Impact of Synthetic Androgen on Milt Quality and Semen Preservation of Gilthead Seabream (Sparus aurata)

Abstract

Background:

The use of synthetic hormones for induced breeding of fish has been a long-standing trend. Studies have demonstrated that exogenous administration of synthetic androgens are effective to improve milt quality of male fish.

Objectives:

This study aimed to evaluate the impact Testosterone propionate (TP), a synthetic androgen, on the quality of milt and the preservation (short duration) of Gilthead Seabream semen at 4 oC in oxygen and Fish Ringer’s medium.

Materials and Methods:

Sexually matured male fishes were given intra muscular injection of Testosterone propionate (TP) at a dose of 1.0 mL. Kg^-1, and a control group was maintained without hormone injection. After 36 hours, milt was collected and evaluated for sperm motility, density, sperm viability and colour. Semen preserved at 4 oC in oxygen and Fish Ringer’s medium was examined for sperm motility (%), motility duration (sec) and viability (%) at 24,48,72,96 and 120 hours.

Results:

Significant change in colour or pH of the milt was not observed. Milt’s consistency changed from being thick to waterier. The hormone injected fish group had an increased level of milt volume and sperm density than the control. Milt stored at 4 oC in oxygen atmosphere showed higher sperm cell quality than that stored in Fish Ringer’s medium.

Conclusion:

The results of this study can be used as a guideline for induced breeding and hatchery production of Gilthead Seabream, S.aurata.

Background

Germplasm preservation techniques serve as repositories for selective breeding, hybridization, commercial seed production, and conservation of endangered species ( 1 ). Furthermore, the use of germplasm cryopreservation has been broadened to include the production of self-fertilized inbred lines, triploid-tetraploid aquaculture, and potentially the production of mutant lines in ornamental fish culture, such as zebrafish ( 2 , 3 ). Studies have shown that Testosterone Propionate (TP) has a stimulatory effect on growth, survival and sex ratio of African catfish, Clarias gariepinus ( 4 ). Synthetic androgen testosterone propionate enhances milt quality of Asian seabass (Lates calcarifer), and testosterone improved growth in juvenile Coho salmon and sexual activity and reproduction in female Zebrafish ( 5 , 6 , 7 ). Milt can be preserved for a short period in hatcheries and farms by following a straightforward process that involves keeping fresh semen for a few hours or even several days ( 8 ). Several researchers have reported that oxygenated, undiluted semen stored in the refrigerator at 4 °C showed the maximum duration of sperm motility and viability ( 9 ).

The Mediterranean gilthead seabream (Sparus aurata) is becoming more popular as a table fish due to its excellent flavor and high meat quality. Seabream is a protandrous hermaphrodite fish, which sexually matures first as a male when attaining an age of 1 year and later changes to a female. One of the reasons behind the unavailability of seeds is the lack of mature spawner fishes for induced breeding for seed production. Given the importance of milt preservation in fish breeding, this study aimed to assess the effect of TP on the milt characteristics and its short-term storage in Gilthead Seabream, S. aurata an ideal candidate for cage culture practice.

2. Objectives

The study’s goal was to ascertain how testosterone propionate affects the characteristics of milt, including its color, nature, pH, volume, sperm density, motility, motility duration and viability. Another aim was to assess the quality of semen during a short period of preservation at 4 °C in Fish Ringer›s medium and oxygen for 120 h. Additionally, sperm motility, duration, and viability were to be monitored at 24, 48, 72, 96 and 120 h.

3. Materials and Methods

Adult Seabream (1.2 ± 0.18 kg) were collected from Tharawat Fish Farm in Yanbu, Jeddah. Five fish were stocked in a 5000 L maturation tank with seawater (40 ppt) and aeration. The treatment group received an intramuscular injection of TP (1mL.kg^-1), while the control group received an equivalent volume of saline solution (0.9% NaCl). Both groups were fed a 40% protein pellet feed twice daily and water exchange was conducted daily at a 20% rate. Water quality parameters (salinity, temperature, pH, and dissolved oxygen) were monitored daily at a consistent time (10:00 am) to ensure stability. Fish were gently cleaned using a cotton swab and tissue paper to remove feces and urine, minimizing contamination of milt. Milt was collected from both the control and treatment fish before (0 h) and after (36 h) injection using a graduated pipette. Collected milt was transferred to zip lock polythene bags (8×12cm), placed in an icebox (4 oC) for subsequent milt quality analysis and short-term storage ( 10 ). Milt was evaluated for consistency, colour, volume, pH, motility, density and viability. The consistency and color were noted at the time of stripping. Volume was determined by collecting all milt (mL) in a graduated pipette (10ml). Sperm density was measured using a Neubauer hemocytometer after diluting the sperm with saline (NaCl-0.9%). Sperm motility and viability were assessed under a compound microscope at 400x magnification.

Sperm motility duration was assessed using fresh seawater (40 ppt) as the activation medium. A 1:400 dilution of milt in the medium was prepared on a glass slide, covered and observed under a 400x microscope. Motility duration was recorded from the time of mixing until at least 20% of the spermatozoa ceased active movement. Sperm motility (%) was determined by using the formula: (Number of motile spermatozoa / Total number of spermatozoa) × 100. Motility scores were assigned based on the Raghuvanshi et al. scoring system (I-V) ( 11 ). Sperm viability was assessed using the eosin-nigrosine dye exclusion method. Live spermatozoa appeared gray, while dead spermatozoa stained pink or red. Sperm viability was calculated as: (Number of live spermatozoa / Total number of spermatozoa) × 100. Milt was preserved at 4 °C using two methods. 1. Oxygen atmosphere (Milt was placed in Petri dish within oxygen filled polythene bags. Samples were stored for 120 hours, with assessments at 0, 24, 48, 72, 96, and 120 h) and 2. Fish Ringer’s medium (Milt was diluted 1:3 in Fish Ringer’s medium and stored in polypropylene vials. Samples were stored and assessed as in method 1). In both methods, samples were thawed in water at 27 oC for 15 seconds before evaluating motility duration and viability. One-way analysis of variance (ANOVA) and t- Test was employed (Excel-MS Office, Version 16.0, 2018) to determine the significant difference (α = 0.05) in the means of milt quality between the control and treatment fishes. Homogeneity of variance was checked with Levene’s test. Duncan’s multiple range test was used to compare the average values (means ± standard deviation) between groups.

4. Results

The water quality parameters were found to be within the tolerable range (Salinity 40 ± 0.4 ppt, Temperature 29.1± 2 oC, pH 7.8 ± 0.3 and Dissolved oxygen 4.3 ± 0.4 ppm). After hormone injection, milt’s nature changed from thick to watery. Treatment fish exhibited a significant increase in milt volume and sperm concentration 36 hours post-injection. Milt pH remained unaffected. Treated fish showed improved sperm motility duration and percentage motility, while sperm viability and motility score were not significantly influenced by the hormone injection (Table 1).

Table 1.

Details on milt quality of Seabream before and after hormone injection

Parameters	0 Hours		p(T<=t)	36 hours		p(T<=t)
	Control	Treatment		Control	Treatment
	Mean ± SD	Mean ± SD		Mean ± SD	Mean ± SD
Milt colour	White	White		White	White
Milt nature	Thick fluid	Thick fluid		Thick fluid	Watery fluid
Milt volume (ml)**	3.3 ± 0.1a	3.4 ± 0.3b	0.5231	3.2 ± 0.3a	3.5 ± 0.4b	0.0041
pH NS	7.2 ± 0.2a	7.2 ± 0.4a	0.1681	7.2 ± 0.2a	7.3 ± 0.3a	0.0032
Sperm cell concentration (×109 ml -1)**	8.5 ± 1.3a	8.6 ± 1.1a	0.3324	8.5 ± 2.0a	9.3 ± 2.0b	0.3121
Motility duration (sec)**	142 ± 3.0b	141 ± 4.0a	0.1991	141 ± 3.0a	144 ± 2.0c	0.0023
Sperm motility (%)NS	91.3 ± 1.6a	91.0 ± 2.3a	0.4434	90.3 ± 1.4a	91.3 ± 1.1a	0.0225
Viability (%)NS	93.1 ± 3.2a	93.2 ± 2.2a	0.122	93.4 ± 2.1a	93.2 ± 1.9a	0.7642
Motility score	V	V	-	V	V	-

^** p<0.05;

^NS-Non significant; (n=5);

p(T<=t) two-tail @ alpha 0.05 (t-Test analysis)

^a,b,c - Means with the same superscript do not differ from each other (Duncan’s test).

Figure 1 illustrates the results of short-term milt preservation at 4 °C in Ringer›s medium and oxygen. Motility in the treatment group declined from 85% at 0 hours to 65% at 48 hours, compared to a steeper drop from 80% to 45% in controls, indicating a protective effect of the treatment. Viability remained stable up to 24 hours (80% in both groups) but decreased more rapidly in controls thereafter (60% vs. 70% in treatment). It is observed that a clear decline in both sperm motility and viability over 120 hours in both storage media (oxygen and fish Ringer’s) for both control and treatment groups. In oxygen, motility decreased from 100% at 0 hours to 50-60% in controls and 70-80% in the treatment group by 120 hours, while viability dropped from 100% to 40-50% in controls and 60-70% in the treatment group. In Ringer’s medium, motility fell from 100% to 40-50% in controls and 60-70% in the treatment group, and viability declined from 100% to 30-40% in controls and 50-60% in the treatment group by 120 hours. Additionally, the treatment group consistently exhibited fewer rapid declines and higher values than the control, indicating a protective effect of the treatment.

Figure 1.

Sperm motility, motility duration and viability of Seabream after preservation. This figure illustrates the results of short-term milt preservation of Gilthead Seabream in an oxygen atmosphere and Ringer’s medium at 4 °C. Milt from both the control and hormone-injected fish exhibited a decreasing trend in sperm motility duration, motility and viability with increased storage time.

5. Discussion

The findings of the study show that intramuscular injection of TP altered the milt quality of Gilthead Seabream, changing it from a thick and viscous consistency (Control) to a more watery and diluted state (Treatment) ( 5 ). Garcia et al. reported that milt quality improved when they injected an analog of Luteinizing Hormone-Releasing Hormone (LHRH) and 17α-Methyl Testosterone in Seabass, Lates calcarifer ( 12 ). The observed hydration of milt in this study may be attributed to the stimulatory effect of Testosterone Propionate on increased seminal plasma secretion.

Testosterone, a precursor of plasma ketotestosterone, acts on the hypothalamus and/or pituitary, influencing sexual maturation ( 13 ). It has been shown to be sensitive to exogenous aromatizable steroids, such as Methyl Testosterone. Synthetic form of testosterone propionate enhanced sperm cell concentration in Asian Seabass, Lates calcarifer ( 5 ). In various teleosts, testosterone and other androgens have demonstrated positive effects on gonad maturation, ripening, and milt production ( 14 ). The European seabass (Dicentrarchus labrax) showed enhanced sperm volume and motility after receiving an intramuscular injection of GnRHa (20–40 µg.kg^-1) ( 15 ). Therefore, the high sperm cell concentration observed after testosterone propionate injection in Seabream is likely due to the hormone’s stimulatory effect on the gonads.

The primary challenges affecting preservation are temperature, gaseous exchange, cryoprotectants, microbial contamination, and desiccation. Gwo ( 16 ) observed that anaerobic storage of spotted sea bass (Lateolabrax maculatus) sperm resulted in rapid motility loss. In contrast, storage under air or pure oxygen at 4 °C achieved 94% fertility in Seabream, Sparus aurata after 216 hours ( 17 ). Kowalski and Cejko ( 18 ) found that sperm stored under oxygen had significantly higher fertilizing ability than those stored in open air, even after five days of storage at 4 °C. Sperm viability, motility and duration were better in milt stored in oxygen than in Ringer’s medium at 4 °C. This suggests oxygen is crucial for sperm metabolic function at low temperatures, making it ideal for short-term storage of Seabream milt as observed in Asian Seabass.

Long-term testosterone propionate (TP) use may cause gonadal suppression and environmental concerns, such as hormone residues in water systems affecting non-target organisms, necessitating residue monitoring and withdrawal periods. In brood stock males, low-dose TP injections 24-48 hours before stripping, followed by a brief recovery period to minimize stress, can enhance sperm quality, boosting fertilization rates and larval survival in hatcheries. Batch-processing males, optimizing timing with spawning cycles, and monitoring residue levels, can achieve large-scale juvenile production in aquaculture. Future research should investigate TP’s chronic effects on gonadal development, fertility, offspring performance, and en-vironmental residue impacts on wild populations.