TY - JOUR
AU - Butts, Ian Anthony, Ernest
AB - Abstract As global aquaculture continues to expand, increasing efforts are focusing on assisted reproductive technologies. This study sought to test whether salmon GnRH [D-Arg6, Pro9NEt]) analogue (sGnRHa) + domperidone injection at 0.25 mL/body weight (BW; 5-μg sGnRHa + 2.5-mg domperidone), 0.5 mL/kg BW (10-μg sGnRHa + 5-mg domperidone), or 1.0 mL/kg BW (20-μg sGnRHa + 10-mg domperidone) affects spawning performance and gamete quality in wild-caught Levantine scraper, Capoeta damascina. The ability of these treatments to elicit a response was further examined by in vivo stimulation of estradiol (E2) and 17α,20β-dihydroxy-4-pregnen-3-one (DHP) and by its in vivo potency to induce oocyte maturation (OM). Females that received saline injection (control) did not spawn, whereas sGnRHa + domperidone induced ovulation and spawning across the hormonal gradient. Spawning success was highest with the 0.5 mL/kg dosage (80%) and female latency period decreased with increasing dosage. Females treated with 0.5 mL/kg had a significantly higher fecundity than those injected with 0.25 or 1.0 mL/kg. Mean oocyte diameter significantly increased in females treated with 0.5 or 1.0 mL/kg. Fertilization success, hatching rate, larvae morphology, and survival were not affected by hormonal treatment. At 12 h postinjection, E2 levels significantly declined in females treated with 0.5 or 1.0 mL/kg, whereas DHP levels significantly increased across the hormonal gradient. This steroidogenic shift is supported by histological analyses, where OM was accelerated by administration of sGnRHa + domperidone in a dose-dependent manner. In conclusion, the 0.5 mL/kg dosage of sGnRHa + domperidone is recommended for assisted reproduction of Levantine scraper. INTRODUCTION The cyprinid fish Levantine scraper, Capoeta damascina belongs to the genus Capoeta, and is one of the most common freshwater fishes for both recreational fishing and wild fisheries across its distribution (Levant, Mesopotamia, Turkey, and Iran; Coad, 2010). Levantine scraper is usually rheophile, but can also be found in lacustrine environments. In native habitats, and depending on geographical origin, their reproductive season occurs from early May to late July at temperatures between 16 to 22 °C (Kamangar et al., 2015). Their maximum total length (TL) and weight are ~52 cm and ~2340 g, respectively (Asadollah et al., 2011), as such this species is considered to have potential for commercial development. However, despite the success of emerging assisted reproductive technologies for spermiation induction in Levantine scraper (Zadmajid et al., 2018), there is no report on induction of ovulation and spawning for females. Thus, it is necessary to continue to study their reproductive physiology under controlled conditions to support breeding efforts and aquaculture development. Broodstock caught from native waters can experience reproductive dysfunctions when induced to spawn in captivity. This reproductive failure is most common in wild-caught female fish (e.g., failure of oocyte maturation [OM], ovulation, and/or spawning), but also exists in males (e.g., low or viscous sperm production) (Mylonas et al., 2017; Zupa et al., 2017). In most cases, assisted reproductive technologies, i.e., using hormonal therapy to stimulate gametogenesis and induce ovulation, can be an effective way to control reproduction in captive fishes (Berlinsky et al., 1996; Berlinsky et al., 2005; Garber et al., 2009; Mylonas et al., 2017). Hormonal manipulations have contributed significantly to the expansion and diversification of the aquaculture industry as a management tool to enhance the efficiency of gamete production and hatchery operations (Berlinsky et al., 1996; Chatakondi et al., 2011; Chatakondi, 2017). In the last decade, there has been growing interest in the development of protocols for captive gamete production in rheophilic cyprinid fish using exogenous hormonal agents (Krejszeff et al., 2008; Krejszeff et al., 2009; Zadmajid, 2016). In most cases, spermiation and ovulation/spawning can be stimulated via hormonal treatment using gonadotropin-releasing hormone agonists (GnRHa), either in the form of salmon GnRH (D-Arg6, Pro9NEt analogue with dopamine antagonist [e.g., sGnRHa + domperidone]) or mammalian GnRH (D-Ala6, Pro9 NEt analogue with metoclopramide [i.e., Ovopel]). This sort of assisted reproductive technology, termed the “Linpe method” (Peter et al., 1998) is commonly used worldwide for cyprinid and noncyprinid fish (Peter et al., 1988; Kowalski et al., 2012; DiMaggio et al., 2013; Acharjee et al., 2017). In this respect, hormones are delivered as an intramuscular or intraperitoneal injection and act directly on the pituitary, stimulating the release of stored gonadotropic hormones while concomitantly preventing dopaminergic inhibition of gonadotropin secretions, ultimately regulating gamete activity and production of sex steroids by the developing gametes. Accordingly, extensive mechanistic studies have successfully obtained viable gametes in rheophilic cyprinid fish, largely by administration of a single dose of sGnRHa + domperidone at the manufacturer’s recommended dose (0.5 mL/kg body weight [BW] for females) (Targońska et al., 2010). Despite its successes, the rationale for choosing this dose and its impact on reproductive performance appears to be largely absent, especially for rheophilic cyprinid fish. This is concerning, as insufficient dosages may prevent ovulation and spawning, whereas overdosing may lead to reduced gamete quality and sometimes even death (DiMaggio et al., 2013). Thus, it is critical to establish appropriate hormonal regimes that are unique to the species of interest. In teleost, gametogenesis (spermatogenesis and vitellogenesis) and final maturation (spermiation and final oocyte maturation [FOM]) are controlled physiologically by a cascade of hormones that are produced in and released from the brain–pituitary–gonadal (BPG) axis (Tokarz et al., 2015). For instance, in fish ovaries, 17β-estradiol (E2) is produced during oocyte growth, to reach peak levels at, or near, the beginning of ovulation, whereas 17α,20β-dihydroxy-4-pregnen-3-one (DHP) peaks during FOM and ovulation. Plasma levels of gonadal steroid hormones have been shown to change in response to exogenous hormonal therapy (Levavi-Zermonsky and Yaron, 1986; Acharjee et al., 2017; Zadmajid et al., 2017). Therefore, in this study, the impact of various doses of sGnRHa + domperidone on ovulation and spawning success in Levantine scraper was evaluated to facilitate its use for supportive breeding and gamete production. Here, the efficacy of induced ovulation protocols was assessed by examining latency time, spawning success, and gamete quality traits. In addition, profiles of serum sex steroid hormones E2 and DHP were monitored to obtain information on in vivo steroidogenic responses, using various doses of sGnRHa + domperidone. To further delineate the effects of hormonal dosage on ovarian development, its effects on oocyte development and their relative abundance were also quantified by histological analysis. MATERIALS AND METHODS Collection and Acclimatization of Broodstock Wild broodstock were caught by cast net from the Qeshlaq River, Sanandaj, Iran, during the reproductive season (early June 2017), when daily mean water temperatures reached 18 to 20 °C. The sex of each fish was determined by visual examination. Specifically, fish were determined as females based on abdominal swelling and males by their slender body, rough dorsal surface of lateral body, and expulsion of a minute drop of sperm upon gentle pressure to the abdomen, anterior to the urogenital opening. The broodstock were in healthy condition and devoid of any visible damage or malformations. The fish were transported in oxygenated tanks (~1 h) to research facilities of the Fish Biology Lab at the University of Kurdistan (Sanandaj, Iran). Fish were stocked in eight 500 L flow-through indoor round fiberglass tanks (127 × 76 cm) and were acclimatized to laboratory conditions under simulated natural 14 h light/10 h dark photoperiod, and ambient temperature (19 °C) with acceptable water quality parameters: 7 mg/L dissolved oxygen, 0.13 mg/L ammonia, 0.0046 mg/L nitrite, and pH of 8.1. All tanks operated independently and were covered with a thin shade mesh to reduce excessive light. Fish were maintained under these conditions for >3 d to acclimate and recover from transport. During the acclimation period, males and females were kept together. The age of each fish was estimated to be 3 to 4 yr based on annuli in cycloid scales (Biswas, 1993). All animal manipulations and handling were approved by the Ethical Committee for Animal Experiments of Iran Veterinary Organization (IVO, protocols 30301 and 30309; 2014). Chemicals and Reagents Salmon GnRH (D-Arg6, Pro9Net analogue [5 mg]) and domperidone (10 mg/mL) (sGnRHa/domperidone) were purchased from Syndel Laboratories Ltd. (British Columbia, Canada). 17β-estradiol and DHP assays were acquired from Demeditec Diagnostics GmbH (Kiel, Germany). Clove oil was purchased from Sigma-Aldrich Inc. (Louis, MO). Analytical-grade organic solvents and histology reagents were obtained from Merck (Darmstadt, Germany). Assessment of Reproductive Status via Ovarian Biopsy After the acclimatization period, ovarian biopsies were conducted in vivo using a flexible catheter (Coloplast Ltd., internal diameter of 1.6 mm, Peterborough, UK) inserted into the genital pore of anesthetized fish and applying gentle aspiration (Berlinsky et al., 1996). To minimize contamination and prevent infection, the catheters were thoroughly cleaned and sterilized by autoclaving prior to inserting into the genital pore. Once anesthetized, specimens were placed ventral side up and the abdomen and the genital pore wiped gently using sterile saline solution (0.9% NaCl). A portion of the sample was taken and placed in Serra solution (6:3:1, ethanol 70%: formaldehyde 40%: glacial acetic acid 99.5%), and after cytoplasm clarification (after 2 to 3 min of exposure), images were taken using a digital camera (Digital Sight DS-L2, Nikon, Gotemba, Japan) to determine stages of maturity based on germinal vesicle (GV) position. To correctly determine the position of the GV, oocytes were rolled and tilted during microscopic observation and the position of the germinal vesicle was determined according to a 4-stage scale: 1) Stage I—germinal vesicle occupies central position (CGV) 2) Stage II—early germinal vesicle migration (early MGV; less than half of the radius) 3) Stage III—late migration of germinal vesicle (late MGV; more than half of the radius) 4) Stage IV—periphery germinal vesicle or germinal vesicle breakdown (PGV or GVBD) Only fish in which >60% of oocytes were at stage of migrating GV (Stages II and III) were considered (diameter from 1.4 to 1.7 mm; i.e., mature vitellogenic oocytes (Vtg) with cytoplasm filled entirely of yolk) eligible for spawning induction. The developmental stage of the migrating GV to the animal pole corresponded to Stage 5 of gamete development in Levantine scraper according to Asadollah et al. (2011) and Kamangar et al. (2015). Hormonal Induction Protocol Measurements of BW (to nearest 0.1 g) and TL (to nearest 1 mm) were taken and females were separated from males and randomly assigned to 8 tanks, each stocked with 5 females. The following injection treatments (each with 2 replicate tanks × 5 fish = 10 fish/treatment; husbandry treatments, as above) were administered under anesthesia (75 to 115 ppm clove oil): 1) saline (0.9% NaCl, 0.5 mL/kg BW; hereafter “control”); 2) 0.25 mL/kg BW of sGnRHa + domperidone; i.e., 5 μg sGnRHa + 2.5 mg domperidone; 3) 0.5 mL/kg BW of sGnRHa + domperidone; i.e., 10 μg sGnRHa + 5 mg domperidone; and 4) 1.0 mL/kg BW of sGnRHa + domperidone; i.e., 20 μg sGnRHa + 10 mg domperidone. All injections were administrated intramuscularly with a single dose using a 1-mL syringe equipped with a 16-gauge needle. The day before in vitro fertilization trials, 3 to 5 males displaying high sperm motility were selected per female and intramuscularly injected with a single dose of 0.25 mL/kg sGnRHa + domperidone (Zadmajid, 2016) under anesthesia (75 to 115 ppm clove oil). Selected males were transferred to four 500-liter tanks with flow-through freshwater at 19 °C. Gamete Quality Traits After 12 h postinjection, all females were assessed for ovulatory response every 3 to 4 h by delicately massaging their abdomens. Fish releasing ova in response to gentle abdominal pressure were immediately stripped into a dry plastic vessel and fecundity (absolute fecundity) was estimated (n = 10) indirectly from their weight by counting 0.1-g egg samples (n = 5/female) from each spawn obtained within each treatment, given a value of ~30 eggs/0.1 g (i.e., number of eggs = dry weight × 30 eggs/0.1 g). The diameter of >8 to 10 unfertilized eggs was measured from each female (10 fish/treatment; n = 100) using a dissecting microscope (Olympus SZ51, Tokyo, Japan) fitted with an ocular micrometer. Spawning success (n = 10; number of ovulated fish/total number of treated fish) and the latency period (n = 10; time between sGnRHa/domperidone/saline administration and ovulation) were determined for each treatment. For in vitro fertilization trails, the males were stripped first. Sperm was collected with syringes (without a needle) by applying gentle pressure to the abdomen. Three egg samples (100 to 150 eggs per sample) from each female were dry fertilized with 0.05 mL of pooled sperm taken from at least 3 to 5 hormonally induced males. The resulting egg to sperm ratio was 1:2 × 107 for all samples (Lahnsteiner et al., 2001). After elimination of stickiness by Woynarovich solution (4-g NaCl + 3-g urea in 1 liter distilled water; Siddique et al., 2016), eggs were incubated separately at 19 °C in Petri dishes in a closed water system and water was changed every 2 h. A minimum of 50 eggs per batch from the random egg sample were assessed for fertilization percentage by observing embryonic development at 16 to 64 (5 to 7 h postfertilization) blastomere cleavage stages under a compound microscope (Eclipse E200-LED, Nikon, Gotemba, Japan). Hatching success was determined by placing 20 eggs in each of 5 replicate 1-liter beakers and recording the number of hatched larvae in each beaker. To measure larval size at hatch, samples of 10 newly hatched embryos/female were randomly collected from each incubator and measured to the nearest 0.1 mm, as described for egg diameter. Larval survival (%) at 6 d posthatch (DPH) and 12 DPH was determined by placing 100 newly hatched embryos into replicate beakers and counting the number of live larvae. Blood Collection At 0 and 12 h postinjection (hpi), blood was collected (1 mL/fish) from the caudal vein from 10 fish per treatment in order to measure E2 and DHP levels. The samples were held at 4 °C for 2 h (1.5-mL eppendorf tubes without anticoagulant) and then centrifuged (3,000 × g, 15 min, 4 °C) for serum separation. The serum samples were stored at −40 °C for further analysis. Quantification of Serum Levels of E2 and DHP Hormone analyses was conducted using commercially available enzyme-linked immunosorbent assay (ELISA) kits. Briefly, serum samples (200 µL) were first extracted with 4-mL diethyl ether under vigorous shaking for 4 min. The aqueous phase was frozen in liquid nitrogen, whereas the organic phase was transferred to a glass tube, evaporated in a water bath at 45 °C, and then reconstituted by addition of 600-µL assay buffer. Concentrations of E2 and DHP were quantified by ELISA, using a microplate reader (model Elx808, Biotek, Winooski, VT). Sensitivity for E2 was 0.0097 ng/mL, and intraassay and interassay coefficients of variation were 6.81% (n = 20) and 7.25% (n = 12), respectively. Sensitivity for DHP was 0.045 ng/mL, and intraassay and interassay coefficients of variation were 5.4% (n = 20) and 9.96% (n = 12), respectively, in the range of 0 to 40 ng/mL. Ovarian Histology and Image Analyses To evaluate the effects of sGnRHa/domperidone/saline manipulation on ovarian development, four transverse sections from the middle region of the ovaries were taken from 5 randomly chosen fish per treatment at 0 and 12 hpi and preserved in Bouin’s fixative for histological analyses. Samples were dehydrated in gradually increasing ethanol solutions (70% to 100%), embedded in paraffin, and sectioned at 4 to 5 μm with a rotatory microtome (DS 8402, DID SABZ CO. Tehran, Iran). Sections were stained with hematoxylin and eosin according to standard procedures and slides were viewed through a light compound microscope (Eclipse E200-LED, Nikon, Gotemba, Japan). Histological sections were photographed with a digital camera (Digital Sight DS-L2, Nikon, Japan) to determine different germ cell types and their relative abundance. The stage-frequency profiles of ovarian follicles were scored according to the presence of primary growth (PG): ovaries with oocytes in chromatin nucleolar and perinucleolar stages (typically oocytes without cortical alveoli [CA], yolk, or lipids droplets); CA: ovaries with a clutch of oocytes in CA stage (one to several layers of small to large cortical vesicles forming along the outer margin of the cytoplasm); Vtg: ovaries with a clutch of oocytes at vitellogenesis stage (oocytes filled with large yolk droplets); and OM: oocyte at stage of FOM with advanced stage of hydration and no visible nucleus (Blazer, 2002). The presence of postovulatory follicles (POF) and atretic oocytes (AO) were also recorded, but unfortunately, the irregular shape of POFs and AOs did not allow us to make an accurate stereological estimation of the numerical density and, hence, they were excluded from scoring. To obtain a representative sample size for each fish, ≥50 oocytes were scored. Statistical Analyses Data were analyzed using a series of one-way and multifactorial ANOVA models (PROC MIXED; SAS Institute, 2003). Alpha was set at 0.05 for main effects and interactions. If a significant interaction was detected, the models were decomposed into a series of reduced ANOVA models. Tukey’s post hoc analyses were used to compare means between treatments. Means are expressed as least square mean ± standard error. Graphs were prepared in SigmaPlot (Version 13.0). RESULTS Body Morphology Mean (±SD) BW and TL of the females were 146.8 ± 29.8 g and 28.8 ± 1.6 cm, respectively. No differences in BW (P = 0.984) and TL (P = 0.983) were detected between the hormonal-treated and saline-injected fish. Latency Period, Total Eggs Spawned, and Egg Size Females that received saline injection did not spawn. However, when injected with 0.25-mL/kg sGnRHa + domperidone, 30% of females spawned. Additionally, when injected with 0.5 and 1.0 mL/kg, 80% and 70% of females spawned, respectively. Hormonal treatment affected female latency period (P = 0.004) such that females injected with 1.0 mL/kg spawned earlier than those injected with 0.25 or 0.5 mL/kg (Figure 1A). Additionally, females injected with 0.5 mL/kg yielded more eggs than those injected with 0.25 mL/kg (Figure 1B), and egg size increased when females were injected with 0.5 or 1.0 mL/kg (Figure 1C). Mean (±SEM) egg size for females injected with saline (taken with biopsy) was 1.63 ± 0.02 mm. Figure 1. View largeDownload slide Effect of sGnRHa + domperidone treatment on female latency period (n = 10), amount of eggs per spawn (n = 10), and egg size (n = 100/treatment) in Levantine scraper, Capoeta damascina. Subscripts with the same letter are not significantly different (P > 0.05). Figure 1. View largeDownload slide Effect of sGnRHa + domperidone treatment on female latency period (n = 10), amount of eggs per spawn (n = 10), and egg size (n = 100/treatment) in Levantine scraper, Capoeta damascina. Subscripts with the same letter are not significantly different (P > 0.05). Fertilization Success, Hatch Success, and Larval Performance Mean (±SEM) fertilization success across the hormonal treatments ranged from 85.1 ± 2.6 (1.0 mL/kg) to 86.3 ± 2.4% (0.5 mL/kg), whereas hatch success ranged from 76.0 ± 3.5 (0.25 mL/kg) to 77.7 ± 2.3% (1.0 mL/kg). No differences in fertilization (P = 0.964) or hatch success (P = 0.888) were detected between the hormonal treatments. Larval size (TL) at hatch did not differ between the treatments, where mean (± SEM) size ranged from 8.8 ± 0.1 (1.0 mL/kg) to 9.1 ± 0.2 mm (0.25 mL/kg). For larval survival, the hormonal treatment (P = 0.761), posthatch time (P = 0.354), and hormonal treatment × posthatch time interaction (P = 0.988) were all nonsignificant, where survival values remained ≥89% from 6 to 12 DPH. Sex Steroids The steroids, E2 (P = 0.039) and DHP (P = 0.0005), were both affected by the hormonal treatment × postinjection time interaction. As such, the models were decomposed to assess the effects of hormonal treatment at each postinjection time. At 0 hpi, no differences were detected for E2 (P = 0.670; Figure 2A) or DHP (P = 0.953; Figure 2B). However, at 12 hpi both steroids were affected such that E2 declined after injection with 0.5-mL/kg sGnRHa + domperidone (P = 0.007; Figure 2B), and DHP increased across the hormonal gradient (P < 0.0001; Figure 2D). Figure 2. View largeDownload slide Effect of sGnRHa + domperidone treatment on estradiol (E2) (n = 10) and 17α,20β-dihydroxy-4-pregnen-3-one (DPH) (n = 10) levels in Levantine scraper, Capoeta damascina. Subscripts with the same letter are not significantly different (P > 0.05). Figure 2. View largeDownload slide Effect of sGnRHa + domperidone treatment on estradiol (E2) (n = 10) and 17α,20β-dihydroxy-4-pregnen-3-one (DPH) (n = 10) levels in Levantine scraper, Capoeta damascina. Subscripts with the same letter are not significantly different (P > 0.05). Ovarian Histology Morphological and stage-frequency profiles of ovarian follicles of fishes treated with either saline or sGnRHa + domperidone at different doses are presented in Figures 3 and 4. Hormonal treatment had no impact on oocyte stage percentage for any oocyte stages at 0 hpi (P ≥ 0.709; Figure 4A). Additionally, the percentages of PG (P = 0.060) and CA oocytes (P = 0.337) were not affected by hormonal treatment at 12 hpi (Figure 4B). However, females injected with 0.5 or 1.0 mL/kg had a lower percentage of Vtg oocytes at 12 hpi than those injected with saline or 0.25 mL/kg (P = 0.0002; Figure 4B). Females injected with 1.0 mL/kg had the highest percentage of OM oocytes at 12 hpi (P < 0.0001; Figure 4B). There was also evidence of a few AO in some ovaries at 12 h following sGnRHa/domperidone treatment. Figure 3. View largeDownload slide Photomicrographs of histological sections of Levantine scraper, Capoeta damascina ovaries from fish treated with saline (control) at 0 (A) and 12 h postinjection (hpi) (B); suboptimal dose of sGnRHa + domperidone (0.25 mL/kg BW) at 0 (C) and 12 hpi (D); optimal dose of sGnRHa + domperidone (0.5 mL/kg BW) at 0 (E) and 12 hpi (F); and supraoptimal dose of sGnRHa + domperidone (1.0 mL/kg BW) at 0 (G) and 12 hpi (H). Oocyte stages and structures are denoted by the following: PG = primary growth; CA = cortical alveoli; Vtg = vitellogenic oocytes; OM = oocyte maturation; AO = atretic oocyte; POF = postovulatory follicle; GV = germinal vesicle. Scale bars: 500 μm. Magnification is 50×. Figure 3. View largeDownload slide Photomicrographs of histological sections of Levantine scraper, Capoeta damascina ovaries from fish treated with saline (control) at 0 (A) and 12 h postinjection (hpi) (B); suboptimal dose of sGnRHa + domperidone (0.25 mL/kg BW) at 0 (C) and 12 hpi (D); optimal dose of sGnRHa + domperidone (0.5 mL/kg BW) at 0 (E) and 12 hpi (F); and supraoptimal dose of sGnRHa + domperidone (1.0 mL/kg BW) at 0 (G) and 12 hpi (H). Oocyte stages and structures are denoted by the following: PG = primary growth; CA = cortical alveoli; Vtg = vitellogenic oocytes; OM = oocyte maturation; AO = atretic oocyte; POF = postovulatory follicle; GV = germinal vesicle. Scale bars: 500 μm. Magnification is 50×. Figure 4. View largeDownload slide Effect of sGnRHa + domperidone treatment on oocyte stage percentage in Levantine scraper, Capoeta damascina at 0 h postinjection (hpi) (A) and 12 hpi (B). Separate one-way ANOVA models were generated at 0 and 12 hpi for each oocyte stage. * represents a significant difference between the hormonal treatments; PG = primary growth; CA = cortical alveoli; Vtg = vitellogenic oocytes; OM = oocyte maturation. Figure 4. View largeDownload slide Effect of sGnRHa + domperidone treatment on oocyte stage percentage in Levantine scraper, Capoeta damascina at 0 h postinjection (hpi) (A) and 12 hpi (B). Separate one-way ANOVA models were generated at 0 and 12 hpi for each oocyte stage. * represents a significant difference between the hormonal treatments; PG = primary growth; CA = cortical alveoli; Vtg = vitellogenic oocytes; OM = oocyte maturation. DISCUSSION Achieving a reliable supply of high-quality gametes is a primary challenge that needs to be overcome when developing assisted reproduction technologies for new species. Specifically, determining the most reliable hormone and hormonal dosage is of critical importance. Here, we focused on optimization of sGnRHa + domperidone to establish criteria for egg production from wild-caught Levantine scraper to support breeding in captivity. The ultimate goal of hormonal therapy for captive reproduction of fishes is induction of FOM, leading to successful spawning. In the present study, females that received the saline injection did not spawn. In contrast, sGnRHa + domperidone injection induced spawning in Levantine scraper, although potencies were different with various doses. Spawning success for the 0.50 and 1.0 mL/kg dosages of sGnRHa + domperidone was 80% and 70% with a latency time of ~28 and ~19 h, respectively. With respect to the 0.25 mL/kg dose, spawning success was sporadic, where only 30% of females spawned with a latency of ~40 h. Thus, this low dosage does not appear to be sufficient for future breeding and aquaculture applications, as it appears that the hormone may not have induced the release of an adequate amount of gonadotropin for optimal spawning and therefore resulted in lower spawning success. On the other hand, reduced spawning success at the highest sGnRHa + domperidone dose (1.0 mL/kg) is difficult to explain. However, we speculate that excessive concentrations of GnRHa may cause antigonadotropic effects, due to pituitary desensitization, but such mechanisms remain unclear (DiMaggio et al., 2013). Generally, high spawning success after a single sGnRHa + domperidone injection at 0.5 mL/kg has been reported for other fishes such as asp, Aspius aspius (Targońska et al., 2010), pinfish, Lagodon rhomboids (DiMaggio et al., 2013), and catfish, Heteropneustes fossilis (Acharjee et al., 2017). It has been shown that the latency period varies greatly among species and depends on both intrinsic (Wendling et al., 2000) and environmental parameters (Yaron, 1995), as well as the type of hormone or dose (Wang et al., 2009; Das et al., 2016). Recorded latency periods in our study were different across the hormonal gradient, with shorter latency periods when sGnRHa + domperidone was administered in higher dosages. This is supported by ovarian histology, where a faster response was documented at higher dosages. Moreover, the latency period in females treated with 0.25 mL/kg was longer than the other doses, which could be related to the fact that the DHP peak occurred later, and therefore, why spawning was delayed. In agreement with our findings, a negative correlation between GnRHa dose and latency period has been reported in other species, such as knifefish, Chitala chitala (Sarkar et al., 2006) and meager (Fernández-Palacios et al., 2014). In the present study, all eggs ovulated after sGnRHa + domperidone injection and were manually stripped from females, resulting in a higher overall mean total fecundity (~7,446 eggs/spawn) in the 0.5 mL/kg treatment. Interestingly, at 1.0 mL/kg, sGnRHa + domperidone accelerated ovulation and induced spawning, but did not offer any perceived increase in total fecundity. It is plausible that this larger dosage may have led to over-ripping of some oocytes resulting in lower fecundity; however, these results should be interpreted cautiously since our data show no deleterious effects of a dose-dependent response on fertilization or hatching. Similarly, in the meager, Argyrosomus regius latency time was shorter and OM was accelerated resulting in lower fecundity in females treated with supra-optimal GnRHa doses vs. optimal GnRHa doses (Fernández-Palacios et al., 2014). In their native habitat, Levantine scraper fecundity varies with size and age. The lowest fecundity (~5,314 eggs/female) was observed by Kamangar et al. (2015) in age 2 fish (~22 cm fork length), whereas the highest fecundity (~13,730 eggs/female) was observed in a larger female at age 5 (34 cm fork length). In our study, age was estimated to be 3 to 4 yr, suggesting that in captive conditions, wild-caught Levantine scraper reach their reproductive potential in response to sGnRHa + domperidone administration at an optimal dose. In the current study, females injected with saline or 0.25-mL/kg sGnRHa + domperidone had similar egg sizes, with most eggs being below ~1.65 mm in diameter, and at various stages of development. In contrast, females injected with 0.5 or 1.0 mL/kg had larger eggs. Such increases are typically correlated with water uptake as a result of high concentrations of inorganic ions inside the oocyte during FOM (Sarkar et al., 2006). Although spawning success, fecundity, and profiles of sex steroid hormones may be better estimators of hormone dose efficacy, observed increases in post-Vtg, even after spawning, confirm the ability of the hormonal dosage to affect oocyte growth at various developmental stages. Increased egg size may convey distinct developmental advantages during the early life stages of fishes as this increased size often results in more egg yolk reserves and better-fit larvae (Greeley et al., 1991). When developing assisted reproductive technologies, it is important to compare captive-bred individuals with their wild and/or naturally spawning counterparts, especially in terms of hormonal therapy and its impacts on gamete quality (e.g., fertilization success, early embryonic development, hatching success (Bonnet et al., 2007; Bobe and Labbé, 2010), and early larval performance (Bobe and Labbé, 2010; Bardon-Albaret et al., 2017)). Exogenous hormone therapies may modify egg mRNA abundance of specific genes and could have delayed consequences for the development of embryos (Bonnet et al., 2007). Thus, it is worth noting that treating wild-caught Levantine scraper with various dose of hormones did not display any negative effects on gamete quality, since mean fertilization rate, hatching success, and larval survival were high in all treatment groups and were not statistically different across the hormonal gradient. Additionally, in the current study, sGnRHa + domperidone treatment leads to large yields of larvae, typically 3,000 to 6,000 larvae per female; however, exact estimates were not calculated. In agreement with our results, other studies have reported no adverse effects of sGnRHa + domperidone on ovulation and spawning in rheophilic cyprinids (Targońska et al., 2010) and noncyprinid fish (Sarkar et al., 2006; Ittzés et al., 2015). Here, we assumed that the better condition of wild females may have influenced the availability of nutrients to constitute embryos endogenous reserves and thus yielded larvae with high survival values. In the present study, at 12 h following hormonal treatment, serum E2 levels declined in females treated with 0.5 and 1.0 mL/kg, whereas serum DHP levels were significantly increased across the hormonal gradient compared with saline-injected fish. Together, this indicates stimulation of gonadotropin-mediated sex steroid modulation by the gonad, such as activation of DHP secretion by the ovarian follicles in response to sGnRHa + domperidone treatment. DHP plays the role of maturation-inducing steroid (MIS) in most teleosts, where it binds to specific receptors in the plasma membranes of oocytes and is directly involved in ovulation processes (Thomas et al., 2004; Thomas et al., 2006). The potency of sGnRHa + domperidone to modify DHP/MIS levels and induce FOM has been evaluated in other fish species. For example, in catfish (Acharjee et al., 2017) and Longspine scraper (Zadmajid et al., 2017), sGnRHa + domperidone treatments stimulated a surge of DHP at 6 and 12 hpi, respectively, and induced spawning. These results indicate that DHP is a natural and major MIS in the genus Capoeta that regulates ovulation and spermiation. A distinct shift in steroidogenesis from E2 to DHP in our study is consistent with other investigations on common carp (Levavi-Zermonsky and Yaron, 1986), chum salmon, Oncorhynchus keta (Park et al., 2007), tench (Podhorec et al., 2016), Longspine scraper (Zadmajid et al., 2017), and catfish (Acharjee et al., 2017). The steroidogenic shift in Levantine scraper ovarian follicles is also supported by our histological analyses and confirms the normality of ovulation rhythm induced by sGnRHa + domperidone injection. Using an aromatase cDNA probe, it has been shown that this steroidogenic shift is due to decreased aromatase activity, which is associated with an acquired capacity of the follicle to respond to pituitary gonadotropins (Yoshikuni and Nagahama, 1991). However, such a steroidogenic shift does not occur in all teleosts (Drori et al., 1994; Pereira et al., 2017). Regardless of the steroidogenic shift mechanism, the decline in E2 is also necessary for the resumption of meiosis during FOM (Pang and Thomas, 2010), and for the release of large numbers of fully matured fertilizable eggs for spawning success (Majumder et al., 2015). The coexistence of various populations of oocytes in Levantine scraper ovaries means that this fish exhibits an asynchronous or group-synchronous reproductive strategy, which is a common characteristic among naturally spawning fish from genus Capoeta (Asadollah et al., 2011; Kamangar et al., 2015; Zadmajid et al., 2017). In our study, the appearance of multiple OM at 12 h following sGnRHa + domperidone treatment reveals that females moved into the spawning subphase; however, this was less evidence in the 0.25 mL/kg dose, since the percentage of OM was lower. An actively spawning subphase can be used to identify those fish that are progressing through OM (i.e., GVBD, or hydration) or ovulation or that are exhibiting newly collapsed POFs, indicating that they are close to ovulation. Vitellogenic oocytes in various stages of development can also be found in ovaries during the actively spawning subphase (Brown-Peterson et al., 2011). Stage-frequency profiles of ovarian follicles from either fish injected with 0.5- or 1.0-mL/kg sGnRHa + domperidone showed a dramatic reduction in the proportion of vitellogenic oocytes at 12 hpi, suggesting the ability of these treatments to promote OM. However, there was evidence of more AO in ovaries of the 1.0 mL/kg dose at 12 hpi, and thus that might be the reason for a lower number of eggs spawned in this group than the 0.5 mL/kg group. In our study, ovaries from the saline group remained unchanged through the experiment and contained all stages of germ cells, predominantly PG and Vtg with a very few OM (Figure 3). The distinct increase in the proportion OM and raised serum DHP levels at 12 hpi across the hormonal gradient support sGnRHa + domperidone dose-dependent responses for induction of ovulation/spawning in Levantine scraper. Similar to our findings, in the meager, OM was accelerated by the administration of GnRHa in a dose-dependent way (Fernández-Palacios et al., 2014). CONCLUSIONS Overall, no prespawning or postspawning side effects were detected with hormone administration, since no mortality was observed among females from the different treatments. Together, this suggests that sGnRHa + domperidone application can be a reliable and safe method for induction of spawning in Levantine scraper, with preference given to the 0.5 mL/kg dosage, based on female spawning performance, serum sex steroids, and ovarian histological results. Ultimately, our hope is that these results will support captive breeding for this important species and will teach us more about reproductive physiology in cyprinids. Conflict of interest statement. None declared. ACKNOWLEDGMENTS We would like to thank the Department of Fisheries Science, University of Kurdistan for providing the necessary facilities. I.A.E.B. was supported by the Hatch project (1013854) which provided by USDA. We acknowledge Dr. Mohammadi (Western Health Lane) for technical assistance with the laboratory analysis. Anonymous reviewers provided great comments/suggestions which improved the manuscript. LITERATURE CITED Acharjee , A. , R. Chaube , and K. P. Joy . 2017 . 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TI - Spawning performance, serum sex steroids, and ovarian histology in wild-caught Levantine scraper, Capoeta damascina (Valenciennes, 1842) treated with various doses of sGnRHa + domperidone
JF - Journal of Animal Science
DO - 10.1093/jas/sky348
DA - 2018-12-03
UR - https://www.deepdyve.com/lp/oxford-university-press/spawning-performance-serum-sex-steroids-and-ovarian-histology-in-wild-0x9PSDBMni
SP - 5253
VL - 96
IS - 12
DP - DeepDyve
ER -