected 10 h after injection by a 5-mL plastic syringe; care was taken to avoid the contamination of urine and blood. Milt was collected before stripping the females and stored in icebox until used. Ovulation was induced by CPE using an initial injection of 0.2 mg kg−1 body weight and a second injection of 2.0 mg kg−1 body weight, 12 h after the first injection. Eggs were collected 10 h after the second injection.

Sperm analysis

Prior to analyzing sperm concentration, milt was diluted 10,000 times with a saline solution (0.07 NaCl) following the methods of Golpour et al. (2013). A 10 μL of diluted sperm was placed onto a Burker cell hemocytometer, and spermatozoa were counted at ×200 magnification (Olympus, BX41, Tokyo, Japan). The number of cells was counted in 20 squares (depth 0.1 mm × length 0.2 mm) of the hemocytometer and expressed as spermatozoa × 109/mL. Spermatozoa were activated in 0.3% NaCl solution at a dilution rate of 1:2000. Sperm motility was triggered directly in activation medium of 0.3% NaCl at ratio 1:2000 and immediately recorded with a CCD video camera (Panasonic 240 Japan) mounted on a dark-field microscope (Leica Camera, Allendale, USA). The duration of sperm motility was measured immediately after initiation of sperm activation until 100% of the spermatozoa were immotile.

Video records were analyzed to estimate the percent of motile cells (motility rate) by micro-image analyzer (Olympus Micro Image 4.0.1. for Windows, Japan) on five successive overlapping video frames. The percentage of motility was defined as the percentage of progressively motile spermatozoa within each activated sample. Progressively motile spermatozoa were defined as actively swimming. Only moving sperm cells either in forward or any direction were judged motile, and sperm cells that vibrated at the place were not considered motile (Golpour et al. 2013). Triplicate samples were measured for each male. For spermatocrit analysis, intact semen was placed into a glass microhematocrit capillary tube (75 mm length, 1.1 to 1.2 mm internal diameter), filling 60–80% of volume. One end of the tube was sealed with clay and then centrifuged for 8 min at 3000 rpm (Sigma, 13 USA). Spermatocrit was measured according to Rurangwa et al. (2004).

Seminal plasma composition

Seminal plasma was obtained by centrifugation of milt immediately after collection at 400×g for 10 min at 20 °C. The seminal plasma pH was measured using a laboratory pH meter (pH meter, Iran 762) and then a 300 μL sample from each male was frozen at −20 °C for further analysis. Calcium (Ca+2), Magnesium (Mg+2), and glucose of the seminal plasma were measured using a spectrophotometer (S2000-UV/VIS, England). Sodium (Na+) and Potassium (K+) were determined using a flame photometer (Jenway PFP, England) (Standard kits from Parsazmoon, Tehran, Iran). The alkaline phosphatase (ACP) and acid phosphatase (ALP) were measured by auto-analyzer (Caretium-XI-921, Germany) using enzymatic procedures with a diagnostic kit (Pars Azmoon Co, Tehran, Iran).

Female properties

Egg diameter (mm), total weight of stripped eggs, number of eggs per gram, total weight of ovary, absolute fecundity, relative fecundity, and gonadosomatic index (GSI) were determined for each female. Fecundity was estimated following the weight method of Bozkurt et al. (2006). Egg size was measured using a slide caliper (at 0.02 mm sensitivity). The relative fecundity was calculated by dividing the total number of stripped eggs by the total body weight of female. The GSI was calculated using the following equation:

$$ \mathrm{GSI}=\mathrm{GW}/ W\times 100\ \left(\mathrm{where},\ \mathrm{GW}\ \mathrm{is}\ \mathrm{gonad}\ \mathrm{weight}\ \mathrm{and}\ \mathrm{W}\ \mathrm{is}\ \mathrm{total}\ \mathrm{weight}\right) $$

Experimental design

The fertilization trials were designed by four treatment groups:

T1

Pooled sperm from 3-year-old males

×

Pooled eggs from 4-year-old females

T2

Pooled sperm from 3-year-old males

×

Pooled eggs from 5-year-old females

T3

Pooled sperm from 4-year-old males

×

Pooled eggs from 4-year-old females

T4

Pooled sperm from 4-year-old males

×

Pooled eggs from 5-year-old females

Eggs were pooled from the two age groups (4 and 5-year-old females) in order to minimize the variation in gamete quality. For each group, ~10 g of eggs were placed into each of 9 plastic dishes (250 mL). Then 100 μL of the pooled milt (from four different treatments as mentioned above) was added to the eggs at a 1:62,000 egg to sperm ratio. Eggs and milt were gently mixed with a plastic spoon and 50 mL of Woynarovich solution (3 g of urea + 4 g of NaCl in 1 L distilled water) was added as an activating medium for fertilizing the eggs. Following fertilization, the eggs were stirred for 5 min, then eggs were rinsed with hatchery water prior to transferring them to the incubator. Fertilized eggs were incubated at 22 to 26 °C with a continuous flow of water and 30% of water exchange per day. A fertilization check was accomplished using a binocular microscope during the eyed-embryo stage at approximately 12 h post-fertilization; non-viable and developing eggs were counted. Hatched larvae were counted 2 days post-fertilization, and larval survivability were observed at 7 days post-hatch. Measurements were taken in triplicate for each sample, and the average of the three measurements was used for the results.

Fertilization, hatching, and larvae survival rate were calculated by the following equations:

$$ \mathrm{Fertilization}\ \mathrm{rate}=\mathrm{number}\ \mathrm{of}\ \mathrm{fertilized}\ \mathrm{egg}/\mathrm{number}\ \mathrm{of}\ \mathrm{total}\ \mathrm{egg}\mathrm{s}\times 100 $$
$$ \mathrm{Hatching}\ \mathrm{rate}=\mathrm{number}\ \mathrm{of}\ \mathrm{hatched}\ \mathrm{larvae}/\mathrm{number}\ \mathrm{of}\ \mathrm{total}\ \mathrm{eggs}\times 100 $$
$$ \mathrm{Survival}\ \mathrm{rate}=\mathrm{number}\ \mathrm{of}\ \mathrm{live}\ 7-\mathrm{day}-\mathrm{old}\ \mathrm{larvae}/\mathrm{number}\ \mathrm{of}\ \mathrm{total}\ \mathrm{hatched}\ \mathrm{larvae}\times 100 $$

Data analysis

All data were analyzed using Statistica v 12 (Statsoft Inc., Tulsa, OK, USA). Residuals were tested for normality (Shapiro–Wilk test) and homogeneity of variance (plot of residuals vs predicted values). To compare male and female quantitative characteristics among the age groups, Student t test was performed. For fertilization, hatching, and larvae survival rate, all proportional data were transformed into arcsin square root prior to analyses. The percentage of fertilization, hatching, and larvae survival rate were analyzed separately using a two-way factorial ANOVA model containing the male and female age main effects as well as the male × female age interaction. Then the model was decomposed into a series of one-way ANOVA models to determine the effects of different age groups of male and female combinations on the fertilization, hatching and larvae survival rate. Alpha was set at 0.05 for main effects and interactions.

Results

There were no significant differences in the sperm motility traits, density and spermatocrit (p > 0.05) between the two age groups of males. While, significant differences (p < 0.05) were found in the ionic compositions (mainly Na+, K+, Ca2+, Mg2+), acid phosphatase and glucose level (Table 1). The quantitative characteristics between the two female groups showed significant differences (p < 0.05) in total weight of stripped eggs, total weight of ovary and relative fecundity (Table 2). However, there were no significant differences between two female age groups in terms of egg diameter, absolute fecundity, and GSI.

Table 1 Quantitative characteristics of different age groups of male bighead carp Hypophthalmichthys nobilis. Data are presented as mean (± SD) values. Columns with the different alphabetic letters are significantly different p < 0.05
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Table 2 Quantitative characteristics of different age groups of female bighead carp Hypophthalmichthys nobilis. Data are presented as mean (± SD) values. Columns with the different alphabetic letters are significantly different p < 0.05
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The first two-way ANOVA model revealed a significant effect of male age on the fertilization rate of eggs (p < 0.05); while female age and male × female age did not show any significant effect on the fertilization success. The second two-way ANOVA model also revealed significant effects of male age on the hatching rate (p < 0.05), but female age and male × female age did not show any significant effect on the hatching success. The third two-way ANOVA model showed significant effects of male and female age and as well as their interaction term (p < 0.001 for all) on the survival rate of larvae.

Then, the factorial models were decomposed into a series of one-way ANOVAs to determine the effects of different age groups of male and female combinations on the fertilization, hatching, and larvae survival rate. Our one-way ANOVA showed significant differences among the treatments on the fertilization (F = 3.797, p = 0.05), hatching (F = 4.802, p = 0.03), and larvae survival rate (F = 32.94, p = 0.001) (Fig. 1).

Fig. 1
figure 1

Fertilization, hatching, and larvae survival rates of different age groups of male and female combinations in bighead carp Hypophthalmichthys nobilis. T1: 3-year-old males vs 4-year-old females; T2: 3-year-old males vs 5-year-old females; T3: 4-year-old males vs 4-year-old females; T4: 4-year-old males vs 5-year-old females

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Discussion

Reproductive success relies on the quality of both male and female gametes. Determination of gamete composition and quality is necessary to understand the basic biochemical processes which occur during fish reproduction (Ciereszko et al. 2000). Reproductive performance between males and females might be expected to vary, because of sex-related differential in age of initial maturity.

Present study showed that male age strongly influenced fertilization, embryonic development, and larval survival rate, while effects of the female and male-female interactions were significant only on larval survival rate at 7 days post-hatch. However, crosses between older males and females showed higher reproductive performance in terms of hatching and larvae survival rate than with crosses between young males and females.

Sperm quality is an important factor that increases artificial fertilization efficiency (Billard et al. 1995; Bromage 1995). In this study, duration of sperm motility in 4-year-old males was slightly longer than in 3-year-old males. A similar result was reported in Atlantic salmon S. salar, where duration of sperm motility in mature male parr was longer than that of adult males (Daye and Glebe 1984). In contrast, no change was observed in the duration of sperm motility of Baikal Omul in the age range of 6–14 years (Khodzher 1981). Similarly, there was no difference in the percentage of motile spermatozoa of 2 and 3-year-old rainbow trout (Liley et al. 2002). Also in young and old guppy, Gasparini et al. (2010) reported the same pattern. In addition, Kanuga et al. (2011) showed no discernable effect of age on sperm motility characteristics in zebrafish, but there was an effect of male age on the number of spermatozoa in the testes and sperm ducts. The discrepancies among specie might be due to feeding conditions, husbandry procedures, age, environmental factors, spawning time, or dilution ratio.

The percentage of motile sperm was higher in 3-year-old males than in that of 1 and 12-year-old males of striped bass M. saxatilis (Vuthiphandchai and Zohar 1999). Higher percentage of sperm motility in younger age classes has been reported in Atlantic salmon (Gage et al. 1995), as well as longer motility duration in younger sockeye salmon (Hoysak and Liley 2001). In addition, sperm density was reported to be increased as the male age is increased in Baikul Omul (Khodzher 1981), rainbow trout (Buyukhatipoglu and Holtz 1984), and steelhead trout (Chechun et al. 1994). Higher spermatocrit values in the 4-year-old males in our study might suggest a higher number of spermatozoa (Vuthiphandchai and Zohar 1999). Similar results were found in 2 and 3-year-old male common carps (Aliniya et al. 2013). An explanation for this is that sperm volume increased in larger fish and as the relationship between sperm volume and sperm density is reverse, therefore with increasing age, sperm volume will increase, but its concentration will decrease (Tekin et al. 2003)

Composition of seminal plasma has been addressed as a reliable tool to understand the reproductive process and has a great influence on the biological quality of the semen and is directly related to the fertilization success (Rurangwa et al. 2004; Alavi and Cosson 2005). In our experiment, the average concentrations of Na+, Ca+2, and Mg+2 in the seminal plasma were significantly higher in the 3-year-old males than the 4-year-old males, while the average concentrations of K+ and acid phosphatase increased with increasing of male age. However, all these values were within the range of reported values for cyprinids (Billard et al. 1995; Alavi and Cosson 2006).

The size and appearance of ova can tentatively be used to evaluate the overall developmental potential of the eggs after fertilization (Bobe and Labbé 2010). Several studies have confirmed that egg diameter increased with increasing female age and size (Bromage et al. 1992; Bromage and Cumaranatunga 1988; Aliniya et al. 2013) as it was observed in the current experiment. However, small eggs have similar rates of fertilization as larger ones under normal conditions of temperature, post-ovulatory aging, and proper husbandry practices (Bromage et al. 1992).

Absolute fecundity and total weight of the ovary generally increase with the age and size of female breeders (Reznick et al. 2002), while relative fecundity (number of eggs per body weight) decrease (Springate et al. 1984; Bromage and Cumaranatunga 1988). Relative fecundity has been reported to decline with increase of age (Siraj et al. 1983; Ridha and Cruz 1989) which supports results of the present study.

In the present study, eggs from 5-year-old female’s sired by 4 year-old males produced significantly higher hatching and larvae survival rate than the other age group combinations, which clearly indicated that older male and female combinations achieve the maximum reproductive success. Results from this study could be useful for broodstock selection in controlled reproduction of bighead carp as well as for hatchery management.