Effects of the time of administration and the concentration of exogenous prostaglandin F on ovulation in pacu

doi:10.21203/rs.3.rs-3235180/v1

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Effects of the time of administration and the concentration of exogenous prostaglandin F on ovulation in pacu

https://doi.org/10.21203/rs.3.rs-3235180/v1

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In this study, we evaluated the effects of applying Ciosin® (containing 0.25 mg/ml cloprostenol, an analogue of prostaglandin F_2α (PGF_2α)) at the time, five or seven hours after hypophysation resolving dose (experiment 1). In the experiment 2, at the time of hypophysation resolving dose, females were induced with 1.0 to 7.0 mL Ciosin /kg. Reproductive parameters and plasma levels of 17α-20β-dihydroxy-4-pregnen-3-one (DHP) and PGF_2α were evaluated at the time of spawning for both experiments. Neither the timing of Ciosin application closer to the spawning nor its higher concentrations resulted in increased reproductive parameters or elevated PGF_2α and DHP plasma levels. The absence of PGF_2α peak at the time of ovulation in both experiments can be attributed to the rapid increase and subsequent decrease in exogenous PGF_2α levels shortly after application. Interestingly, multivariate analysis in experiment 2 revealed two main clusters: one characterized by spawning failure, poor-quality spawns and low DHP levels; and the other by showing successful spawning, high fecundity and elevated DHP levels. Since it is known that the DHP binding to the nuclear progestin receptor induces prostaglandin receptor expression, these facts may explain why better reproductive performances were associated with higher endogenous DHP levels, but not with changes in exogenous PGF_2α application. Although positive correlations were observed between DHP levels and good reproductive performance, the reasons behind the substantial variation in DHP levels among females subjected to the same treatments remain unknow. An emerging focus is to elucidate the pattern of expression of PGF_2α receptors and its relationship with DHP in Piaractus mesopoatamicus.

ovulation failures

migratory fish

hormonal induction

prostaglandin synthetic

1. The hypothesis that administering exogenous PGF_2α closer to ovulation would improve pacu reproductive performance was not supported by this study.

2. The hypothesis that higher exogenous PGF_2α concentration would improve pacu reproductive performance was not supported by the study results.

3. Endogenous PGF_2α plasma levels at the time of ovulation neither vary regard to time nor to exogenous PGF_2α concentration.

4. Endogenous DHP levels at the time of ovulation are correlated with good spawning performance but vary among animals by unknown reasons.

5. PGF_2α and DHP levels at the time of ovulation are strongly positively and negatively correlated with fecundity and latency, respectively.

Pacu (Piaractus mesopotamicus) is a highly valued fish species in the Neotropical region for human feed and fishery. It is favored for its desirable traits in aquaculture, including hardness, resistance to low temperatures, and rapid growth under various conditions (Jomori et al. 2003; Souza et al. 2003; Gelman et al. 2004). Pacu, tambaqui (Colossoma macropomum), pirapitinga (Piaractus brachypomus), and their interspecific hybrids belong to the commercially significant “round fish” group. In Brazil, this group ranks second in terms of fish production, accounting for approximately 27% of farmed fish production in 2021 (IBGE, 2022), with a substantial increase in their exportation rates (Peixe BR 2021).

Pacu is a rheophilic species that exhibits a batch-spawning reproductive strategy characterized by synchronous ovulation, where mature eggs are released in a single reproductive event (Kuradomi et al. 2016). The reproductive season of pacu typically occurs from October to January in Brazil (Schorer et al., 2016), coinciding with the rainy season and increasing water temperatures. Pacu needs hormonal induction for spawning in captivity. The hypophysation with carp pituitary extract is the primary protocol for inducing spawn of native migratory fishes in Brazil, including pacu (Criscuolo-Urbinati et al. 2012; Kuradomi and Batlouni 2018; Mechaly et al. 2023). This protocol, however, still presents failures related to female ovulation response, consequently causing spawning success unpredictability (Criscuolo-Urbinati et al. 2012; Kuradomi and Batlouni 2018).

Prostaglandins have been demonstrated as effective inducers of ovulation in various teleosts (Jalabert and Szöllösi 1975; Fujimori et al. 2012; Joy and Singh 2013; Tang et al. 2017). Previous studies have shown that the addition of exogenous prostaglandin F_2α (PGF_2α) (2 mL or 5 mL of Ciosin® (MSD Animal Health) per fish, containing 0.25 mg/ml cloprostenol, a synthetic analogue of PGF_2α alongside the hypophysation improved spawning rate in pacu females (Criscuolo-Urbinati 2012; Criscuolo-Urbinati et al. 2012). However, the conclusions of that pioneering studies have highlighted the need for further investigations to determine the most effective doses and administration periods. The studies published by Criscuolo-Urbinati (2012) and Criscuolo-Urbinati et al., (2012) were conducted over two reproductive seasons, with two experiments in the first season (testing hypophysation and 2 mL of Ciosin) and a third experiment in the second season (testing hypophysation and 5 mL of Ciosin). In both seasons 100% of fish treated with Ciosin spawned, while the control fish (only hypophysation) had spawning rates of 53.0%, 83.3% and 25.0%, respectively. Evaluating the data in detail, two key points remained to be investigated. Firstly, oscillations in reproductive performance among spawnings, since evaluating all fish together, regardless of the treatments, there was a notable decrease in fertilization and hatching rates comparing the first (earlier in November) and second spawn (later in December) (Criscuolo-Urbinati 2012). Secondly, for unknown reasons and regardless of treatment, females that spawned earlier (reduced latency period) exhibited the highest fertility rates, while those spawning later showed the lowest fertility rates (Criscuolo-Urbinati 2012).

Meanwhile, we have shown that endogenous levels of PGF_2α and 17α-20β-dihydroxy-4-pregnen-3-one (DHP) at the time of ovulation are associated with successful ovulation of this species, but values of the former were similar among spawned females and some rare females that did not spawn (Kuradomi and Batlouni, 2018). Later, we showed that the application of exogenous PGF_2α, at resolving dose during hypophysation, provoked a plasma peak of PGF_2α one hour after application, however such a peak was 50% reduced 4 hours after injection. Finally, at the time of spawning (around 12 hours after resolving hypophysation doses), an endogenous PGF_2α plasmatic peak was registered for both PGF_2α treated and untreated (control) hypophysed females (Sato et al. 2020). So, in summary, the observations indicated that exogenous PGF_2α stimulates ovulation, but the peak of PGF_2α that occurs at the time of ovulation (12 hours after the resolving dose) is endogenous; and the injected exogenous PGF_2α peaks one hour after application but declines eight hours before ovulation (Sato et al. 2020).

With this in mind, in this study we tested two new approaches to increase the consistency of reproductive performance among females and to try to make females spawn earlier, increasing the chances of better reproductive performance. We evaluated whether applying exogenous PGF_2α closer to ovulation or in higher doses at the time of resolving doses would increase reproductive performance, especially by potentially increasing PGF_2α plasma levels close to ovulation time.

This study was conducted in agreement with the precepts of the National Council for the Control of Animal Experimentation (CONCEA) and was approved by the Animal Ethics and Welfare Committee from Universidade Estadual Paulista (UNESP), Jaboticabal, SP, Brazil, under permission number 003837/17.

2.1 Origin, characteristics and management of fish

The experiments were carried out during the pacu reproductive season, in December 2017 (experiment 1) and December 2018 (experiment 2) at the UNESP Aquaculture Center (CAUNESP), located in Jaboticabal, São Paulo, Brazil (21º15'17"S and 48º19'20"W). The broodstock was maintained throughout the year in 200 m² earthen ponds (at a density of 0.80 kg/m²), supplied with a constant flow of approximately 20 L/min. The fish were fed ad libitum twice a day (approximately 1-4% of the biomass) with commercial feed Omnivores Growth - Fri-Acqua, containing 28% crude protein, 3.5% ether extract, 12% ash and 9% of fibrous matter, according to the manufacturer's information. The fish used were produced at CAUNESP from crossings carried out with the broodstock already existing at the Institution.

Temperature and conductivity were measured using a HANNA probe, HI-98311; pH was measured using a KASVI probe, K39-0014P and dissolved oxygen with a BERNAUER-424 AQUACULTURE probe, F-1550A. The mean ± standard deviation of pH, dissolved oxygen concentration, conductivity and water temperature, were, at 5.71 ± 0.88, 6.43 ± 0.13 mg/L, 34.45 ± 10.60 μS/cm and 24.50 ± 2.70 °C, and, respectively, in experiment 1 and, 5.82 ± 0.65, 5.98 ± 0.33 mg/L, 34.26 ± 12.05 μS/cm and 25.35 ± 2.86 °C respectively, in experiment 2.

2.2 Experimental Design

2.2.1 Experiment 1

In a previous pharmacokinetics study (Sato et al. 2020), we demonstrated that the application of 2 mL of Ciosin per fish at the time of resolving hypophysation dose (referred to as group 2D in the present study) led to a PGF_2α plasmatic peak within one hour post-resolving dose, which decrease after four hours post-resolving dose. Considering that ovulation in this species occurs 12 hours after the second dose (i.e., eight hours after the decline in the PGF_2α peak caused by exogenous administration), our hypothesis was that by shifting the PGF_2α peak closer to the moment of ovulation, five (5H) or seven hours (7H) after the hypophysation resolving dose, the ovulation-inducing potential of PGF_2α would be potentiated.

To test this hypothesis, twenty-four adult females aging five years old and weighing 3.098 ± 0.181 kg, were distributed in a completely randomized design with four groups (n = six females per group): hypophysed females received 2 mL/fish of Ciosin/Kg either at 2D, 5H or 7H. The exogenous PGF2α used was Ciosin®, which contains 0.25 mg/ml of cloprostenol (a synthetic analogue of PGF_2α). For hypophysation, carp crude pituitary extract (CPE) was macerated and diluted in saline solution (0.9% NaCl) and applied intramuscularly, diluting in 0.2 ml/kg of fish. Hypophysation was composed of two doses of CPE (0.6 and 5.4 mg/kg) with a 24-hours interval between doses (Criscuolo-Urbinati et al. 2012; Kuradomi and Batlouni 2018; Sato et al. 2020). In the control groups (C), the same volume of saline solution (0.2 ml/kg) was administered at the time of hypophysation doses (Figure 1). To establish baseline conditions, an initial group (IN) was included, in which six females were randomly selected from the same batch for blood sampling, without receiving hormonal induction. Males used for fertilization weighed 2.520 ± 0.147 kg.

Ovulation in this species occurs, for the most part, between 276 and 323 accumulated thermal units (ATU) after the second dose (i.e., between 10 and 12 hours after the resolving dose at 27 °C) (Criscuolo-Urbinati et al. 2012). The value of ATU was obtained by summing the water temperature (°C) each hour between the second hormonal dose and the moment of spawning. To mitigate interference related to collection handling, blood samples were carried out only at the time of ovulation. The sampling process included spawned females that had spawned up to 340 ATU, as well as unspawned females that had not spawned up to 340 ATU (Kuradomi and Batlouni 2018) (Figure 1).

2.2.2 Experiment 2

Figure 1 clearly illustrates that in the experiment 1, in order to apply Ciosin in 5H and 7H groups, additional handling of the fish was required (a third handling after priming and resolving hypophysation doses). Conversely, fish from 2D group were handled only twice (Figure 1). To explore an alternative method of enhancing the inducing effect of exogenous PGF_2α without the need for a third handling procedure, the experiment 2 focused on maintaining the 2D moment for Ciosin application (i.e., the time of the resolving dose), while increasing the injected PGF_2α concentration. Since Sato et al., (2020) demonstrated a prominent plasma peak of Ciosin one hour after injection, followed by a reduction but still elevated levels four hours post-injection, we hypothesized that increasing the dose at the time of the resolving dose could yield better results compared to altering the PGF injection schedule and introducing a third handling procedure.

For this purpose, ten adult females, aged six years old and weighing 2.871 ± 0.113 kg, were included in a completely randomized design. They were divided into five groups, with each group consisting of two females. The groups were injected with increasing and equidistant doses of exogenous PGF_2α in conjunction with the resolving dose of hypophysation. The dosage levels and respective groups were as follows: T1 - hypophysation with 1.0 mL/kg of Ciosin(Ciosin® containing 0.25 mg/ml cloprostenol (a synthetic analogue of PGF_2α), MSD Animal Health); T2.5 - hypophysation with 2.5 mL/kg of Ciosin, T4 - hypophysation with 4.0 mL/kg of Ciosin, T5.5 - hypophysation with 5.5 mL/kg of Ciosin, and T7 - hypophysation with 7.0 mL/kg of Ciosin. The range of doses was based on previous attempts to determine an effective dose reported by Criscuolo-Urbinati et al., (2012). Males used for fertilization weighed 2.621 ± 0.182 kg.

Sampling was carried out only at the time of ovulation. The sampling process included spawned females that had spawned up to 340 ATU, as well as unspawned females that had not spawned up to 340 ATU (Kuradomi and Batlouni 2018). Each individual fish was treated as an experimental unit.

2.3 General methods in both experiments

2.3.1 Broodstock selection

Females were selected based on specific external characteristics, including a swollen abdomen (Schorer et al. 2016) and hemorrhagic urogenital papilla (Kuradomi et al. 2017), and by the degree of development of the intra-ovarian oocytes sampled with a plastic catheter (≥ 30% oocytes with germinal vesicles shifted towards the periphery) (Schorer et al. 2016; Kuradomi and Batlouni 2018). Males were selected by obtaining semen though slight abdominal pressure (Kuradomi et al. 2016). Once selected, the animals were transported to the laboratory in transport boxes and placed in 750 L tanks coupled to a water recirculation system. The tanks were maintained at a constant temperature throughout the experiments. In experiment 1, the temperature was set at 26.08 ± 0.27ºC, while in experiment 2, it was maintained at 26.82 ± 0.26ºC. The fish were exposed to the natural photoperiod, which consisted of approximately 13 hours and 20 minutes of light exposure per day in both experiments.

2.3.2 Reproductive performance

After observing the onset of reproductive behavior of the females in the tanks (i.e., restlessness and muscle spasms in the abdomen) and/or observing the first oocytes released at the bottom of the tanks, the process of extruding the oocytes was carried out through abdominal massage (between 270 and 340 ATU). The oocytes were extruded into a dry container and the oocyte mass and spawning time were recorded.

Fertilization was obtained by adding a pool of semen from three males (in the proportion of 0.5 mL of pooled semen for each 50 g of oocytes). The sperm concentration in pacu ranges from approximately 4.7 to 6.9 x 10¹⁰ cells/mL (Kuradomi et al. 2016). Females with fecundity lower than 35,000 oocytes/kg of fish were considered females with poor-quality ovulation. The oocytes were fertilized and hydrated according to conventional methodology, adding water after mixing the gametes. Then, 10 mL of hydrated eggs from each female were transferred to 8 L acrylic incubators at an average temperature of 26.8 ± 0.3 ºC. The estimated rates of fertility (blastopore closure) and hatching (caudal fin moving and fully unfolded) were performed about 12 and 18 hours after eggs fertilization, respectively (Schorer et al. 2016; Kuradomi and Batlouni 2018). To obtain fertility and hatching rates roughly 100 eggs) were classified as viable or non-viable embryos per female.

The following parameters were recorded:

Ovulation rate (%) = number of ovulated females / total number of females x 100

ATU = mean of water temperature (ºC) x latency period (h)

Relative fecundity = number of oocytes released / female body mass (kg)

Fertility rate (%) = number of viable embryos / total number of eggs counted x 100

Hatching rate (%) = number of eggs with embryos at caudal fin detachment / total number of eggs counted x 100

2.3.3 Blood sampling

For sample collection to assess the level of PGF and DHP at the time of ovulation (Figure 1), fish were anesthetized with 100 mg/L of benzocaine; blood samples were collected from the caudal vein using heparin-treated syringes. The plasma was separated by centrifugation at 1000×g for 15 min at 4 °C and then stored at -80 °C until quantification of the steroid hormone concentrations. For PGF_2α dosage, 10 μM indomethacin was added to the blood immediately after collection to inhibit prostaglandin synthesis (Kuradomi and Batlouni 2018). The plasma levels of 17α-20β-dihydroxy-4-pregnen-3-one (DHP) and PGF_2α were quantified by Enzyme-Linked ImmunoSorbent Assay (ELISA) using commercial kits (Cayman Chemical Company, Ann Arbor, MI, USA) following the manufacturer’s instructions. The readings of DHP and PGF_2α plates were performed at an absorbance of 412 and 405 nm, respectively, using an Epoch2 plate reader (BioteK Instruments, Inc., Highland Park, Winooski, USA), and all samples were read in duplicate. To validate the analysis, intra- and inter-assay variability were assessed, and we obtained the respective following coefficient of variations (CV %): 0.30 to 17.27% and 1.71 to 18.53% for DHP; 2.88 to 19.31% and 4.71 to 20.53% for PGF_2α.

2.4 Data analysis

In experiment 1, all statistical tests were performed using the STATISTICA software (StatSoft, Inc., Tulsa, OK, USA) and Excel (Microsoft, Redmond, USA). Normality and homogeneity of the variances were tested using the Shapiro-Wilk’s test and Levene’s test, respectively. Parametric data (i.e., reproductive performance) were analyzed using the variance test (one-way ANOVA) followed by Tukey’s test. Kruskal-Wallis’s test was used for non-parametric data (DHP and PGF_2α plasma levels) followed by Dunn’s test for multiple comparisons. A Fisher's Exact Test for count data was used to compare ovulation and poor-quality ovulation rates among the experimental groups that have spawned. The plasma levels of DHP and PGF_2α were correlated with the reproductive performance parameters (regardless of group) using the non-parametric Spearman's rank correlation test. In the correlation event, negligible correlation is considered with ρ (rô) values from 0.0 to 0.10, weak from 0.10 to 0.39, moderate from 0.40 to 0.69, strong from 0.70 to 0.89 and very strong from 0.90 to 1.0, according to a conventional method of interpreting correlation coefficients (Schober et al. 2018). Significant differences were accepted with a P value <0.05. Data were expressed as mean ± standard error or median (first quartile; third quartile) and correlation in ρ value.

In experiment 2, a multivariate analysis was performed using Factoshiny package in RStudio. Two multivariate statistical methods were applied to separate fish into groups: principal component analysis (PCA), and clustering on the PCA results by the hierarchical method using as a clustering metric the Euclidian distance and the Ward’s algorithm to detect the number of clusters based on the inertia gain. Firstly, variables were standardized by default, to give the same importance to each variable, of which the mean and variance were 0 and 1, respectively. For handling missing values an impute was used with 2-dimensional PCA-model through the missMDA library. The algorithm estimates the missing data values with values that have no influence on the PCA results, taking simultaneously into account: similarities between individuals and links between variables. The dataset for performing PCA and clustering contained 10 individuals, six quantitative variables (ATU, relative fecundity, fertility and hatching rates, DHP and PGF_2α plasma levels), and one categorical variable (treatment).

3.1. Experiment 1

3.1.1. Reproductive performance

Both 2D and 5H groups exhibited an ovulation rate of 4 out of 6 fish (66.7%), while the 7H group had an ovulation rate of 3 out of 6 fish (50%) with no significant differences among groups (p > 0.05). No ovulation was recorded in the control group (Table 1). Regarding the quality of ovulation, the 2D group did not exhibit any instances of poor-quality ovulation, defined as fecundity below 35,000 oocytes/kg fish. In contrast, the 5H and 7H groups showed rates of poor-quality ovulation at 25% and 66.7%, respectively. However, no significant differences in poor-quality ovulation rates were observed among the groups (p > 0.05) (Table 1). The average values of ATU, fecundity, fertility and hatching rates were similar among all groups that spawned (2D, 5H and 7H) (p > 0.05) (Table 1). This indicates that the different timings of exogenous PGF_2α application did not significantly influence these reproductive parameters.

Table 1

**Experiment 1.** Reproductive performance of *Piaractus mesopotamicus* females submitted to hypophysation associated with exogenous PGF_2α applied in different periods.
Groups	Ovulation rate (%)	Poor-quality ovulation (%)*	ATU	Relative fecundity	Fertility rate (%)	Hatching rate (%)
C	0	-	-	-	-	-
2D	66.7^a	0^a	288.6 ± 6.2^a	70,065 ± 10,369^a	52.5 ± 11.8^a	47.4 ± 7.7^a
5H	66.7^a	25^a	289.3 ± 11.0^a	73,050 ± 21,634^a	63.3 ± 15.4^a	55.8 ± 14.0^a
7H	50^a	66.7^a	309.2 ± 9.8^a	53,092 ± 26,428^a	44.9 ± 19.0^a	29.9 ± 14.1^a

The “IN” group was not submitted to spawning and there are no data for reproductive performance for this group. C: 0.9% saline control; 2D: hypophysation + 2 mL/fish of prostaglandin F_2α (PGF_2α) in the resolving dose; 5H: hypophysation + 2 mL/fish of PGF_2α five hours after the resolving dose; 7H: hypophysation + 2 mL/fish of PGF_2α seven hours after the resolving dose. ATU: accumulated thermal units. Relative fecundity: number of oocytes released/kg fish. The variables ATU, relative fecundity, fertility rate and hatching rate were similar among groups (p > 0.05). Data expressed as mean ± SE.

* Female considered as poor-quality ovulation presented fecundity below 35,000 oocyte/kg fish

3.1.2. Plasma levels of PGF_2α and DHP at the time of ovulation

Mean PGF_2α values were similar among 2D, 5H and 7H groups. All groups exhibited considerable individual variation in plasma levels of PGF_2α at the time of ovulation. In group 2D, levels ranged from 1.81 to 150.45 ng/mL, in group 5H from 3.61 to 555.52 ng/mL, and in group 7H from 3.16 to 261.43 ng/mL. The PGF_2α levels were significantly higher (more than 200-fold) in groups 2D, 5H, and 7H compared to group C and the IN group (p < 0.05) (Fig. 2A).

Mean DHP values were similar among 2D, 5H and 7H groups. DHP levels ranged from 0.08 to 0.66 ng/mL in group 2D, from 0.11 to 0.50 ng/mL in group 5H, and from 0.10 to 1.15 ng/mL in group 7H. Similarly, the plasma levels of DHP were more than ten times higher in groups 2D, 5H, and 7H compared to group C and group IN (p < 0.05) (Fig. 2B).

3.1.3. Correlation

Correlations between variables were analyzed regardless of group. Following the method by Schober et al. (2018), strong positive and negative correlations were observed (p < 0.05). (Table 2). A strong positive correlation was observed between fecundity and PGF_2α levels, fertility and hatching rates. On the other hand, ATU values were strong negatively correlated to fecundity, fertility and hatching rates, suggesting that increased ATU is associated with reduced reproductive success.

Table 2

**Experiment 1.** Spearman's correlations (regardless of group) using the non-parametric Spearman's rank correlation test.
Correlation	ρ	Rate
PGF_2α x Fecundity	0,83	Strong
Fecundity x Fertility rate	0,81	Strong
Fecundity x Hatching rate	0,88	Strong
Fecundity x ATU	-0,78	Strong
ATU x Fertility rate	-0,79	Strong
ATU x Hatching rate	-0,84	Strong
PGF_2α: prostaglandin F_2α; Fecundity: number of oocytes released/kg fish; Fertility and Hatching rate: number of viable embryos/total number of eggs×100; ATU: accumulated thermic units. Rate: following (Schober et al., 2018)

3.2. Experiment 2

Individual values of reproductive performance and plasma levels of PGF_2α and DHP are presented in the Table 3.

Table 3

**Experiment 2.** Individual reproductive performance of *Piaractus mesopotamicus* females submitted to hypophysation associated with exogenous PGF_2α applied in different concentrations.
Individual - Group	Relative fecundity	ATU	Fertility rate (%)	Hatching rate (%)	PGF_2α (ng/mL)	DHP (ng/mL)
1 - T1	142,080	299.0	71.0	60.1	66.67	0.21
2 - T1	-	-	-	-	449.04	0.08
3 - T2.5	184,920	322.4	68.6	67.8	161.37	0.30
4 - T2.5	33,720*	338.0	15.9	5.1	48.92	0.08
5 – T4	-	-	-	-	3.61	0.03
6 – T4	50,160	299.0	73.2	71.6	38.20	0.21
7 – T5.5	195,480	325.0	21.5	19.0	516.39	0.22
8 – T5.5	77,400	312.0	91.9	88.0	296.14	0.20
9 – T7	132,360	306.8	25.2	25.1	99.54	0.26
10 – T7	119,760	301.6	37.9	36.4	63.10	0.17

The table shows data of the two fish used for each treatment. T1: hypophysation + 1.0 mL/kg of PGF_2α in the resolving dose; T2.5: hypophysation + 2.5 mL/kg of PGF_2α in the resolving dose; T4: hypophysation + 4.0 mL/kg of PGF_2α in the resolving dose; T5.5: hypophysation + 5.5 mL/kg of PGF_2α in the resolving dose; T: hypophysation + 7.0 mL/kg of PGF_2α in the resolving dose. Dash (-) represents the non-spawning female. Relative fecundity: number of oocytes released/kg fish. ATU: accumulated thermal units. PGF_2α: Prostaglandin F_2α. DHP: 17α-20β-dihydroxy-4-pregnen-3-one.

* Female considered as poor-quality ovulation presented fecundity < 35,000 oocyte/kg fish

3.2.1. Multivariate statistical analysis

3.2.1.1. Principal component analysis (PCA)

The PCA allowed for a unique distribution of females according to Kaiser's criterion (1959), as only two eigenvalues were greater than 1, with the largest eigenvalue being 3.31 (Principal Component 1 - PC1) and the second largest eigenvalue being 1.54 (Principal Component 2 - PC2) (Table 4). The first two dimensions of the analysis account for 80.68% of the total inertia of the dataset, indicating that 80.68% of the total variability of the variables in the dataset is explained by the plane. A bidimensional ordination of the fish and variables was conducted using PC1 and PC2, enabling the construction of a biplot graph (Fig. 3). Most variables have a high correlation with PC1, except for PGF_2α, which has a positive correlation with PC2 (Table 4).

Table 4

**Experiment 2.** Information of the principal component analysis (PCA) of *Piaractus mesopotamicus* females submitted to hypophysation associated with exogenous PGF_2α applied in different concentrations.
	Principal component 1	Principal component 2
Explained variance %	55.09	25.59
Eigenvalue	3.31	1.54
Variable correlation
Relative fecundity	0.61	0.68
ATU	− 0.77	0.34
Fertility rate (%)	0.89	− 0.23
Hatching rate (%)	0.92	− 0.17
PGF_2α	− 0.24	0.80
DHP	0.82	0.49

The PCA revealed interesting patterns in the distribution of individuals based on their characteristics. The Wilks test did not show any significant association between the categorical variable (treatment) and the distance between individuals (p = 0.7697), indicating that the treatment did not explain the observed differences. Evaluating PC1, a clear separation was observed between females 1, 3, and 6 (located on the right side of the graph with positive values in this dimension) and females 2, 4, and 5 (situated on the left side of the graph with negative values in this dimension), despite belonging to the same treatments (T1, T2.5, and T4) (Fig. 3). Females 1, 3, and 6 exhibited high values for variables such as DHP levels, fertility rate, and hatching rate (Table 3). In contrast, females 2, 4, and 5 had low values for DHP and relative fecundity. Furthermore, PC2 showed a stronger positive correlation with PGF_2α levels, which was particularly evident in female 7. This female stood out from the others based on the higher levels of PGF_2α (Table 3).

The relationships among variables are represented by the angles between the vectors. For instance, the fertility rate and hatching rate showed a positive correlation, while both variables were inversely related to ATU, suggesting that increased ATU values were associated with lower fertility and hatching rates (Fig. 3). Another correlation observed was between relative fecundity and DHP levels, indicating that higher levels of DHP were associated with higher relative fecundity (Fig. 3).

3.2.1.2. Hierarchical cluster analysis

Using the similarity coefficient method based on Euclidean distance and Ward’s algorithm for obtaining the similar access groups, it was possible to separate the individuals into three clusters (Fig. 4). Cluster 1, consisting of females 2, 4, and 5, is characterized by low DHP plasma levels and relative fecundity (Table 3). Cluster 2, composed of female 7, is characterized by high PGF_2α plasma levels. Cluster 3, consisting of females 1, 3, 6, 8, 9, and 10, is characterized by high DHP levels and relative fecundity.

The observation that clusters 1 and 3 exhibit opposite characteristics in CP1 is notable, with cluster 1 characterized by low DHP levels and cluster 3 characterized by high DHP levels (Fig. 5). A significant difference in DHP levels between these two clusters was detected using the Wilcoxon test (p-value = 0.02688), although this difference does not appear to be related to the treatments (Fig. 6). Interestingly, low DHP levels seem to be associated with females that did not spawn (females 2 and 5) or with poor-quality spawns (female 4).

The findings of our study on the reproductive performance of pacu females raise important insights regarding the efficacy of the proposed modifications to the hormone induction protocol previously suggested by Criscuolo-Urbinati et al. (2012). In an effort to enhance the reproductive performance, we conducted two independent and consecutive experiments that involved combining hypophysation with the application of exogenous PGF_2α at varying timings and concentrations. However, our results suggest that these modifications did not yield significant improvements, leading to the questioning of the validity and effectiveness of the altered protocol.

Previously, the administration of exogenous PGF_2α has shown promising effects on the reproductive performance of pacu females, resulting in a significantly higher spawning rate compared to hypophysation alone (Criscuolo-Urbinati et al. 2012). Furthermore, the use of exogenous PGF_2α has been associated with a reduction in the number of follicle-enclosed oocytes at the GVBD stage remaining in the ovaries post-spawning, indicating its potential role in oocyte release, even though GVBD oocytes were still present in the ovaries (Criscuolo-Urbinati et al. 2012). Subsequent investigations have demonstrated that the application of exogenous PGF_2α at the time of hypophysation resolving dose leads to prolonged elevations in circulating PGF_2α levels compared to conventional hypophysation, which exhibits a single peak at 12 hours after the resolving dose (Sato et al. 2020). Building upon these findings, we formulated the hypothesis in the present study that modifying the timing of exogenous PGF_2α administration and/or adjusting the dose concentration could potentially induce a peak of circulating PGF_2α closer to ovulation, thereby eliciting a more potent and predictable ovulatory response.

However, our attempts to modify the timing of exogenous PGF_2α administration closer to ovulation (groups 5H and 7H) did not result in any improvement in the evaluated reproductive parameters. In fact, these treatments showed a higher incidence of females experiencing poor-quality ovulation, indicated by fecundity below 35,000 oocyte/kg of fish, compared to the group receiving PGF_2α application at the time of hypophysation resolving dose (group 2D). Surprisingly, we observed that the plasma levels of PGF_2α and DHP at the time of spawning were similar regardless of the timing of exogenous PGF_2α application. Furthermore, females that received the third intervention (5H and 7H) did not spawn earlier than those in the 2D group, despite the expectation that earlier spawning (lower ATU) would potentially result in higher embryo viability, as we observed that the ATU had a strong negative correlation with fertility and hatching rates. Nevertheless, postponing PGF_2α application closer to ovulation did not anticipate the spawning moment and resulted in poor-quality ovulation. These observations raise the possibility that the effectiveness of exogenous PGF_2α application may rely on a synergistic interaction with luteinizing hormone (LH) released during hypophysation, in order to exert positive effects on ovulation.

It is well established that LH plays a critical role in regulating key molecular pathways involved in ovulation, including the expression of nuclear progestin receptors (npr) and prostaglandin receptors (ptger4b) (Hagiwara et al. 2014; Tang et al. 2016, 2017). In an in vivo study using zebrafish, it was observed that the relative expression of ptger4b increased 0.5 hours after human chorionic gonadotropin (hCG) injection, returning to baseline levels 2 hours after injection (Tang et al. 2016). This suggests that the availability and activation of prostaglandin receptors may occur rapidly in response to LH stimulation. In our study, we applied exogenous prostaglandin at different time intervals following hypophysation resolving dose: 5 hours (5H) and 7 hours (7H). Despite limited knowledge regarding these processes in neotropical species, we cannot rule out the possibility that the action of LH in activating prostaglandin receptors and their subsequent availability may be as rapid as in zebrafish.

Based on these observations, we speculate that applying exogenous PGF_2α outside the timeframe of LH action may result in negligible effects, as the prostaglandin receptors may not be readily available for activation. Thus, the timing of exogenous PGF_2α administration relative to LH-induced ovulation is of crucial importance. Our results indicate that applying exogenous PGF_2α at intervals of 5 and 7 hours after hypophysation did not yield significant improvements in reproductive parameters, suggesting that the receptors for prostaglandin may have already returned to baseline levels or were not adequately activated within this timeframe. These findings emphasize the need for further investigation into the intricate molecular processes underlying ovulation in neotropical species. Future studies examining the dynamics of LH-mediated prostaglandin receptor activation in pacu females will contribute to the development of more precise and effective hormone induction protocols for optimizing reproductive success.

The considerable variation in endogenous levels of PGF_2α observed in pacu females undergoing hypophysation has been previously reported (Kuradomi and Batlouni 2018), and our study corroborates these findings. In parallel, the absence or inconstancy of a response pattern to the use of exogenous prostaglandins has been reported since the first studies on the role of prostaglandins in ovulation induction in several species of fish (Jalabert and Szöllösi 1975; Stacey and Pandey 1975; Goetz and Theofan 1979; Berndtson et al. 1989). Determining the optimal dose-response relationship, specifically the amount of prostaglandin injected versus plasma concentration, remain a challenge due to individual variations in endogenous production, which strongly influence this ratio.

In our study, we examined the levels of DHP, which is associated with prostaglandins and their receptors (Berndtson et al. 1989; Goetz 1997; Kagawa et al. 2003; Knight and Van Der Kraak 2015). Hagiwara et al. (2014) demonstrated that the expression of the PGE₂ receptor, EP4b (ptger4b), is regulated by a mechanism involving the nuclear progestin receptor (Pgr) in medaka (Oryzias latipes). This mechanism involves DHP binding with the Pgr and subsequent association with the promoter region of the ptger4b gene, leading to its transcription and expression. However, in our study, the similar levels of DHP observed in the 2D, 5H, and 7H groups at the time of ovulation suggest that the third intervention (application of PGF_2α in the 5H and 7H groups) did not significantly affect DHP levels. This finding aligns with previous studies by Sato et al. (2020) and Batlouni and Kuradomi (2018).

In light of these observations, maintaining the application of PGF_2α at the time of the resolving dose of hypophysation appears to be the most favorable option. This finding supports the previous study by Sato et al. (2020), suggesting that the protocol involving PGF_2α application at the resolving dose may demonstrate greater predictability in spawning and ovulation success likely due to the earlier and prolonged increase in circulating levels of PGF_2α compared to hypophysation alone. This extended action interval of PGF_2α has been observed in other fish species, such as goldfish (Stacey and Pandey 1975) and medaka (Fujimori et al. 2011). Based on these findings, we conducted experiment 2, testing different doses of PGF_2α to achieve higher levels after application and potentially prolong its action interval.

In experiment 2, we observed significant variability among individuals, irrespective of the treatment received. To better understand these individual-specific variations, we conducted a multivariate analysis. Interestingly, the treatment factor did not account for the observed differences, indicating that the concentration of the exogenous PGF_2α dose was not a limiting factor in the reproductive processes of pacu females. This is evident in the separation of replicates from the same treatment on opposite sides of the principal component 1. These individuals exhibited contrasting responses in terms of DHP levels, indicating that DHP levels did not show a dose-dependent relationship with exogenous prostaglandin doses.

The clustering analysis further supported this characteristic. Higher doses of PGF_2α did not influence the release of DHP, as evidenced by the presence of females with high DHP levels across all dose groups, from the lowest dose of PGF_2α (female 1) to the highest dose (females 9 and 10). Conversely, higher levels of DHP were associated with higher fecundity, while females that did not spawn or exhibited low-quality spawning displayed low levels of DHP. These findings align with previous literature demonstrating the role of DHP in ovulation, as it binds to the nuclear progestin receptor, which subsequently acts as an inducer of prostaglandin receptor expression (Takahashi et al. 2013).

Similarly, endogenous PGF_2α levels were not found to be dose-dependent on exogenous prostaglandin dose concentrations. This suggests that applying higher concentrations of PGF_2α at the time of the hypophysation resolving dose did not result in elevated circulating PGF_2α levels at the time of spawning or extend its range of action. However, it is worth noting that a previous study reported exogenous PGF_2α levels increase shortly after its application and decrease rapidly in a few hours (Sato et al. 2020), whereas in our study, we only quantified PGF_2α levels at the time of spawning. Therefore, we cannot exclude the possibility that a dose-dependent peak may have occurred before the time of quantification. Thus, our findings indicate that individual variability plays a significant role in the reproductive response of pacu females, independent of the applied treatments. DHP and endogenous PGF_2α levels did not demonstrate a direct dose-response relationship with exogenous PGF_2α doses. The relationship between DHP and fecundity supports its role in ovulation, acting through the nuclear progestin receptor and influencing prostaglandin receptor expression. While endogenous PGF_2α levels did not appear to be dose-dependent, further investigations are needed to determine the optimum curve for PGF_2α action, including in vitro tests and information about the pattern of prostaglandins and DHP receptors in this species.

In summary, our study sheds light on the limitations associated with the proposed modifications to the hormone induction protocol. The findings suggest that maintaining the application of PGF_2α at the time of the hypophysation resolving dose remains the most practical option, as it offers better predictability regarding spawning success. However, further research is necessary to refine the protocol and enhance reproductive performance. Specifically, exploring the kinetics of PGF_2α and DHP receptors throughout induced reproduction may hold promise for improving the effectiveness of the induction protocol.

Acknowledgements

This work was supported by process nº 2016/20741-3, Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP). Rafael Tomoda Sato was supported by a doctoral scholarship from the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior – Brazil (CAPES) – Finance Code 001. Sergio Ricardo Batlouni was supported by a research productivity grant from the National Council for Scientific and Technological Development associated with this proposal.

Declaration of Competing Interest

We declare that we have no conflict of interest.

Author contribution statement

Rafael Tomoda Sato: Conceptualization, Methodology, Formal Analysis, Investigation, Writing – Original Draft. Mariana Roza de Abreu: Formal Analysis, Investigation, Writing – Original Draft. Rafael Yutaka Kuradomi: Methodology, Validation, Writing – Review & Editing. Laíza Maria de Jesus-Silva: Investigation, Writing – Review & Editing. Sergio Ricardo Batlouni: Supervision, Writing – Review & Editing

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Effects of the time of administration and the concentration of exogenous prostaglandin F on ovulation in pacu

Status:

Version 1

Abstract

Figures

Highlights

1. Introduction

2. Material and methods

3. Results

3.1. Experiment 1

3.1.1. Reproductive performance

3.1.2. Plasma levels of PGF_2α and DHP at the time of ovulation

3.1.3. Correlation

3.2. Experiment 2

3.2.1. Multivariate statistical analysis

3.2.1.1. Principal component analysis (PCA)

3.2.1.2. Hierarchical cluster analysis

4. Discussion

5. Conclusion

Declarations

References

Additional Declarations

Status:

Version 1

Effects of the time of administration and the concentration of exogenous prostaglandin F on ovulation in pacu

Status:

Version 1

Abstract

Figures

Highlights

1. Introduction

2. Material and methods

3. Results

3.1. Experiment 1

3.1.1. Reproductive performance

3.1.2. Plasma levels of PGF2α and DHP at the time of ovulation

3.1.3. Correlation

3.2. Experiment 2

3.2.1. Multivariate statistical analysis

3.2.1.1. Principal component analysis (PCA)

3.2.1.2. Hierarchical cluster analysis

4. Discussion

5. Conclusion

Declarations

References

Additional Declarations

Status:

Version 1

3.1.2. Plasma levels of PGF_2α and DHP at the time of ovulation