Effects of Exogenous Melatonin and Zinc Amino Acid on Male Clarias macrocephalus Broodstock

MATERIALS AND METHODS

Animals: The Clarias macrocephalus broodstock were obtained from the Fisheries Station of Kham PhengPhet, Department of Fisheries, Ministry of Agriculture and Cooperative, Thailand and the trial experiments were done in the Laboratory of Nutrition and Aqua feed, Department of Aquaculture, Faculty of Fisheries, Kasetsart University, Bangkok, Thailand. The 8 week old catfish were acclimatized in 500 L tanks at the density of 15 ind m^–2/fish/tank and fed with control feed for 2 weeks prior to experiment. The water supply for the experiment was aerated to maintain the oxygen supply in the experiment tank.

Experimental diets: A feeding trial comprised of one control and two combinations of melatonin-ZnAA diet. The basal diet was formulated from practical ingredients, containing 22% fishmeal, 35% soybean, 1% spirulina, 12% wheat flour, 11.8% tapioca, 5% ricebran, 2% fish oil, 3% soy oil, 1.2% mineral premix, 2% soy lecithin, 1.5% calcium phosphate, 1% attractant, 2% binder and 0.5% vitamin premix. The diet also consisted of 37% crude protein and 9.3% crude lipid. Diets containing zinc and melatonin were prepared by adding graded levels of zinc and melatonin to the basal diet. The ZnAA and melatonin concentrations: 0 ppm ZnAA and 0 mg kg^–1 melatonin (Control), 100 ppm and 50 mg kg^–1 melatonin (MZn1) and 200 ppm and 50 mg kg^–1 melatonin (MZn2)^18,19.

Experimental condition: A total of 45 male catfish of mean weight 88.07±9.57 g (Mean±SD) were starved in tanks for 2 days prior to the experiment. All the population was subjected to a normal photoperiod (12L:12D) prior to the treatment. The fish were fed at an amount equivalent to 3% of their body weight and this amount of diet was divided into two equal feedings per day. The fish were randomly distributed in three treatments (Control, MZn1 and MZn2) replicated three times. Following acclimation, the fish were exposed to the treatments for 8 weeks. This study project including experimental trial and laboratory analysis was conducted from early January, 2013 to end of July, 2013 (7 months).

Histology: The testis from each treatment was fixed in 10% buffered formalin. The fixed ovaries were placed in the cassettes for embedding process in paraffin wax. Serial sections were made at four micrometer and were processed using the standard method of histology for sample cutting. For staining method, the samples were stained using Haematoxylin-eosin method. The histology method was according to previous study²⁰. The samples’ cells were counted by microscope.

Gonadosomatic index: The Gonadosomatic Index was determinedas²¹:

GSI = 100 (Gm/Tm)

Characteristics of semen and spermatozoa: Testes from the treated fish were surgically removed and were cut in small pieces to allow sperm release after a gentle squeezing. Semen was stored with extender solution in sterile Appendorf tubes on ice before use. The composition of extenders was as followed: 8.760 g NaCl in 1000 mL distilled water²².

Live sperm rate and sperm concentration: Eosin-nigrosin stained semen smears were evaluated with brightfield microscopy for the live cells by counting 400 cells per sample. The sperm concentration was determined in duplicate and expressed as number of spermatozoa ×10⁶ mL^–1. The number of spermatozoa was counted in improved Neuber haemocytometer under a light microscope.

Sperm kinetic characteristic: Kinetic characteristics were evaluated immediately after semen collection. Ten microlitres of the samples were diluted 1:1 with the extender solution and were laid over a pre-warmed Hamilton Thorne Biosciences chamber at 27.8°C. The analyses were done by the Computer-Assisted Sperm Analysis (CASA). The sperm parameters recorded were motility (%) = The percentage of motile spermatozoa, progressive (%) = The percentage of spermatozoa with a progressive motility, path velocity (μm sec^–1) = A smoothed path constructed by averaging several neighbouring positions on the track (five points) and joining the averaged positions, which reduced the effect of lateral head displacement, prog. velocity (μm sec^–1) = The straightline distance between the first and last tracked points, divided by the acquisition time, track speed or curvilinear velocity (μm sec^–1) = the total distance between adjacent points, divided by the time elapsed, lateral amplitude (μm) = The mean width of the head oscillation as the sperm cells swim, beat frequency (Hz) = The frequency of sperm head crossing the average path in either direction, straightness (%) = An index of the departure of the sperm path from a straight line, linearity (%) = An index of the straightness of the path^23,24.

Sperm abnormality: Fresh semen in the extender solution was stained within ten min post-collection. Eosin-nigrosin stained semen smears were evaluated using brightfield microscopy at ×1000 magnification under oil immersion and the percentages of abnormal and normal sperm cells were determined.

Statistical analysis: All data were analysed by one-way ANOVA (analysis of variance), followed by the Tukey’s honest significance test to analyse the significance between the treatment means. All means comparisons significance was tested at p<0.05 using SPSS software²⁵.

RESULTS AND DISCUSSION

Histological changes in testes during the melatonin-ZnAA treatment were observed in C. macrocephalus (Fig. 1). In the present study, histological analysis showed high spermatogonium percentage in the control treatment (20% (control), 9.9% (MZn1) and 3.8% (MZn2)) and high spermatozoa percentage for both combinations of melatonin-ZnAA treatment (51.3% (control), 75% (MZn1) and 70.5% (MZn2)) (Table 1).

Fig. 1(a-c):	Effects of melatonin-ZnAA treatment on testis histology of the C. macrocephalus. Cross section of testis treated with ZnAA and control, (a) Control, (b) MZn1 and (c) MZn2. Spermatogonia (SG), spermatids (SD) and spermatozoa (SZ) scale bar: 100 μm

Spermatogonia and spermatozoa were observed for the testicular development and staging²⁶. It also showed that the gonadosomatic index was a significant increase with the mean ranging from 0.45-0.96 %, p = 0.005 (Table 1). Spermatozoa cells are the smallest and the most mature cells among the other sperm cells in the sperm developmental stages²⁷. In addition, the testicular development is generally divided into four stages: Spermatogonia, spermatocytes, spermatids and spermatozoa²⁶. Spermatogonia are the undifferentiated and immature sperm cells, having large ovoid cells, central and vesicular nucleus, distinct nuclear membrane and perinuclear cytoplasmic granules, prominent nucleolus enclosed in a membrane of the spermatogonial nucleus²⁶.

Table 1:	Maturation analysis of male C. macrocephalus with melatonin and different ZnAA levels (Mean±SE)
a,bValues with different superscripts in a row differ significantly (p<0.05)

Table 2:	Sperm analysis for C. macrocephalus with melatonin and different ZnAA levels (Mean±SE)
a,bValues with different superscripts in a row differ significantly (p<0.05)

Fig. 2(a-b):	Some of the sperm abnormality traits in melatonin-ZnAA treatment of the male C. macrocephalus, (a) Normal C. macrocephalus spermatozoa and (b) Abnormal C. macrocephalus spermatozoa. Normal (n), bend tail (bt), giant head (gh), midpiece defect (mpd) and small head (sh)

Meanwhile, spermatozoa are the mature sperm cells and found in abundance in the lumen of the seminiferous lobules. In male teleost, melatonin plays a significant role in regulating the testicular event. Melatonin treatment may act through interneurons to influence the GnRH neural activity and regulate follicle stimulating hormone (GTH-I) in male teleost⁶. On the other hand, zinc plays an important role in the synthesis of GTH-I and GTH-II gonadotropins hormone in male teleost. Even though the zinc mechanism on synthesizing the gonadotropins is not fully understood, it is known that zinc deficiency may lead to decreasing GTH-I and GTH-II production²⁸. In the teleost testicular event, GTH-I regulates Leydig cell and Sertoli cell activities²⁸. Another pituitary hormone that regulates the testicular physiology is gonadotropin GTH-II that co-regulates Sertoli cell function²⁸. Sertoli cells’ main roles are to sustain germ cell survival, growth and physiological function. The development of sperm cells mostly depends on the Sertoli cells to provide the somatic elements of the testis²⁸. In the current study, melatonin and ZnAA mechanism in the testicular event indicated that melatonin and zinc were essential components for spermatogenesis.

In the current study, sperm abnormality was observed (Fig. 2) and it was noted that it significantly decreased in the melatonin-ZnAA treatment (p = 0.022, Table 2) and some sperm kinetic parameters were found highly significant (motility p = 0.013, sperm concentration p = 0.037, sperm lateral amplitude p = 0.037) (Table 2, 3). Both melatonin and zinc was effective in protecting the sperm cells from free radical (reactive oxygen species) and lipid peroxidation via antioxidative properties²⁹. The unsaturated fatty acids (PUFA) in the plasma membranes of the spermatozoa are highly sensitive to free radicals that may lead to lipid peroxidation³⁰. In addition, cell proliferation, differentiation and migration for intracellular signaling may benefit from the activity of free radicals^{31, 32}. However, high levels of free radicals especially the reactive oxygen species (ROS) may produce excessive peroxides that affect the spermatozoa membrane. Lipid peroxidation may lead to membrane damage, especially in the acrosomal area. The membrane damage may cause low motility and viability of the spermatozoa and oxidize the DNA, ultimately resulting to defective sperm cells that are unable to fertilise the oocytes³³.

Table 3:	Motility parameters measured by CASA for male C. macrocephalus with melatonin and different ZnAA levels (Mean±SE)
a,bValues with different superscripts in a row differ significantly (p<0.05)

In the current study, melatonin and ZnAA showed that melatonin and zinc were able to reduce abnormality rate and increase the sperm kinetic in male C. macrocephalus.

CONCLUSION

For the first time, this study showed that a combined melatonin and zinc amino acid feeding treatment to male C. macrocephalus has significantly improved the maturation in MZn2 treatment. The results indicated that this feeding treatment could be applicable to teleost reproductive biology.

SIGNIFICANCE STATEMENT

This study discovers that combined melatonin and zinc amino acid in the feed able to enhance the maturation of male Clarias macrocephalus broodstock with improved sperm qualities instead of conventional feeds. This study will help the researchers uncover the mechanism of melatonin and zinc amino acid in reproductive physiology of Clarias macrocephalus and finally, the improved quality of male Clarias macrocephalus may increase the production of this species due to high demand by the local consumers.

ACKNOWLEDGMENTS

The authors would like to thank the Ministry of Higher Education, Malaysia that had funded this study through scholarship bench fees (Reference No: KPT(BS) 840409085436).