Hormonal Induction Impacts on Grass Carp (Ctenopharyngodon

idella) Broodstock Health and reproduction capacity and Progeny

Survival and growth performance"

Hani M. Abdelsalam1*, Amr A. Shalaby1*, Mohamed W. Ali2*, Aya Magdy 3*, Wafaa G. El-Nagar 1*
1- Department of Zoology, Faculty of Science, Zagazig University, Egypt.
2- department of Spawning and Fish Physiology, Central laboratory Aquaculture Research, A.R.C
3- Clinical Pathology Laboratory, Faculty of Medicine, Zagazig University, Egypt.
Corresponding author: Email: [email protected]

Abstract
One of the main considerations for cultivating grass carp (Ctenopharyngodon idella)
is its herbivorous feeding habit that makes it important to overcome excess aquatic
vegetation. Therefore, grass carp culture can be used as biological control of aquatic
vegetation in addition to its commercial value as an edible fish species. therefore this study
has checked the effect of hormonal stimulation on egg quality, broodstock health and growth
performance of fries through investigating some parameters as Egg number and diameter,
Serum Urea, total cholesterol and triglycerides for male and female broodstock besides
weight and length gain of fries along eight weeks of study and survival rate of fries. Females
and males of grass carp were injected by ovaprim, pituitary extract and a mixture of ovaprim
and pituitary extract in three treatments then the former investigations were done. Ovaprim
(T1) showed highest length increment of fries 0.75 cm/week average in 5 of 7 weeks, also it
had superior performance in female weight gain (9.22 ± 0.25 kg post injection), higher egg
production (1.51 ± 0.3 million eggs/female), and greater egg density (680,000 ± 1,200
eggs/kg) compared to other treatments. So this study had revealed that the best result for
production of fries including their weight, length, survival rate, mortality rate and growth
performance were highly significant in first treatment followed by second treatment followed
by third treatment .So it is recommended to use ovaprim for hormonal stimulation of
artificial spawning of grass carp.

Introduction
The grass carp (Ctenopharyngodon idella), also referred to as White Amur, is a
herbivorous freshwater species endemic in the Amur River basin in Asia. Due to its
herbivorous nature of feeding, this fish has gained significant attention as an effective
biological control agent for managing excessive aquatic vegetation, making its cultivation
crucial for ecological balance in water bodies. While carp is among the few fresh water fish
species that have undergone domestication, notable differences exist between wild and
farmed populations in terms of reproductive efficiency, growth rates, and feed conversion.
Wild grass carp typically exhibits slower growth and full-scale coverage, whereas
domesticated forms (including both scaled and mirror carp strains) show accelerated growth
rate due to their ability to efficiently utilize both artificial cereal-based feeds and natural food
sources.
Additionally, carp exhibits remarkable resistance to stress factors such as handling
during harvesting, sorting, and transport, as well as fluctuations in water temperature and
dissolved oxygen levels. Their high market demand persists across Eastern Europe and much
of Asia, where they remain a preferred food fish due to their excellent taste and texture
(Horváth et al., 2015). Successful cultivation of grass carp (Ctenopharyngodon idella)
presents unique challenges as natural reproduction only occurs in their native habitats.
Enhancing reproductive performance is therefore essential for effective aquaculture. Fish
reproduction is governed by environmental cues that activate internal physiological
processes. Spawning - the culminating reproductive event involving gamete release - can be
induced through environmental manipulation or hormonal intervention that alters internal
regulatory mechanisms.
The pituitary gland serves as a crucial endocrine organ, synthesizing and storing
gonadotropin hormones (Gth) that regulate both ovulation and spermiation. Direct pituitary
extract administration circumvents the brain-pituitary axis, acting immediately on gonadal
tissues to produce the characteristic Gth surge preceding natural spawning [Rottmann et al.,
1992; Brzuska & Bialowas, 2002]. Artificial propagation remains particularly challenging
for wild cyprinids, as successful hatchery reproduction consistently requires hormonal
induction (Żarski et al., 2009).
Pituitary extract injection represents not only the pioneering technique for induced
fish reproduction but has also demonstrated enduring efficacy as it has stood the test of
incubation time, remaining the gold standard for many aquaculture operations. In numerous
cases, this method proves superior for achieving reliable final gamete maturation and
spawning induction, forming the foundation of many commercial production systems
(Erdahl, 1996). Alternative approaches using synthetic analogs like Ovaprim have shown
promise across multiple species. Research documents successful ovulation induction in
Common carp (Amer et al., 2009), African catfish (Akar, 2006), Nase fish (Szabó et al.,
2002), Northern pike (Szabó, 2014) and European catfish (Brzuska and Adamek, 1999)

Materials and Methods

The present study was conducted using the research facilities of Central Laboratory for
Aquaculture Research (CLAR) Abbassa Abo-Hammad Sharkia and faculty of Science
Zagazig University. Fish used in this study was grass carp fish (Ctenopharyngodon idellla),
species were collected from Abbassa Fish Farm Sharkia, Egypt. The experimental fish was
transported to hatchery. Fish were acclimatized for fourteen days. The experiment was
applied in wet lab aquaria. The rearing culture consisted of 9 aquaria units. Twenty seven 27
fish (18 females and 9 males with average weight 7200±30g) were distributed in nine tanks
of fiberglass of grass carp (Ctenopharyngodon idella) by sex ratio 2 females: 1 male for each
tank with three replicates for each treatment; Treatment 1 (T1), females and males of grass
carp were injected by ovaprim. Treatment 2 (T2), females and males of grass carp were
injected by pituitary extract. Treatment 3 (T3) females and males of grass carp were injected
by a mixture of ovaprim and pituitary extract.

Preparation of injection:
To prepare the preliminary dose of pituitary extract, one dried pituitary gland is carefully
ground into a fine powder in a mortar. The resultant powder then well be mixed with 1ml of
0.65 percent saline solution. As some fluid is lost in the mortar during extraction 10 percent
more gland and solution should be calculated and made up for each batch. The grinding is
more effective, if a dry mortar is used and the gland ground into a fine powder. A few drops
of saline are added and the mixture ground into a paste. The remaining saline is added, mixed
and the solution then drawn up into a hypodermic needle and syringe, while ovaprim was
solution ready for injection directly where it is Salmon gonadotropin releasing hormone
analog, dopamine antagonist, and domperidone, obtained from Syndel, Laboratories, Ltd.,
Vancouver, British Columbia..

Method of injection:
Both ovaprim solution, pituitary extract and the mixture were injected deeply
intramuscularly below the top of the dorsal fin of the fish by suitable dose for each according
to weight using a medium size needle. The needle is kept in the muscle and a finger should
be used to massage the area, this finger should then be placed over the entry point as the
needle is withdrawn slowly to avoid losing any of the injected solution. this was done for the
three fish treatments respectively T1, T2 and T3, where ovaprim was injected at the general
dose of (0.5 ml/Kg of body weight) and After obtaining pituitary extract treatments, fish were
injected by suitable dose of pituitary extract according to weight (4 mg/Kg of body weight),
finally fishes of the third group were injected by the same doses of both ovaprim and
pituitary extract, then all fish were put in specific tank for each treatment.

Brood stock measurements:

Female weight per Kg before and after injection , Egg number and Egg diameter were
determined, and some serum biochemical parameters in male and female were measured such
as Serum Urea, Total cholesterol and Triglycerides

Acclimatization experiment:
Stoking density of larvae was 1000 fry of grass carp / tank, from different treatments for
mothers was rearing throughout 8 weeks post hatching, this experiment was divided into
three groups according to the three previous treatments and larvae were fed on natural food
from micro algae with artificial food (40% crude protein). The following measurements were
taken every week and collected as means in tables illustrated in results for evaluating growth
performance beside fecundity and health of broodstock:
Initial weight of larvae (g), average weekly weight and weight gain is calculated (g), initial
and average length (cm) of fries after hatching up to eight weeks (cm) and Total length gain,
Average Survival rate (%) of fries after hatching up to eight weeks.

Methods of calculation:
Egg number (Bagenal and Tesch, 1978).
Egg diameter : was measured to the nearest mm by ocular micrometer fixed in the eye piece
of a light microscope. Mean egg diameter (mm) =long axis length+ short axis length/2.

Serum Urea Analysis: Modified Berthelot Method (Patton & Crouch 1977)

Total Cholesterol Analysis: CHOD-PAP Method (Allain et. al 1974)

Triglycerides Analysis: GPO-PAP Method (Bucolo & David 1973)

The following growth performance parameters were calculated as follows:

Survival rate (SR %) = (Number of fish at end/ Number of fish at start) × 100
Total length gain, mm (Ricker, 1975): LG = L2-L1, Where L2: is the final length of fish in
cm., L1: is the initial length of fish in cm.
Total weight gain (g) : WG = W2-W1, Where W2: is the final weight of fish in grams, W1: is
the initial weight of fish in grams.

Statistical Analysis:
The data obtained in this study were analyzed by one-way ANOVA. Means at
gnificance level of 0.05 and standard error of group means were also estimated. All statistics
were carried out using Statistical Analysis Systems (SAS) program SAS, (2009)®

Results
Table 1: Average Survival rate (%) of fry (Means ± S.D.) after hatching up to eight weeks
at three treatments, ovaprim (T1), Pituitary extract (T2) and mixture of ovaprim and Pituitary
extract (T3) of grass carp.
Treatment T1 T2 T3
Items
Second week (%) 88.2 ±0.9a 85.3±0.7 c 86.3± 0.6b
Third week (%) 91.7±0.4 a 89.5 ±0.3 c 90.2±0.2 b
Fourth week (%) 93.8 ±0.5 a 90.6±0.3 c 91.3±0.6 b
Fifth week (%) 95.2±0.7 a 94.6±0.6 a 93.6±0.6 b
Sixth week (%) 94.7±0.6 b 95.2± 0.2 a 94.6±0.5 b
Seventh week (%) 96.2±0.2 a 94.6± 0.2c 95.5±0.3 b
Eighth week (%) 94.8±0.2 a 94.5± 0.3 a 94.5±0.2 a
The means have the same latter in the same raw are non-significant (P>0.05).

Average Survival rate (%) of fry after hatching up to eight weeks at three treatments
was very highly significant in T1 (88.2 ±0.9%), highly significant in T3 (86.3± 0.6%) and
significant in T2 (85.3±0.7%), T1 (Ovaprim) showed the highest survival rates in weeks 2–4
and week 7, with statistically significant superiority (denoted by superscript "a") over T2 and
T3 in most cases. T3 (Mixture) performed intermediately between T1 and T2, while T2
(Pituitary Extract) had the lowest survival rates in early weeks (2–4) but increased by weeks
5–8, even surpassing T1 in week 6. Finally at the eighth week there was no significant
difference in the three treated groups,T1(94.8±0.2%), T2 (94.5±0.3%) and T3 (94.5±0.2%).

Table 2: Average weekly weight gain (gm) of grass carp fries (Means ±S.D.) after hatching
up to eight weeks at three treatments, ovaprim (T1), Pituitary extract (T2) and mixture of
ovaprim and PE (T3).
 Treatment T1 T2 T3
Items
Second week (gm) 4.20±0.05b 3.91±0.06 b 4.78±0.03 a
Third week (gm) 4.45±0.04 a 4.41±0.04 a 3.97±0.03 b
Fourth week (gm) 4.46±0.05 a 4.35±0.06 a 4.33±0.06 a
Fifth week (gm) 4.49±0.03 a 4.02±0.04 c 4.20±0.01 b
Sixth week (gm) 4.41±0.03 a 4.10±0.04 b 4.55±0.06 a
Seventh week (gm) 4.00±0.05 a 4.12±0.03 b 3.95±0.02 b
Eighth week (gm) 4.46±0.05a 4.18±0.03 b 4.00±0.05c

The means have the same latter in the same raw are non-significant (P>0.05).
Weekly weight gain (gm) of fries of grass carp in second week was significant in T3
(4.78±0.03gm) compared with T1 (4.20±0.05gm), and T2 (3.91±0.06gm), whereas there was
no significant difference in T2 and T1.Weekly weight gain (gm) in third week was highly
significant in T1 (4.45 0.04gm) and T2 (4.41±0.04gm) compared with T3 (3.97±0.03gm). In
fourth week there was no significant difference between three treatments, whereas T1
(4.46±0.05gm), T2 (4.35±0.06gm) and T3 (4.33±0.06gm).Weekly weight gain (gm) in fifth
week was very highly significant in T1 (4.49±0.03gm), highly significant in T2
(4.02±0.04gm), and significant in T3 (4.20±0.01gm).Weekly weight gain (gm) in sixth week
was highly significant in T3 (4.55±0.06gm) ,and T1(4.41±0.03gm) compared with T2
(4.10±0.04gm). In seventh week, weekly weight gain (gm) was significant in T1
(4.00±0.05gm) compared with T2 (4.12±0.03gm) and T3 (3.95±0.02gm) whereas there was
no significant difference in T2 and T3.Weekly weight gain (gm) in eighth week was very
highly significant in T1 (4.46±0.05gm), highly significant in T2 (4.18±0.03gm), and
significant in T3 (4.00±0.05gm).
Table 3: Average length (cm) of fries of grass carp (Means±S.D.) after hatching up to eight
weeks at three treatments, ovaprim (T1), Pituitary extract (T2) and mixture of ovaprim and
PE (T3) .
Treatments T1 T2 T3
Items
First week (cm) 1.29±0.05a 1.30±0.04 a 1.32±0.03 a
Second week (cm) 2.10±0.05 a 2.00±0.03b 2.10±0.05 b
Third week (cm) 2.90±0.06 a 2.7±0.04c 2.80±0.05 b
Fourth week (cm) 3.6±0.09 a 3.40±0.05 b 3.30±0.03 c
Fifth week (cm) 4.42±0.07 a 4.20±0.04 b 4.10±0.03 c
Sixth week (cm) 5.03±0.04 a 4.80±0.5 b 4.70±0.03 c
Seventh week (cm) 5.72±0.06 a 5.52±0.05 b 5.42±0.04 c
Eighth week (cm) 6.55±0.07 a 6.22±0.04 b 6.10±0.05 c
The means have the same letter in the same row are not significantly (p>0.05).

In first week length (cm) of fry had no significant variation in three treatments, they
ranged from (1.29±0.05cm to 1.29±0.05cm). In second week of fries there was significant in
T1 (2.10±0.05cm) compared with T2 (2.00±0.03cm) and T3 (2.10±0.05cm), whereas T2 and
T3 had no significant difference. In third week length (cm) of fries was highly significant in
T1 (2.90±0.06cm), highly significant in T3 (2.80±0.05cm) and significant in T2
(2.7±0.04cm). In fourth week, length (cm) of fries was highly significant in T1
(4.42±0.07cm), highly significant in T2 (3.40±0.05cm) and significant in T3 (3.30±0.03cm).
In fifth week, length (cm) of fries was highly significant in T1 (3.6±0.09cm), highly
significant in T2 (4.20±0.04cm) and significant in T3 (4.10±0.03cm). In sixth week, length
(cm) of fries was highly significant in T1 (5.03±0.04cm), highly significant in T2
(4.80±0.5cm) and significant in T3 (4.70±0.03cm). In seventh week, length (cm) of fries was
highly significant in T1 (5.72±0.06cm), highly significant in T2 (5.52±0.05cm) and
significant in T3 (5.42±0.04c). In eight week, length (cm) of fries was highly significant in
T1 (6.55±0.07cm), highly significant in T2 (6.22±0.04cm) and significant in T3
(6.10±0.05cm).
Table 4: length increment (cm/week) (Means±S.D.) of grass carp fries from the first week up
to week eight at three treatments, ovaprim (T1), Pituitary extract (T2) and mixture of ovaprim
and PE (T3) .
Week T1 (cm/week) T2 (cm/week) T3 (cm/week)

1→2 0.81 0.70 0.78

2→3 0.80 0.70 0.70
3→4 0.70 0.70 0.50
4→5 0.82 0.80 0.80
5→6 0.61 0.60 0.60
6→7 0.69 0.72 0.72
7→8 0.83 0.70 0.68
Increments
(Means±S.D.) 0.75 ± 0.086 a 0.70 ± 0.060 ab 0.68 ± 0.096 b
Treatments sharing a common letter are not significantly different.

T1 had the highest weekly increments in most weeks (especially Weeks 1→2, 4→5,
and 7→8) while T2 and T3 were similar in mid-weeks, but T3 lagged in later weeks. Data
reveals important differences in growth patterns among the three hormonal treatments.
Ovaprim (T1) demonstrated superior mean growth velocity (0.75 ± 0.086 cm/week)
compared to Pituitary Extract (T2: 0.70 ± 0.060 cm/week) and the Mixture (T3: 0.68 ± 0.096
cm/week). The standard deviation values indicate T2 produced the most consistent growth
outcomes (SD = 0.060), while T3 showed the highest variability (SD = 0.096), suggesting
less predictable performance.

Table 5: Female weight before and after injection, egg number per female, egg number per
kg female, egg number per gm female, and egg diameter (Means ±S.D.) in grass carp in
different treatments during spawning season.
Treatment Items T1 T2 T3
Female weight per Kg before injection 7.52±0.54a 7.54±0.53 a 7.51±0.42 a
Female weight per Kg after injection 9.22±0.25 a 8.77±0.15 b 8.62±0.18 b
Egg number /female 1.51±0.3 a 1.13±0.2 b 0.92±0.11 b
Egg number per Kg 680000±1200 658000±1020b 651000±1100c
Egg number /gm 666±3a a 658±3b 651±3c
Egg diameter (mm) 4.31±0.18 a 4.28±0.16 a 4.51±0.19 a
The means have the same latter in the same raw are non-significant (P>0.05)

Female weight before injection had non-significant variation in the three treatments
which ranged from (7.51±0.42kg to 7.54±0.53kg). Female weight per kg after injection was
significant in T1 (9.22±0.25kg) compared with T2 (8.77±0.15kg) and T3 (8.62±0.18kg),
while there was no significant difference between T2 and T3. Also eggs number per female
was significant in T1 (1.51±0.3) compared with T2 (1.13±0.2) and T3 (0.92±0.11), whereas
no significant difference between T2 and T3. Egg number per kg in grass carp was very
highly significant in T1 (680000±1200), highly significant in T2 (658000±1020), significant
in T3 (651000±1000). In female grass carp, eggs number per gm has ranged from (666±3 to
651±3) where the number of egg per gm was very highly significant in T1 (666±3), highly
significant in T2 (658±3), significant in T3 (651±3) respectively. Finally eggs diameter had
no significant difference between different treatments and ranged from (4.51±0.19mm to
4.28±0.16mm).

Table 6: Some serum biochemical changes in male and female grass carp through 30 days of
artificial injection by using ovaprim in first group (G1).

Ovaprim Before injection After 2 weeks of injection After 4 weeks of injection

group(G1) )control)
♂ ♀ ♂ ♀ ♂ ♀
Kidney function:
Serum Urea 19.8±0.86b 20.0±1.58c 22.2±1.6b 26.0±1.0b 25.4±1.29a 30.0±1.52a
Lipid profile: 214.4±0.86a 214.2±5.58c 240.6±1.86b 227.4±1.52b 245.4±1.29c 241.6±2.66a
Total cholesterol 140.2±1.66a 150.2±1.77b
Triglycerides 141.6±1.8b 150±5.1a 155.2±1.463c 153.2±1.8a
The means have the same latter in the same raw are non-significant (P>0.05).

Biochemical changes in male grass carp through 30days of artificial injection by

using ovaprim in first group during three stages, before injection, after 2weeks of injection
and after 4weeks of injection. Serum urea was very high significant after 4weeks, high
significant after 2weeks, significant in T1, ranged from 19.8±0.86 to 25.4±1.29. Cholesterol
was very high significant after 4weeks, high significant after 2weeks, significant in T1
ranged from 214.4±0.86 to 245.4±1.29. Triglycerides were very high significant after
4weeks, high significant after 2weeks, significant in T1ranged from 140.2±1.66 to
155.2±1.463.

Serum biochemical changes in female grass carp by using ovaprim in first group
showed that urea was very high significant after 4weeks, high significant after 2weeks,
significant before injection. Cholesterol was very high significant after 4 weeks, ranged from
214.2±5.58to 241.6±2.66. Triglycerides were significant after 4week and after 2weeks,
ranged from 141.6±1.8 to 153.2±1.8.
Table 7: Some serum biochemical changes in male and female grass carp through 30 days
of artificial injection by using of pitutary extract in second group (G2).
Pituitary extract Before injection After 2 weeks of After 4 weeks of injection
group(G3) )control) injection
♂ ♀ ♂ ♀ ♂ ♀
Kidney function:
Serum Urea 20±0.71c 23.6±0.93c 23.8±1.07b 28.2±1.16b 29.2±1.07a 32.2±1.72a
Lipid profile: 211.8±0.71b 238.9±2.7a
215.6±5.69c 228.4±2.27b 238.0±3.97a 238.2±2.334a
Total cholesterol 141.6±3.58c 149.4±2.11b
142.8±3.49c 158±2.65a 157.0±1.84a 151.4±1.57b
Triglycerides
The means have the same latter in the same raw are non-significant (P>0.05).

Biochemical changes in male grass carp through 30days of artificial injection by

using pituitary extract in second group during three stages of injection. Urea was very high
significant after 4 weeks ranged from 20±0.71 to 29.2±1.07. Cholesterol was high significant
after 2weeks and after 4 weeks, ranged from 211.8±0.71 to 238.9±2.7. Triglycerides was
very high significant after 4 weeks of injection, ranged from 141.6±3.58 to 157.0±1.84.
Biochemical changes in female grass carp by using pituitary extract in second group
during three stages showed serum urea was high significant after 4weeks ranged from
23.6±0.93 to 32.2±1.72. Cholesterol was high significant after 4 weeks ranged from
215.6±5.69 to 238.2±2.334. Triglycerides were high significant after 2 weeks ranged from
142.8±3.49 to 158±2.65.

Table 8: Some serum biochemical changes in male and female grass carp through 30 days
of artificial injection by using of Mixture of ovaprim and pitutary extract in third group (G3)
Mixture of ovaprim Before injection After 2 weeks of injection After 4 weeks of
and pituitary extract )control) injection
group(G3) ♂ ♀ ♂ ♀ ♂ ♀
Kidney function:
Serum Urea 19.0±1.14c 23.2±1.07b 25.0±1.3b 31.0±1.4a 28.8±1.43a 29.4±1.17a

Lipid profile:
Total cholesterol 209.6±4.98b 207.2±1.07c 233.6±2.82a 225.2±2.154a 205±2.55b 221.2±3.4b
Triglycerides 140.6±2.11b 140.6±2.38c 148.6±2.11a 151.1±1.28b 147.6±1.9a 154.6±1.36a

The means have the same latter in the same raw are non-significant (P>0.05).
Biochemical changes in male grass carp through 30days of artificial injection by
using mixture of ovaprim and pituitary extract in third group during three stages showed
serum urea was very high significant after 4 weeks, ranged from 19.0±1.14 to 28.8±1.43.
Cholesterol was high significant after 2 weeks, ranged from 205±2.55 to 233.6±2.82.
Triglycerides were high significant after 2 weeks of injection, ranged from 140.6±2.11 to
148.6±2.11.
Biochemical changes in female grass carp by using mixture of ovaprim and pituitary
extract in third group during three stages, showed serum urea ranged from 23.2±1.07 to
31.0±1.4. Cholesterol was high significant after 2 week, ranged from 207.2±1.07 to
225.2±2.154. Triglycerides were high significant after 2 weeks of injection, ranged from
140.6±2.38 to 154.6±1.36.

Discussion:
 The present study has investigated the effects of three hormonal treatments Ovaprim,
pituitary extract, and a mixture of them on female grass carp fecundity and post injection
weight gain indicating the superiority of ovaprim treatment on the other treatments in egg
quality represented in egg diameter and egg number. Regarding fries growth performance,
the results of this study also support the use of ovaprim in carp spawning induction for a
higher survival rate, weight gain and length increment of fries.
Marosz and Bieniarz, (1979) mentioned that the cyprinidae gonadotropins possess at
least a partial specificity ,manifested difference in potency of action on carp oocyte
maturation of cyprinidae can be used to stimulate carp reproduction while the use of
pituitaries from other fish families for carp reproduction may fail to give any positive results.
The stimulatory action of various gonadotropins which may be connected with the specificity
of ovarian receptors Burzawa-Gerad and Fontaine, (1972), or with differences in hormonal
structure of different fishes lie the gonadotropins potency of the same species was higher
than that of the other species. Woynarovich and Horvath, (1980) recorded that even for fish
species which can undergo natural spawning in captivity, induced ovulation followed by
artificial fertilization was generally preferred in order to achieve a greater control over fry
production. Brzuska and Grazywachzewski (1999). The data obtained by Brzuska 1999a,
b and 2000b distinctly show that ovapel applied in two doses considerably increased the
percentage of spawning feriales as compared with hypohysed fish. The fishery practice is
sew up crosswise the skin on the sides of sexual papilla to prevent losing of eggs by fish
(Okoniewski and Klodzinska, 1975).The operation is carried out together with the second
injection of the ovulation stimulator (Kozlowski, 1994).
Our study showed that the injection of ovaprim was the most effective treatment,
these results were In conflict with the findings of (Brzuska, 2003 and 2004 ,and Akar &
Ali, 2006), which indicated that the use of pituitary extract in combination with ovaprim is
considered.
Various scientists have conducted investigations on various aspects of synthetic
hormones to check their efficacy over the fertilization rate, fish growth rate and fish
physiology etc. In the last few decades the artificial stimulation of fish fertilization rate has
gained great attention of scientists. As fishes are major source of instant proteins in the local
Asian fish market. Commercially important tropical freshwater and marine finfishes were
investigated by Marte et al., (1989).
A study obtained by Javed Khan and Fawad Ali, (2021) on the pituitary gland and
Ovaprim showed that the Ovaprim is better product than pituitary gland when a single
coelomic injection of pituitary gland into Rohu (Labeo rohita) and Grass carp
(Ctenopharyngodon idella) indicated that the pituitary gland is less effective as compared to
Ovaprim. They were spawned with pituitary homogenate, human chorionic gonadotropin
(HCG) and semi-purified fish gonadotropins. These hormones were injected into two doses, a
lower priming dose followed a few hours later by a higher resolving dose. The present study
on the pituitary gland and Ovaprim showed that the Ovaprim is better product than pituitary
gland.
Certain hormones are used individually like different analogous of LH and RH
without pituitary gland which result in failure of spawning and clearly indicates that the
dopamine block the action of LHRH on the secretion of gonadotropin (Billard et al., 1983,
Naeem and Salam, 2005a, Naeem et al., 2005c, Naeem et al., 2011). In Canada the
researchers introduced the Ovaprim containing the analogue of salmon gonadotropin
releasing hormone (DArg6, Pro9 Net) and dopamine antagonist. The studies conducted in
India (Nandeesha et al., 1990) and Pakistan (khan et al., 1992, khan et al., 1990 and
Naeem et al., 2005) revealed the superiority of Ovaprim in induced spawning. The study of
Javed Khan and Fawad Ali, (2021) was conducted to work out the efficacy and usefulness
of both the hormones using Rohu (Labeo rohita) at Government Fish Seed Hatchery,
Bahawalpur and Punjab. A comparison was carried out for both drugs for fecundity,
fertilization, and hatching rate during the induced spawning of Labeo rohita by a single dose
of each hormone. Ovatide performed much better than Ovaprim. Javed Khan and Fawad
Ali (2021) mentioned that 4 mg pituitary gland for one kg fish showed a fertilization rate of
53.6% for Grass carp (Ctenopharyngodon idella) and 75% for Rohu (Labeo rohita). The
study of Nandeesha et al., 1990 and Sokolowska et al., 1986 reveals that the Ovaprim use is
more economical in commercial carp speed production as it saves a considerable amount of
time and avoids the excessive handlings of brooder fishes. Grass carp (Ctenophyaryngodon
idella) and Rohu (Labeo rohita) preferably spawn during mid of April to June with
fertilization rate of 53.6% and 75% respectively with hatchlings percentage of 63.88% at
favorable temperature of 20ºC to 25ºC. The present study indicates that pituitary gland
injection gives low production than Ovaprim.
A study made by Salman et al., (2023) discussed the effect of hormonal treatment on
egg production by comparing the efficiency of the HCG, PG extract and Ovaprim hormones
to promote egg production in grass carp, it was found that females injected by PG extract
produced the highest number of eggs (40 g/kg) followed by ovaprim and the lowest was in
female received HCG (7.3 g/kg). These findings are comparable to other studies which
proved PG extract as the best hormonal treatment for egg production in grass carp (Szabo et
al., 2019). Ovaprim is characterized by the presence of Domperidone which is Dopamine
inhibitor and hence it could stimulate gonadotropins. It was first used in Iraq by Al-Mukhtar
et al., (2004). According to Nandeesha et al. (1990) Indian carp receiving PG and ovaprim
may increase their egg production. In another study made by Dhawan and Kaur (2004) the
superiority was for using Ovaprim for carp spawning induction in terms of Higher
ovulation/fertilization rates, Shorter latency period (time to spawning post-injection) and
Better egg quality which agrees with the present findings for egg diameter and egg number.
The ovaprim treatment was used also by Metwally and Fouad (2008) to increase the
gonado-somatic index (GSI) in grass carp. As for the Peter et al. (1988) reported that using
HCG alone is not effective for grass carp unless used in high dose or mixed with PG extract
as mentioned by Opuszyhski and Shireman (1995). The same authors stated that the
activation level by HCG increase in grass carp after 6h then dropped sharply after 12 h due to
reduction in steroid hormones. According to Naeem et al. (2011), HCG usage as a substitute
for PG extract may be effective for certain types of fish species such as silver carp but not for
grass carp.

The present study evaluated the physiological effects of three hormonal treatments
Ovaprim, pituitary extract, and a mixture of them on kidney function (serum urea) and lipid
metabolism (total cholesterol, triglycerides) in grass carp (Ctenopharyngodon idella) over a
four-week period. In concern with Serum urea, the present study recoded much pronounced
elevation in serum urea displayed by the PE-treated group, This sharp rise suggests that
the complex hormonal composition of PE (FSH, LH, and other gonadotropins) imposes a
greater metabolic burden on the kidneys, this was supported by previous studies
demonstrating that crude pituitary extract can increase nitrogenous waste due to enhanced
protein turnover (Harvey & Hoar, 1980). In contrast, the Ovaprim group exhibited a more
gradual increase in urea levels, demonstrating a less abrupt metabolic shift, possibly due to
its targeted stimulation of pituitary LH release without the additional hormonal load present
in PE. The combined treatment resulted in intermediate urea levels, This suggests that the
synergistic effects of the two hormones may partially offset the renal stress induced by PE
alone, possibly by optimizing the hormonal cascade and reducing metabolic waste
production, this was nearly identical to the findings of Wang, et al. (2020) in his study on
grass carp urea dynamics during induced spawning, also (Alavi et al., 2014) findings were in
agreement where he reported that PE caused higher urea/creatinine levels (renal stress) on cat
fish due to uncontrolled hormonal release while Ovaprim showed stable metabolic profiles,
supporting its safety.

Concerning Lipid metabolism notably, the third treatment (mixture) resulted in lower
final cholesterol levels compared to Ovaprim or PE alone. The three groups in this study
exhibited elevated triglycerides levels consistent with the energy demands of spawning.
However, the PE group reached the highest reinforcing the idea that PE induces a more
aggressive metabolic response. Likely (Yaron et al., 2003) found that GnRH analogues (like
Ovaprim) induced gradual lipid mobilization, while PE caused sharp triglyceride spikes.

Recommendations for Grass Carp Management:

This study revealed that ovaprim is the Preferred Treatment for female grass carp
highest Egg Production and post-Injection weight Gain. For fry growth, Ovaprim is also the
top choice due to its superior and consistent performance in early-stage survival (critical for
fry health), highest weight gain indicating reliable and sustained growth and longest fries
suggesting optimal developmental conditions for grass carp fries. Concerning broodstock
physiology, pituitary Extract (PE) elicits the strongest metabolic response, making it
potentially stressful for long-term use, while the milder effect of Ovaprim on urea levels and
lipid metabolism supports its use in induction of spawning where minimizing renal stress is a
priority. Overall, prioritize ovaprim for maximum productivity of grass carp.

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