ASSISTED REPRODUCTION IN NILE TILAPIA OREOCHROMIS NILOTICUS:

MILT PRESERVATION, SPAWNING INDUCTION AND ARTIFICIAL

FERTILIZATION
Received date: 26 October 2018
Revised date: 12 February 2019
Accepted date: 4 April 2019

Patharapol Piamsomboon, Nicole Sirisopit Mehl, Sudson, Sirivaidyapong, Janenuj

Wongtavatchai
ABSTRACT
Methods for milt collection and chilled preservation, hormone-induced spawning and
artificial fertilization were studied in Nile tilapia Oreochromis niloticus. Milt samples were
selected from 5 male brooders. Each milt sample was divided into 4 treatments; 3 treatments
were centrifugewashed with 0.85% normal saline solution (NSS), and then added with the
extenders (1) freshwater fish saline (FFS) or (2) modified fish Ringers solution (MFS), or
without an extender as (3) NSS (control), while the fourth treatment was the unwashed
control. Milt samples treated with either extender at 4 oC presented a sufficient sperm motility
score (≥ 3) and sperm viability (≥ 70%) for up to 20 h post collection. For female brooders,
gonadotropin-releasing hormone analog (GnRHa) was used to induce spawning. The highest
number of females spawned and a pseudo-gonadosomatic index were observed in groups that
received two GnRHa injections (15 and 30 μg kg1, 8 h interval) with an oral administration
of a dopamine antagonist (5 mg kg1).
Artificial fertilization was performed with fresh and 20 h chilled-storage milt. Mean
fertilization rates for the fresh and chilled milt fertilized groups were 83.6 ± 7.2% and 42.3
± 5.1%, respectively, with hatching rates of 67.5 ± 3.5% and 28.3 ± 6.8%, respectively.
Nevertheless, the development of fish larvae from both groups was comparable. This study
indicated that artificial fertilization using chilled milt and GnRHa-induced-spawn produced
healthy tilapia fry. Thus, the procedures facilitate breeding programs of Nile tilapia in pair
mating selection and distribution of genetic materials.
Keywords: breeding management; chilled milt; GnRH analog; tilapia.

1. Introduction
Nile tilapia Oreochromis niloticus is a major freshwater fish cultured in Thailand and
worldwide. In 2016, the total world production of Nile tilapia was approximately 6.2 million
metric tons, with over two-thirds of this volume produced in Asia (Fitzsimmons, 2017).
Enhancement of the Nile tilapia genetic line has been widely implemented to serve increasing
global demand. However, pair mating of Nile tilapia brood fish causes injuries or death in
many instances due to the aggressive behavior of the males. Milt preservation and spawning
induction, together with artificial fertilization, may be applied to overcome this problem.
These techniques have been developed for many fish species (Cabrita et al., 2010), but so far
are not welldocumented in Nile tilapia. Cool short-term milt storage has been reported in
rainbow trout Oncorhynchus mykiss (Scott and Baynes, 1980), Mozambique tilapia
Oreochromis mossambicus (Harvey and Kelley, 1984), common carp Cyprinus carpio (Saad
et al., 1988) and Indian major carp Labeo calbasu (Hassan et al., 2013). The spawning
induction in tilapia was achieved using human chorionic gonadotropin (HCG) injection, but
the method of administration was inconsistent amongst different studies. For example,
synchronous spawning in Nile tilapia was demonstrated using double intramuscular
injections of HCG with a 24 h interval at 25 and 50 IU g1 dosages (El-Gamal and El-Greisy,
2005), and dosages from 1 to 5 IU g1 given twice at an 18
h interval, where the first dose was 10% of the total dose (Fernandes et al., 2013). A single
intramuscular injection of HCG at 0.5, 1.0 and 1.5 IU g1 successfully synchronized
spawning in blue tilapia Oreochromis aureas (Owusu-Frimpong, 2008). Because of the
variable approaches that have been applied for breeding management in farm fish, the present
study aimed to develop appropriate methods for milt preservation, induced spawning by
administration with a releasing hormone analog (GnRHa) and artificial fertilization, in order
to facilitate breeding selection for Nile tilapia.

2. Materials and methods

2.1 Nile tilapia
The animal use was approved by the ethics committee of Chulalongkorn University Animal
Care and Use (CU-ACUC; Approval No. 11310046). Ten healthy tilapia broodstock at 2024
months of age were stocked in 6.25 m2 net pens with 1.2 m water depth for each experimental
group. A total of 10 male brooder (average weight 1.5 ± 0.3 kg) and 180 female brooder
(average weight 1.1 ± 0.2 kg) were used in the experiment. Commercial feed was given twice
a day (1% bodyweight per day) and withheld 24 h prior to the experiment. Water quality
parameters were maintained throughout the study as follows: 2831 oC water temperature, 0
salinity and dissolved oxygen > 5 mg L1. Fish were sedated for restraint during the
experiment using anesthetic solution (Aquanes®, Better Pharma, Thailand).

2.2 Milt collection

The anesthetized fish were covered with a moistened towel from head to genital opening and
pressure was applied in a craniocaudal direction along the fish abdomen. A 1.5 mL microfuge
tube was used to collect the milt sample from each fish. Contamination of urine or feces was
avoided during collection. Milt volume, pH, sperm motility and sperm viability were
immediately (within 1 min) assessed. Sperm motility was evaluated using a subjective score
from 1 to 4, as described by Viveiros et al. (2003). Briefly, 10 μL of milt was added to 20 μL
pond water to activate sperm movement and samples were observed under light microscopy
at 100x magnification. Sperm movement was observed and categorized as 14 by the
proportion of showing progressive movement of 125%, 2650%, 5175% and ≥ 75%,
respectively. Eosin-nigrosin staining was used to determine the viability of sperm (Zilli et
al., 2004), where 5 μL of milt was mixed with 5 μL eosin-nigrosin stain on a glass slide and
observed under light microscopy at 400 x magnification. A total of 200 sperm were counted
at 1 min after addition of the stain, and the percentage of unstained (live) sperm was recorded.
Milt samples showing a subjective motility score of 4 and ≥ 95% live sperm were selected
for the milt preservation trial.

2.3 Milt preservation

Different chill storage methods were evaluated on each selected milt sample. A sample was
divided into 4 tubes, at 0.2 mL per tube. Three tubes were centrifuge-washed with 0.85%
normal saline solution (NSS) at 1000 g, 4 oC, for 30 s and then the supernatant was removed.
Then 0.4 mL of one of (1) freshwater fish saline (FFS; modified from Choa et al., 1987), (2)
modified fish Ringers solution (MFS; modified from Rana and McAndrew, 1989) or (3) NSS
was added, while the last sample contained unwashed milt to which was added 0.4 mL of
NSS. The composition of the FFS and MFS extenders is listed in Table 1. The test samples
were then stored at 4 oC and the sperm motility and viability were evaluated at 0, 4, 8, 12,
16, 20, 24, 28, 32 and 36 h postcollection (hpc).

Table 1
Composition of milt extenders used in this study, freshwater fish saline (FFS) and modified
fish Ringers solution (MFS).
Antibiotics, 8 IU ml-1 penicillin and 1 mg ml-1 streptomycin, were added. pH was adjusted to 7.5

2.4 Spawning induction

The GnRHa, buserelin acetate (Suprefact®, Sanofi Aventis, Canada), was applied to induce
spawning in female brooders. “Ready to spawn” females with an enlarged, tender abdomen
and a prominent, hyperemic genital papilla were chosen for the trials. Three hormonal
administrations were performed; “single injection”, “double injection” or “double injection
supplemented with an oral dosage of dopamine antagonist”. Each trial was divided into 6
groups, 10 fish per group, including 5 different dosages of GnRHa and 1 sham injection with
NSS. GnRHa solutions were prepared using NSS to obtain the selected dosage in a final
volume of 0.5 mL. Anesthetized fish were given the dosage via intramuscular injection at the
base of the dorsal fin.
The single injection trial included 0, 20, 40, 60, 100 and 200 μg kg1 GnRHa. For the double
injection trail, GnRHa was injected twice at an 8 h interval apart with a dosage of (1st
dose2nd dose) 00, 515, 1030, 2040, 3070 and 40160 μg kg1. For the third trial, the
optimal dosages derived from the previous two trials were repeated with an oral supplement
of the dopamine antagonist, domperidone (Motilium®-M, Janssen-Cilag, Thailand), at 5 mg
kg1. For this, a 10 mg Motilium®-M tablet was ground and dissolved in 5 mL sterile distilled
water. The solution was administered using a feeding tube connected with a syringe and
inserted into the fish’s mouth while the operculum was held closed for a few seconds to allow
appropriate ingestion. Spawning was observed at 12, 24, 30 and 36 h after the dosage. The
number of spawned females, pseudo-gonadosomatic index (PGSI; [the weight of stripped egg
mass / body weight of the female before stripping] x 100) (Szabó et al., 2015), number of
eggs per gram and egg size were recorded.

2.5 Artificial fertilization and incubation of fertilized eggs

The protocol that provided the best result for milt preservation (motility score ≥ 3, sperm
viability > 70% and a maximum storage duration) and spawning induction in the preliminary
study was implemented in this test. Fresh milt immediately collected from 5 males and chilled
milt from 5 males were used in the trial. Induced-spawn eggs were obtained from a single
female and the eggs were divided into 10 replicates (5 each for the fresh and chilled milt
fertilization).
After the milt was removed from chilled storage, the microfuge tube was placed in pond
wáter (20–22 oC) for approximately 5 min, and then added into a canister containing 2 g of
eggs and 20 mL of pond water. Eggs and milt were gently stirred using a clean duck feather
for 23 min and the eggs were then incubated in a 2.5 L round-bottom container supplied
with a flow-through water (flow rate of 5 L min-1) recirculating system. The water
temperature, salinity and pH were maintained at 30 ± 1 °C, < 3 and 77.5, respectively.
Fertilization rate, hatching rate and postfertilization development were compared between
groups fertilized with fresh and chilled storage milt. The fertilization rate was computed at 1
h post fertilization (hpf). A total of 50 eggs were randomly collected and observed for the
appearance of cytoplasmic accumulation at the animal pole of the eggs, using a stereo
microscope (Olympus, Tokyo, Japan). Hatching rate at 72 hpf was determined by the
appearance of a head or tail. The morphological development of fertilized eggs was observed
at 0, 1, 2, 4, 8, 12, 16, 20, 24, 28, 32 and 40 hpf and thereafter every 8 h until 200 hpf. Stages
of tilapia larval development were defined from the study by Fujimura and Okada (2007).

2.6 Statistical analysis

The continuous data (sperm viability, female PGSI, number of eggs per gram, and egg size)
those presented normal distribution were analyzed with Analysis of Variance (ANOVA) and
Tukey’s HSD post hoc test. Whereas the non-parametric data (sperm motility score,
fertilization and hatching rates) were presented as descriptive data. All statistical procedures
were performed using the “R” software (www.r-project.org). Statistical significance of
differences was accepted at the P < 0.05 level.

3. Results
3.1 Milt sample
Milt samples that showed a sperm motility score of 4 and > 95% sperm viability were chosen
for the milt preservation trial, and 5 milt samples were selected. The average quality
parameters of the 5 milt samples were: 1.3 ± 0.4 mL milt per fish, pH 7.5 and 97.6 ± 2.2%
sperm viability.
The motility score in the washed, NSS-control group dropped during the 1228 hpc, whereas
in the unwashed control it decreased at 4 hpc and no sperm movement was observed at 20
hpc. A higher sperm motility score was observed in both extender-treated groups
(Supplementary Table S1) and a higher percentage of live sperm were found in the extender-
treated groups than in the control groups at all time points, becoming significantly higher at
1236 hpc (Fig. 1). Both extender-treated groups showed a similar sperm motility score and
live sperm percentage.

Fig. 1. Mean percentage of sperm viability of the chilled-storage milt at 4 oC evaluated 0-

36 hpc. Milt samples were centrifuge-washed and then added with freshwater fish saline
(FFS), modified fish Ringers solution (MFS), or normal saline solution (NSS); whereas the
unwashed samples were used as control.

3.2 Spawning induction

The number of spawned females, PGSI, number of eggs per gram and egg size in each trial
is presented in Table 2. Spawning induction was not competent with a single GnRHa
injection (1st trial), whereas spawning occurred within 24 h after the last GnRHa dose in both
the second and third trials. In the second trial, approximately 20% (11 out of 50) of female
brooders spawned and the PGSI values were not significantly different between the different
GnRHa dosages. In the third trial, a total of 32 out of 50 fish spawned, and the PGSI values
significantly increased with higher GnRHa dosages (1530, 3030 and 2040 μg kg1). In
every trial, the mean number of eggs per gram and egg size were not significantly different.
Table 2
Number of spawned Nile tilapia (N=10 fish per dose) and mean values of the pseudo–
gonadosomatic index (PSGI), number of eggs per gram and egg size.
PSGI; (weight of stripped egg mass / body weight of the female before stripping) x 100.
Egg size; diameter of the major axis. Different letters indicate statistically significant
differences (P < 0.05).

3.3 Artificial fertilization and post fertilization development

The chilled milt in the FFS extender after 20 h storage was used in this study. Eggs were
obtained from a female brooder that received double GnRHa injections (1530 μg kg1, 8 h
interval) with an oral dopamine antagonist (5 mg kg1). The observed fertilization and
hatching rate were higher when fertilized with fresh milt (83.6 ± 7.2% and 67.5 ± 3.5%,
respectively) tan with chilled milt (42.3 ± 5.1% and 28.3 ± 6.8%, respectively). The
development of fish embryos to the early larval phase was as described in the natural
fertilized egg, where the first 7 stages of embryonic development were observed within 80
hpf, 2 further stages at 81–185 hpf and they reached the early larval phase at 200 hpf
(Supplementary Fig. S1).

4. Discussion
This study demonstrated the chilled preservation of milt, GnRHa-induced spawning and
artificial fertilization using fresh and chilled milt in Nile tilapia. Milt preservation by cool
storage is simple to perform and requires less special skill or expensive equipment. It allows
sperm to be preserved for periods of time sufficient for the shipment of genetic materials in
an intermediate distance, such as within a country (Vuthiphandchai et al., 2009). Milt samples
treated with a centrifuge wash and supplemented with either FFS or MFS as an extender
showed higher sperm motility and viability than non-treated samples when stored at 4 oC.
This technique aims to decontaminate the collected milt and maintain the osmolality of the
milt suspension.
Washing the collected milt with NSS or either extender was performed to remove
contaminated water and urine that can alter the milt osmolality (Gwo, 2009). For example,
an alteration of spermatozoa morphology, reduction of ATP content in spermatozoa and
decreased sperm motility were observed in common carp milt when contaminated with urine
or distilled wáter (Perchec-Poupard et al., 1996, 1998).
This study also showed that the tilapia sperm motility and viability of the chilled, unwashed
milt was decreased as early as after 4 h of storage. In teleost fish, sperm movement is
activated by an abrupt osmolality change that triggers K+ channels on the sperm plasma
membrane, causing membrane hyperpolarization, depolarization and Ca2+ influx. The Ca2+
ions then actívate sperm movement via the calmodulin system (Alavi and Cosson, 2006). It
was reported that spermatozoa in Nile tilapia milt were motile over a range of favorable
osmolalities, with high motility being observed at around 200 mOsmol kg1 and
progressively decreasing at osmolalities above 200 mOsmol kg1 of NaCl, KCl and mannitol
buffer solution (Mochida et al., 1999).
Among the two extenders used in the present study, no difference in the sperm motility and
viability were found. This may be associated with the osmolality of both extenders (MFS at
290 mOsmol kg1 and FFS at 270 mOsmol kg1) that are close to the osmolality of Nile tilapia
milt.
Although the glucose composition in the MFS extender may function as an energy source for
sperm and maintain sperm osmolality by stabilizing the spermatozoa liposomal membrane
during storage (Orfão et al., 2011), our study did not observe any significant differences in
sperm quality when using either extender. This may imply that the optimal osmolality is more
crucial in maintaining the quality of Nile tilapia milt than glucose supplementation. In
addition, chill storage and cryopreservation of sperm were reported to cause sperm DNA
damage in European seabass Dicentrarchus labrax (Zilli et al., 2003), rainbow trout (Pe´rez-
Cerezales et al., 2009), gilthead seabream Sparus aurata (Cabrita et al., 2011) and Siberian
sturgeon Acipenser baerii (Shaliutina et al., 2013). The chill storage procedures induce
injuries affecting sperm motility, viability and cell structure (Pe´rez-Cerezales et al., 2009),
and this might contribute to the reduced sperm motility and viability observed in this study.
Egg spawning induction in Nile tilapia is challenging due to the naturally asynchronous
spawning behavior, which is influenced by several environmental factors, such as
temperature, photoperiod and rainfall (Yoshida et al., 2015). This asynchronous spawning
character may restrain genetic improvement of broodstock because it consumes a large
amount of time and intensive labor to obtain selective breeding. The present study developed
a method for the induction of egg spawning in Nile tilapia with the combined administration
of two GnRHa injections and oral administration of a dopamine antagonist. A single injection
with GnRHa was not sufficient to induce spawning while a double injection with GnRHa
resulted in a higher spawning rate, but the amount of obtained eggs was still insufficient for
further applications.
Theoretically, final oocyte maturation is induced by an initial hormone injection, while the
second injection stimulates egg release from the ovary (Yu et al., 2017).
The low success rates of GnRHa-induced egg spawning in this study could be due to the
presence of an inhibition in the reproductive axis, known as neuron secreting dopamine. A
study in Senegalese sole Solea senegalensis showed that dopamine reduced GnRH synthesis
in the brain and pituitary gland, interfered with the GnRH-signal transduction pathways and
downregulated GnRH receptors (Guzmán et al., 2011). This inhibition was also reported in
several freshwater fish species, such as cyprinids (Trudeau, 1997), tilapia (Melamed et al.,
1998), salmonids (Saligaut et al., 1999) and silurids (Silverstein et al., 1999). In the third trial
of our study, the fish that received an oral dopamine antagonist showed a significant
improvement in number of spawned females and egg quantity. A dopamine antagonist is
commonly used by local farmers in several fish species, such as climbing perch Anabas
testudineus and broadhead catfish Clarias microcephalus. Dopamine antagonists, such as
domperidone tablets, are ground, dissolved in water and then injected into the fish
intramuscularly (Alok et al., 1997). However, this method causes damage to the fish muscle
and consequently reduces the high value of the broodstock. This study revealed that oral
administration of the dopamine antagonist can be performed without the need for injection,
which is preferable since it requires only a minimal technique to sedate the fish for a short
period of time, reduces injury and stress caused by the injection of a foreign substance and
may be applied in other fish species.
Factors governing egg size in tilapia are claimed to be the food availability and quality,
species, maternal size and age (Coward and Bromage, 2000) and photoperiod (Campos-
Mendoza et al., 2004), while the influence of GnRH treatment on egg size in tilapia has not
been reported.
Despite the higher numbers of females that spawned and the increased PGSI in groups that
received both the GnRH injections and domperidone administration, the egg size was not
significantly different in all treatments. This finding was in agreement with a previous study
(El- Gamal and El-Greisy, 2005), which showed that the egg diameter in Nile tilapia was not
affected by HCG treatment. The similarity in the egg size from synchronized females
receiving different dosages of HCG has also been observed previously in Nile tilapia
(Fernandes et al., 2013).
Fertilization and hatching rates were found to be approximately 15% lower in the chilled milt
compared to the fresh milt fertilized groups, while the characteristics of embryonic
development were typical and similar in both groups. Development of Nile tilapia embryos
from fertilized eggs to the early larval stage took 200 h at 30 ± 1 oC in this study. It was
previously reported at a 1.44-fold longer period of 288 h, but at 28 ± 1 oC (Fujimura and
Okada, 2007), and so the 2 oC increase in the incubation temperature in this study may have
resulted in a faster embryonic development. Development of fish eggs is accelerated by the
heat energy that enhances their biochemical activity and metabolism (Valeta et al., 2013),
and increasing the incubation temperature is known to shorten the duration required for fish
embryonic development in several fish species, such as Nile tilapia (Bhujel et al., 2000),
Atlantic cod Gadus morhua (Geffen et al., 2006), common carp (El-Gamal, 2009) and Lake
Malawi tilapia Oreochromis karongae (Valeta et al., 2013).

5. Conclusion
Milt preservation, spawning induction and artificial fertilization are reproductive
techniques that aim to assist breeding management. Tilapia milt washed with the MFS or
FFS (as an extender) and stored at 4 oC provided an adequate quality of sperm and could be
applied practically. Two GnRHa injections in conjunction with oral administration of the
dopamine antagonist induced successful spawning. Artificial fertilization with chilled milt
was achieved and the larva developed to tilapia fry as usually observed in natural fertilization.
Thus, the reproductive techniques outlined in this study can facilitate the distribution of
genetic materials and pair mating selection for genetic improvement of captive Nile tilapia.

Highlights of the manuscript

- A practical technique was developed to provide competent quality of tilapia sperm
preserved at 4 oC for up to 20 h.
- Effective spawning synchronization was accomplished with 2 GnRHa intramuscular
injection in conjunction with an oral administration of the dopamine antagonist.
- The reproductive technique demonstrated in this study did not cause injury to the
broodfish, and Nile tilapia embryo obtained from artificial fertilization using chilled milt
and induced spawn eggs showed typical characteristics of embryonic development.