https://orcid.org/0009-0001-3678-1932
Figures
Abstract
To reduce reproduction costs, broodstock undergo multiple stripping, during which semen quality gradually declines toward the end of the spawning season. One potential solution is the use of slow-release delivery systems such as mGnRHa-PLGA (Poly[lactic-co-glycolic acid]) microparticles to prolong the spermiation period. In this study, mGnRHa microparticles with an average size of 14.77 ± 8.09 µm were prepared using an oil-in-water emulsion that release more than 72% of the mGnRHa within 61 days at 12°C. For the in vivo trial, during the end of the natural spawning season, 50 mature rainbow males (738.60 ± 104.86 g and 40.09 ± 2.03 cm) were randomly divided into 5 groups. The control group (C) received 1 mL kg-1 physiological saline. The second group (10A) received 10 μg kg-1 mGnRHa. The third to fifth groups (10M, 20M, and 40M) received 10, 20, and 40 μg kg-1 mGnRHa in the form of mGnRHa-PLGA microparticles, respectively. Semen and blood samples were collected at 0, 9, 21, 47, and 61 days days post injection (dpi) for semen quality and sex steroid hormones assessments. Only 40M group continued to release semen until 61 dpi. The 40M group showed the highest semen volume and sperm density throughout the experimental period, while no significant differences were observed between C, 10A, and 10M groups. At 21, 47, and 61 dpi, the 40M group exhibited the highest sperm density, semen volume, and plasma testosterone levels among all groups. At 61 dpi, 40M group showed the highest GSI and the largest surface area occupied by spermatids and spermatozoa. Finally, while the acute mGnRHa failed to enhance spermiation parameters under multiple stripping conditions, the 40M were capable of increasing the duration of semen release and boosting semen volume at the end of spawning season. This study provided aquaculturists with novel information to improve reproductive performance in rainbow trout hatcheries.
Citation: Mokhles Abadi Farahani A, Paykan Heyrati F, Dinari M, Habibi HR, Dorafshan S (2026) The biodegradable mGnRHa-Poly[lactic-co-glycolic acid] microparticles enhances semen quality in multi-stripped rainbow trout. PLoS One 21(3): e0339688. https://doi.org/10.1371/journal.pone.0339688
Editor: Sayyed Mohammad Hadi Alavi, University of Tehran, IRAN, ISLAMIC REPUBLIC OF
Received: September 16, 2025; Accepted: December 10, 2025; Published: March 10, 2026
Copyright: © 2026 Mokhles Abadi Farahani et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are within the paper and its Supporting Information files.
Funding: FPH funded by Isfahan University of Technology (Grant No. 1404.4.15; https://iut.ac.ir), and the Center for International Scientific Studies and Collaboration (CISSC), Ministry of Science, Research, and Technology, Islamic Republic of Iran (Grant No. 4000099; https://cstid.ir/). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
1. Introduction
The success of artificial reproduction in fish depends on their semen and egg quality [1]. In males, spermatogenesis is also a tightly regulated process influenced by endocrine and local testicular factors that determine semen volume, sperm motility, and overall fertilizing capacity [2]. Spawning duration in rainbow trout usually lasts for 4–6 weeks but it can be prolonged by photoperiod or hormonal therapy [3]. On the other hand, male rainbow trout have a distinct yearly cycle of spermatogenesis, which expresses semen for a relatively short period [3]. By the end of the spawning season, semen quantity and quality decrease rapidly such, that very low amounts of low quality semen will be available at the end of the spawning season [4]. A promising method that has demonstrably led to significant reductions in breeding costs is to keep a limited number of genetically superior breeders for multiple stripping. However, repeated semen collection (multiple stripping) has been shown to negatively affect fish semen quality, leading to reduced motility, velocity, and sperm quantity as well as decreased semen volume and density [5].
The negative impacts on semen characteristics can be reduced by hormonal treatment [1]. Gonadotropin-releasing hormone and its analogs have been widely used for spawning success in teleost fish; but they are rapidly removed from the fish body, which poses a challenge against their long-term action [6]. GnRHa sustainable delivery systems have shown promising results regarding spermiation, enhanced semen hydration, and extended spremiation period [7]. In addition, Freund’s incomplete adjuvant [2], fatty acid dimer copolymer microparticles [8], chitosan-gold nano conjugates [9] and ethylenevinyl acetate copolymer (EVAc) [9] have also been tested as delivery systems. Some of these methods have drawbacks, such as non-biodegradability and potential contamination [10], which limit their use; for example, adjuvants have such potential disadvantages as inflammatory reactions, severe systemic reactions, autoimmune diseases, weakening immune systems, safety concerns, species sensitivity, anaphylactic shock, chemical toxicity, and potential contamination [10]. Chitosan-gold nano conjugates have some disadvantages in medical use because remaining formic acid and other small by-products can cause cell toxicity [11]. Finally, ethylene vinyl acetate copolymer (EVA) is disadvantageous due to its large size (more than 2 mm3) and non-biodegradability [9].
In contrast, poly [lactic-co-glycolic acid] (PLGA) microparticles have exhibited promising results in delivering drugs in both humans and animals thanks to their properties such as biocompatibility, small size, and ability to control dosage and release duration [11]. For instance, a sustained-release system using mGnRHa incorporated into PLGA microparticles was successfully applied in pikeperch (Sander lucioperca), where a low dose of mGnRHa-PLGA (5 µg kg-1) significantly improved ovulation induction and maintained adequate reproductive performance under controlled conditions [7]. This demonstrates the potential of PLGA-based hormonal delivery systems to enhance reproductive outcomes in fish species. However, little is known about mGnRHa-PLGA microparticles despite their many useful effects on boosting semen quality and quantity in multiple stripping of rainbow trout. The release kinetics of PLGA-based microparticles can be modulated by adjusting the ratio of lactic acid to glycolic acid, as well as by altering the physicochemical properties of the polymer such as molecular weight and end-group functionality. These parameters determine the degradation rate, morphology, and hydration behavior of PLGA particles, thereby controlling the rate of drug diffusion and polymer erosion [7]. In general, formulations with a higher glycolic acid content degrade faster due to their higher hydrophilicity, whereas increasing the lactic acid fraction results in slower degradation and prolonged release [8]. The objective of the present study is to synthesize and characterize the mGnRHa-PLGA microparticles and evaluate their application to improve semen quality and quantity under multiple stripping.
2. Materials and methods
2.1. mGnRHa-PLGA microparticles preparation
PLGA delivery systems were prepared using an oil-in-water (o/w) emulsion process based on Andhariya [12] with minor modifications. Briefly, 450 µL of dichloromethane was used to dissolve 100 mg of PLGA (Poly [lactic-co-glycolic acid] (lactic/glycolic 50:50; Mw 7000−17000Da, Sigma-Aldrich, USA)) and 0.2 mg of mGnRHa (Sansheng, China); the solution thus obtained was designated as the organic phase that was subsequently added gradually to an aqueous solution containing 900 µL of 0.35% (w/v) polyvinyl alcohol (PVA; MW 72000, Sigma-Aldrich, USA). The final solution was stirred for 10 minutes at 12,000 rpm to be followed by stirring for six hours at 25°C and 250 rpm. The microparticles formed were washed three times with distilled water, freeze-dried (Martin Christ, Germany) for 24 hours, and finally stored at −20°C.
2.2. Examination of the microparticles surface and particle size distribution
The microparticles were subjected to SEM (scanning electron microscope) investigations for surface analysis. Briefly, mGnRHa microparticles were placed on carbon-taped aluminum stubs before being gold sputtered in a high-vacuum argon evaporator. The samples were observed using a scanning electron microscope (XL30 S-FEG; Philips, Amsterdam, the Netherlands). ImageJ (Version 1.53t; imagej.net) was used to assess the morphology of the microparticles and the sphericity factor (SF) was determined SF = 4πA/P2, where, A is the microparticles area in mm2, p is the microparticles perimeter in mm, and π is equal to 3.14 [12].
Particle size distributions were determined by laser light scattering using Fraunhofer diffraction (Mastersizer X, Malvern, Worcestershire, UK, equipped with a 100-mm lens) as described by Andhariya [12].
2.3. Drug loading and encapsulation efficiency
Drug loading and encapsulation efficiency were determined by dissolving 30 mg of mGnRHa microparticles in dichloromethane and analyzing the aqueous phase using HPLC (Hitachi L-7110 pump, L-7420 UV detector, C18 column). The mobile phase consisted of 48% acetonitrile and 0.1% trifluoroacetic acid, with detection at 215 nm, a flow rate of 1.0 mL min ⁻ ¹, and an injection volume of 20 µL [13]. To ensure accuracy and reproducibility, all measurements were performed in four replicates under identical analytical conditions.
The following equations were used to calculate microparticles drug loading, (DL = [weight of drug entrapped/ weight of microparticles] × 100). Encapsulation efficiency (EE = [weight of drug entrapped/ drug weight at preparation] × 100), and yield (Yield = [total weight of prepared microparticles/ total amount of drug and polymer used] × 100) [14].
2.4. In vitro drug release
To evaluate In vitro mGnRHa release from a microparticles, 10 mg of mGnRHa microparticles and 2 mL of phosphate-buffered saline (PBS; pH 7.4) were mixed and placed on a rotator set at 100 rpm for 61 days at 12°C. At 0, 9, 21, 47, and 61 days, the release medium was collected and centrifuged at 4000 × g for 3 minutes. After being filtered through 0.22 µm, GnRHa concentration was determined by HPLC [12]. To ensure the reliability of the results, the in vitro release experiment was performed in 4 replicates under identical conditions.
2.5. Animal maintaining and experimental design
This study was carried out during the late spawning season. Fifty mature rainbow trout males (738.60 ± 104.86 g and 40.09 ± 2.03 cm; Table 1) were individually tagged in the body cavity with passive integrated transponders (PIT, Trovan, China). The study was conducted at Abzi Nagin Shayan Fereydunshahr Co., located in Fereydunshahr, Isfahan, Iran (33°01’17.7“N, 49°53’02.2”E). Fish were randomly divided into 5 groups. The control group (C) received 1 mL kg-1 physiological saline. The second group (10A) received 10 μg kg-1 mGnRHa. The third to fifth groups (10M, 20M, and 40M) received 10, 20, and 40 μg kg-1 mGnRHa in the form of mGnRHa-PLGA microparticles, respectively. All injections were administered into the body cavity, and semen and blood samples were collected at 0, 9, 21, 47, and 61 dpi. The fish were maintained in a single raceway tank under ambient photoperiod, at 10–12°C with water oxygen saturation levels between 90–100%. The study was approved by the Educational Office of the Department of Natural Resources, Isfahan University of Technology, Iran (#121.1400.20000, 8 August 2021).
2.6. Semen characteristics volume, density and viability
At each sampling, semen volume of all fish were measured (±0.1 mL) using syringes. Sperm density (×109 cells kg ⁻ ¹ b.w.) was determined following Heyrati [3]. The cell membrane integrity of sperm samples was assessed following the eosin–nigrosin staining method described by İnanan and Yılmaz [15]. A 10 µL semen was mixed with eosin–nigrosin solution (5% eosin, 10% nigrosin; Sigma-Aldrich, Steinheim, Germany; pH 6.9) at a 1:4 (v/v) semen/ dye ratio. A 5 µL drop of the stained mixture was smeared on a glass slide and air-dried. Slides were examined under a light microscope (400 × ; Leica DM500, Heerbrugg, Switzerland). Spermatozoa with colourless cytoplasm were considered intact, whereas those with stained cytoplasm were considered damaged. A total of 100 sperm cells were counted per slide, and the percentages of intact and damaged sperm were calculated.
2.7. Plasma level testosterone
Blood samples were collected from the caudal vein of three fish per treatment at each sampling time using heparinized syringes (22G) after sedation with MS-222 (100 mg L ⁻ 1).The samples were centrifuged at 4000 × g for 10 minutes at 8°C before plasma levels of testosterone were measured in duplicates using the relevant kits (T; KAPD1559; DIA source ImmunoAssays SA, Louvain-la Neuve, Belgium) according to the ELISA technique and The dynamic range of the assay was 0.15–16 ng mL-1 [16].
2.8. Testis morphology
At the end of the experiment at 61 dpi, five fish from each experimental treatment were randomly selected and euthanized with an overdose of MS-222 (300 mg L ⁻ 1) for gonadosomatic index (GSI) measurement and testis histology studies. The following formula was used to determine GSI (GSI = [gonad weight/Body weight] × 100).
For histological analysis, testis tissue was stained with hematoxylin-eosin (HE) according to the method described by Toledo-Solís and et al [17]. Spermatids and spermatozoa areas were determined by analyzing the area (per 85 mm2 testis section) of the testicular tissue using ImageJ (Version 1.53t; imagej.net) software [17]. For every parameter evaluated, at least 3 measurements per section were performed.
2.9. Statistical analysis
All data are presented as mean ± standard error (SE). Prior to analysis, data were tested for normality using the Kolmogorov–Smirnov test. A one-way analysis of variance (ANOVA) was conducted to evaluate differences among treatments, with a significance threshold of p < 0.05. Duncan’s multiple range test (DMRT) was applied for post hoc pairwise comparisons: lowercase letters indicate significant differences among treatments at the same sampling time, while uppercase letters indicate significant differences across sampling times within the same treatment. Statistical analyses were performed using IBM SPSS Statistics (version 25) and Microsoft Excel (2013).
3. Results
3.1. Examination of the microparticles surface and particle size distribution
The surface morphology of mGnRHa microparticles was examined using scanning electron microscopy (SEM). Fig 1A and B show SEM images of the microparticles produced via the solvent evaporation method. The drug-loaded microparticles were spherical with smooth surfaces, exhibiting uniform morphology without any visible polymer residues or crystalline drug particles (Table 2). The mGnRHa microparticles exhibited average diameter particle sizes of 14.77 ± 8.09 µm (Table 2; Fig 2).
The drug-loaded microparticles are observed as spherical particles with a smooth surface (scale bars: 500 μm in A and 100 μm in B).
All preparations were collected in purified water by laser light scattering using Fraunhofer diffraction.
3.2. Drug loading and encapsulation efficiency
Table 2 presents the drug loading, encapsulation efficiency, yield, mean size, and sphericity factor of the mGnRHa microparticles. The microparticles exhibited a drug loading of 0.083 ± 0.031% and an encapsulation efficiency of 44.06 ± 2.28%. The production yield was 81.21 ± 2.03%, and the mean particle size was 14.77 ± 8.09 μm. The particles were highly spherical, with a sphericity factor of 0.99 ± 0.00 (Table 2).
3.3. In vitro drug release
The drug release test helps estimate release behavior. Fig 3 illustrates the drug release rate of the mGnRHa microparticles in phosphate buffer at 12°C over 61 days. The initial release was 54.53 ± 1.21% within the first 5 days, followed by a steady, slow release until day 21 (Fig 3). From day 21 to day 61, a more rapid and nearly linear discharge was observed, with 72.70 ± 0.75% of the mGnRHa released by the final day of the experiment (Fig 3).
3.4. Semen characteristics volume, density and viability
Fish subjected to 1 mL kg-1 0.9% NaCl (C), acute mGnRHa 10 μg Kg-1 b.w., (10A), 10, 20, and 40 μg Kg-1 b.w. mGnRHa microparticles represented as 10M, 20M, and 40M, respectively (n = 50). As shown in Table 3, all fish in each group were able to release semen at day 0. From day 47 onward, the number of stripable fish in the control and M10 groups decreased sharply and reached to zero. Only the 40M group maintained the highest number of stripable fish until the end of the study.
The mean expressible semen over the entire study period in the control group (C) was 1.54 ± 0.47 mL kg ⁻ ¹ b.w., which was not significantly different from the A10 and M10 groups (Fig 4A; P > 0.05). However, the M20 (4.54 ± 0.76 mL kg ⁻ ¹ b.w.) and M40 (5.15 ± 0.66 mL kg ⁻ ¹ b.w.) groups showed significantly higher amounts of expressible semen (Fig 4A; P < 0.05). Additionally, regarding the mean sperm density, only the M40 group showed a significantly higher value compared to the control group (Fig 4B; P < 0.05).
At 0 dpi, no significant differences were observed among the treatment groups (Fig 5A; P > 0.05). At 9 dpi, all groups showed higher levels of expressible semen compared to the control group (Fig 5A; P < 0.05). However, at the subsequent sampling points on days 21, 47, and 61, the 40M group exhibited the highest amount of expressible semen among all groups (Fig 5A; P < 0.05). Furthermore, the 40M group exhibited the highest mean sperm density among all experimental treatments at all sampling points from day 9 onwards (Fig 5B; P < 0.05).
No significant differences were observed among the treatments with respect to the percentage of sperm viability at 0 dpi (P > 0.05; Fig 6). In contrast, this value exhibited significant increases on dpi 9 across the treatments (P < 0.05; Fig 6) while it remained almost the same between the 9 and 61 dpi albeit it was higher than 70% at all sampling times (P > 0.05; Fig 6).
Fish subjected to 1 mL kg-1 0.9% NaCl (C), acute mGnRHa 10 μg Kg-1 b.w., (10A), 10, 20, and 40 μg Kg-1 b.w. mGnRHa microparticles represented as 10M, 20M, and 40M, respectively. Significant differences between treatments indicated by capital case (one-way ANOVA, DMRT, P < 0.05, Total n = 250).
Fish subjected to 1 mL kg-1 0.9% NaCl (C), acute mGnRHa 10 μg Kg-1 b.w., (10A), 10, 20, and 40 μg Kg-1 b.w. mGnRHa microparticles represented as 10M, 20M, and 40M, respectively. Significant differences between treatments at each sampling time are indicated by lower case, while the capital letters are used to show significant differences within an experimental treatment over the period (one-way ANOVA, DMRT, P < 0.05, Total n = 250).
Fish subjected to 1 mL kg-1 0.9% NaCl (C), acute mGnRHa 10 μg Kg-1 b.w., (10A), 10, 20, and 40 μg Kg-1 b.w. mGnRHa microparticles represented as 10M, 20M, and 40M, respectively. Significant differences between treatments at each sampling time are indicated by lower case, while the capital letters are used to show significant differences within an experimental treatment over the period (one-way ANOVA, DMRT, P < 0.05, Total n = 147).
3.7. Plasma level testosterone
At 0 dpi, no significant differences were observed among the treatment groups (Fig 7; P > 0.05). At 9 days post-injection (dpi), all groups showed higher plasma testosterone levels compared to the control group, but the 40M group exhibited a significantly higher level than the all treatments (Fig 7; P < 0.05). At 21 dpi, no significant differences were observed between the A10, M10, and C groups, while the 20M and 40M groups showed significantly higher testosterone levels (Fig 7; P < 0.05). At 47 dpi, the A10 group exhibited significantly lower testosterone levels compared to the 40M group, while the other treatment groups displayed intermediate values between A10 and 40M (Fig 7; P < 0.05). At 61 dpi, no significant differences in testosterone levels were observed among the groups (Fig 7; P > 0.05).
Fish subjected to 1 mL kg-1 0.9% NaCl (C), acute mGnRHa 10 μg Kg-1 b.w., (10A), 10, 20, and 40 μg Kg-1 b.w. mGnRHa microparticles represented as 10M, 20M, and 40M, respectively. Significant differences between treatments at each sampling time are indicated by lower case, while the capital letters are used to show significant differences within an experimental treatment over the period (one-way ANOVA, DMRT, P < 0.05, Total n = 75).
3.8. Morphology of testis
At the end of the study period, the gonadosomatic index was evaluated. Only the 40M group showed a significantly higher GSI compared to the control group (Fig 8A; P < 0.05), while no significant differences were observed between the other treatment groups and the control (Fig 8A; P > 0.05). Different sperm cell types were observed in testicular histology including: spermatogonium, primary spermatocyte, secondary spermatocyte and spermatid (Fig 9). The distribution of spermatids and spermatozoa in the testicular tissue was analyzed using histological images (HE) across the treatments on 61 dpi (Fig 9). The lowest spermatid and spermatozoa surface (%) was observed in the control group. This value increased significantly in the A10, M10, and M20 groups, while the highest value was recorded in the 40M group (Fig 8B; P < 0.05).
Fish subjected to 1 mL kg-1 0.9% NaCl (C), acute mGnRHa 10 μg Kg-1 b.w. (10A), 10, 20, and 40 μg Kg-1 b.w. mGnRHa microparticles represented as 10M, 20M, and 40M, respectively (one-way ANOVA, DMRT, Total n = 25).
Fish subjected to 1 mL kg-1 0.9% NaCl (C), acute mGnRHa 10 μg Kg-1 b.w. (10A), 10, 20, and 40 μg Kg-1 b.w. mGnRHa microparticles represented as 10M, 20M, and 40M, respectively. Descriptions: spg: spermatogonium, sc1: primary spermatocyte, sc2: secondary spermatocyte, st: spermatid, sz: spermatozoa, Ley: Leydig cell. Scale bar = 50 μm. (one-way ANOVA, DMRT, Total n = 25).
4. Discussion
The structural features of mGnRHa-PLGA microparticles and their effects on the quality and quantity of rainbow trout’s semen were investigated. The most important structural feature was the microparticles’s size, with particle sizes below 125 µm most appropriate for syringe injection and those below 250 µm still regarded as acceptable [18]. In this study, the size of mGnRHa-PLGA microparticles was 14.77 ± 8.09 µm, making them suitable for injection using a 22-gauge syringe. Compared to other drug delivery systems such as ethylene vinyl acetate copolymer (≈2 mm3) [9] or cholesterol implants (≈3 mm3) [16], the small size of microparticles helps reduce stress on breeders during injection.
A three-stage release pattern of mGnRHa was observed over the 61-day study period. The first phase occurred from day 0 to day 5, primarily due to drug hydrogel particles located near the surface, inter-channel spaces, and inner pores of the microparticles, which were formed by solvent evaporation during solidification [13]. The second phase was driven by the gradual degradation of the polymer matrices; most microparticles structures remained intact for up to 47 days, likely due to adhesion and coalescence during the test. The experiment was performed at 12°C, the expected temperature of rainbow trout in the farm, which may have influenced the degradation rate compared to other studies conducted at different temperatures [6]. The third phase, from day 47 to day 61, involved an accelerated release caused by the erosion of the polymer matrices.
It is frequently claimed that a pellet sphericity factor of 0.9 is optimal [15]. This is much desired since high sphericity improves dry powder flow characteristics [6]. In this study, sphericity factors close to 1, mostly near 0.99, were observed that showed a complete circular shape of the particles.
The mGnRHa-PLGA microparticles are free from the toxic properties of adjuvants [16] or chitosan-gold nanoconjugates [17]. Moreover, compared to ethylene vinyl acetate copolymer [5], they are smaller in size and biodegradable. The combination of these properties makes mGnRHa-PLGA microparticles suitable for in vivo test of rainbow trout.
By the end of the spawning season, or after multiple semen collections, only a limited number of genetically superior breeders remain, and semen quantity and quality decline rapidly [19]. In such situations, mGnRHa microparticles can be effectively used not only to improve semen volume and expressible semen but also to enhance plasma testosterone levels and increase GSI by the end of the study period. Therefore, we conducted this in vivo test on rainbow trout at the end of the spawning season for this reason.
Only 40M males continued to release semen until 61 dpi. The 40M group showed the highest semen volume and sperm density throughout the experimental period, while no significant differences were observed between the C and rest groups. At 21, 47, and 61 dpi, the 40M group exhibited the highest sperm density, semen volume, and plasma testosterone levels.
Semen quantity in meagre (Argyrosomus regius) has been significantly improved following administration of a GnRHa slow-release implant, as opposed to acute GnRHa treatment [9]. This has been attributed to the short GnRHa lifetime, as a single acute injection can enhance semen quantity and quality by inducing an LH surge in rainbow trout, but its effect is transient [4]. In the starry flounder (Platichthys stellatus), which is a species with typically viscous semen output, implants with GnRHa have also been successfully used to improve semen volume [20]. Similar results have been reported for yellowtail flounder)Pleuronectes ferrugineus) [21] and the Atlantic cod (Gadus morhua) [22].
GnRHa injection reportedly improves the hydration of the testes by facilitating seminal fluid secretion, which is influenced by the maturation-inducing steroid [23]. More sperms are frequently present in the testes (i.e., intra-testis sperms) that are released and can be stripped as a result of the increased fluid content of the testes. This method “washes out” more readily the sperms available in the testes. As a result, the sperm density thus collected gradually declines, as already observed in the present study, such that only acute mGnRHa was capable of temporarily increasing the semen volume [5].
Reportedly, encapsulation renders a more stable GnRHa [18]; otherwise, it only lasts for a short time in fish blood [4]. The advantages associated with novel slow-released methods include lower adverse impacts on the GnRHa process and enhanced efficacy. On the other hand, sustained release of mGnRHa enjoys such advantages, over the conventional method, as relaxing the need for repeated injections and continuous manipulation [14].
In the slow release GnRH system groups, blood plasma testosterone level is higher than those in the control and acute mGnRHa treatments [14]. This finding is confirmed by the results obtained in the present study. The hormones, 11-KT, and testosterone are found to be in charge of triggering and regulating spermatogenesis. Meanwhile, 11-KT reportedly stimulates the development of secondary sexual characteristics, spermatogonial proliferation, and spermiation [21]. This is while testosterone, a biosynthetic precursor of 11-KT, stimulates spermatogenesis. Increased levels of steroids indicate their beneficial function in promoting spermiation in fish and demonstrate the advantages of the PLGA microparticle technology with continuous mGnRHa release in sperm production and quality [21]. Finally, the release ratio may be regulated depending on the method used to construct the delivery system [8]. It may, therefore, be concluded that this method of hormone administration will help manage the reproduction of male rainbow trout under different environmental conditions.
At the end of the experiment, the control group was found to have very low spermatozoa and spermatids surface areas with most of the lobule lumen being nearly empty. Despite the fact that no semen was obtained from the 10A and 10M groups at 61 dpi, a large tissue area containing spermatozoa and spermatids was still observed. In contrast, histological sections of the testes of the 20M and 40M on 61 dpi exhibited active spermatogenesis with a large surface area of spermatids and free spermatozoa, with expressible semen detected only in 40M. As also demonstrated in a previous study, the treatment with slow release GnRHa implants resulted in the maintenance of spermatogenesis, generating more spermatozoa over a long period of time [19].
5. Conclusions
This study demonstrates that mGnRHa microparticles, with their small and uniform size (14.77 ± 8.09 µm), high sphericity, and controlled release over 61 days at 12°C, offer a practical and efficient approach for improving semen quality in male rainbow trout during the late spawning season, when semen quality in some males may occasionally decline. The sustained release of mGnRHa significantly enhanced semen volume, plasma testosterone levels, GSI, and maintained active spermatogenesis, particularly in the 40M treatment. These findings highlight the potential of PLGA-based sustained-release systems as a valuable tool for managing broodstock reproduction and reducing production costs in aquaculture. Further studies evaluating the long-term performance of these microparticles under varying environmental conditions are recommended, particularly in species inhabiting different temperature regimes or exhibiting spawning periods of different durations. Such investigations would help determine how temperature and spawning season length influence hormone release profiles and the optimal duration of hormonal stimulation required for successful application in commercial breeding programs.
Acknowledgments
We would also like to express our gratitude to the manager, P. Daneshmandi, and his colleagues at Abzi Negin Shayan Fereydounshahr Co. for their kind assistance and support. We also thank Center for International Scientific Studies and Collaboration (CISSC), Ministry of Science, Research, and Technology, Islamic Republic of Iran for its support.
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