WO2017127949A1

WO2017127949A1 - Novel injectable veterinary composition for synchronising spawning in fish

Info

Publication number: WO2017127949A1
Application number: PCT/CL2017/000002
Authority: WO
Inventors: Johanna Margarita GAJARDO CORDOVA; Mauricio Alberto VENEGAS BENITEZ; James Frederick FRANCIS POWEL
Original assignee: Farmacologia En Aquacultura Veterinaria Fav S.A
Priority date: 2016-01-27
Filing date: 2017-01-11
Publication date: 2017-08-03
Also published as: CL2016000217A1

Abstract

The invention relates to a novel oily composition comprising salmon gonadotropin-releasing hormone analogue, in order to induce gamete maturation in fish. The composition comprise a mixture of two or more oils, allowing efficient sustained release, thereby synchronising spawning in ponds, with a high yield of viable ova and, hence, a high yield of fry.

Description

NEW INJECTABLE VETERINARY COMPOSITION FOR SYNCHRONIZATION OF FISHING IN FISH

Field of the Invention

The object of the invention is in the field of aquaculture, in the fish farm industry. More specifically oriented to the industry of fish ova production, such as salmon, carp, tilapia, croaker, sturgeon and rainbow trout.

Summary of the Invention

The Chilean aquaculture industry, particularly the salmon farm, has had rapid development in recent decades, achieving an intense and industrialized productivity according to high quality standards, which has positioned it among the main producing countries worldwide.

However, one of the main problems that this industry has had to face is the optimization of the production and availability of salmonid eggs, resulting from the contamination of imported eggs, indicated as the main cause of the presence of the ISA virus in the country.

The crisis experienced caused the momentary cancellation of the entry of eggs from other countries, developing various regulations that allowed a salmon-free productive chain of viruses. The first regulations established that the process should be carried out on land, raising costs by producing eggs only from parents who were always in fresh water and under strict biosafety systems.

However, after the virus crisis, the increase in the cost of production of eggs, led producers to import them again after strict epidemiological evaluations, verifications and technical dresses from Iceland or Denmark. This dependence has once again put on the table the need for a supply throughout the year of national eggs at a low cost and thus eliminate dependence on other countries.

To achieve this process, the industry has had to optimize the control of the reproductive function (ovulation and spawning) of the species under captive conditions, which was resolved through the use of gonadotropic hormones.

The use of natural fish GnRH (US patent 4,443,368) and subsequently that of its synthetic analogues (United Kingdom patent GB 2152342), has been a useful tool for this purpose. This is because the hormone allows the induction of spawning in a controlled way by synchronizing all gametes allowing the maturation of oocytes and spermatozoa during the spawning season and thus, considerably increasing the productivity in fish farms.

The methods traditionally used to induce spawning in fish consist of injecting a saline solution containing GnRH or analogues thereof into the fish (US Patent 4,410,514), but unfortunately the half-life of GnRH in the blood is very short, occurring a minimal effect after treatment. In order to solve this problem, other strategies have been used lately to obtain a prolonged action in the time of gonadotrophin (GtH) in the blood, or sustained-release implants (US Patent 5,288,705), which are found in the national and international market since the nineties. This consists of a sustained release discharge system of the gonadotropin release hormone analogous to salmon sGnRHa through the use of pellet implants or microcapsules (Ovaplant®). However, this type of implants, when presenting fixed doses of the analogous hormone, are not very effective in synchronizing spawning in batches or ponds where there is variability in fish weight, such as salmon in culture. It should be noted that when placing implant devices in many fish, according to prior art, specialized personnel are required for handling, where in addition to the administration of the hormone, it is necessary to determine: the state of the oocytes through techniques such as biopsy ovarian and evaluation of the diameter of the oocytes or palpitation of the females, allowing to detect the presence of free ovules in the abdominal cavity of the breeders. All these factors become important since if the administered dose is not adequate, the process loses all efficiency. In the case of breeding males, prior administration of the hormone, the release of seminal fluid is verified, being possible to increase the volume and mobility of sperm with the administration of the hormone.

Therefore, there is a need to provide an alternative veterinary pharmaceutical composition to the provisions of the prior art, whose vehicle or matrix allows a prolonged release of the hormone in salmon, more efficiently and thus induce gamete maturation ( oogenesis and spermatogenesis), where in turn the release of the hormone is not delayed by entrapment in the matrix of the formulation thus allowing control of the production of eggs by lowering costs and therefore avoiding the import process.

The present invention provides an injectable veterinary pharmaceutical composition, which comprises sGnRHa in an oily vehicle, to be used in fish found in ponds as producers of eggs.

The veterinary pharmaceutical composition object of the present invention is composed of an oily vehicle comprising a mixture of a fatty acid ester and vegetable oil, and pharmaceutically acceptable excipients. As another object of the invention is that the oily mixture of fatty acid ester and vegetable oil can be in proportions such ^'as 1: 3 to 3: 1, 1: 2 to 2: 1, or 1: 1.

Another object of this invention is also to provide a pharmaceutical composition composed of esters of fatty acid selected from triglyceride and ethyl oleate.

Another object of this invention is that the pharmaceutical composition is composed of vegetable oil selected from cotton oil, carnauba oil, jojoba oil, marigold oil, sesame oil, soybean oil and cane oil.

The use of the new pharmaceutical composition for the synchronization of spawning in fish composed of sGnRHa, aluminum stearate, mixture of ethyl oleate: sesame oil, vitamin E and benzyl alcohol is also an object of the present invention.

It is also the object of this invention to provide a process for controlling the synchronization of spawning in fish, by administering the composition of the invention, which consists of the steps of: a) Identifying mature females by palpation. b) administering an injectable composition comprising sGnRHa, aluminum stearate, oily mixture of ethyl oleate and sesame oil, vitamin E and benzyl alcohol. c) Identify immature females by palpation for a period of 3 to 4 days until they are identified as mature. d) administer the hormone composition to those females of stage c). e) Wait 6 to 10 days for group spawning of females. f) Desorve, separate and label batches of produced eggs. Another object of this invention is to provide a commercial kit for administering the injectable veterinary pharmaceutical composition of sGnRHa comprising a syringe, a vial or container and a mobile device (cardboard box). Other objects and industrial applications of the present invention will be detailed in the Descriptive Memory shown below. It will be understood that what is detailed in the description and its specific examples, such as the preferred objects of the invention, are described only as a form of illustration, after several others will be part of the detailed description of the invention for those skilled in the art, but in no way limit the scope of this patent.

REFERENCES: 1. Lawrence X. YUX, Todd P. Foster, Ron W. Sarver, and William M. Moseley; Preparation, Characterization, and Vivo Evaluation of an Oil Suspension Of Bovine Growth Hormone releasing Factor Analog.

2. US Patent No. 3,869,549. Geller et al. 3. GB patent number 2,237,571. Millar et al.

4. GB patent number 2,152,324. Gulyas et al.

5. US Patent No. 4,443,368. Sherwood et al.

6. US Patent No. 5,288,705 Zohar et al.

DESCRIPTIVE MEMORY

According to the invention, it was found that as a breeding management tool, it is possible to synchronize spawning of spawning fish in ponds located on land, by administering an oily mixture formulation with extended-release sGnRHa. This administration of the formulation achieved that the spawning was completed during the next 6 days post injection.

The present invention provides an oily composition for the synchronization of fish spawning in ponds, which comprises an effective amount of sGnRHa in an oily mixture of two or more oils as a prolonged release vehicle or matrix.

In the present invention, a commercial product of sGnRHa, analog of the release hormone similar to gonadotropin from salmon, is employed. Whose amino acid sequence is Glp-His-Trp-Ser-Tyr-DArg-Trp-Leu- Pro-NHEt.

The term extended release is understood as a gradual release in time of sGnRHa in the fish.

The term of reproduction management is understood as the induction of ovulation and spawning in the fish, which allows both processes to be arranged synchronously outside the seasons of the year determined naturally.

As mentioned above, the disposition of the GnRH for the control of the reproduction in the aquaculture was of great help for the productive yield. The susceptibility of the native GnRH for its rapid degradation in the blood by the cut in the decapeptide in positions 5-6 and 9-10, led to the development of analogous hormones, with less susceptibility to enzymatic degradation. As these analogs were disclosed in US Patent 4,410,514, which is mentioned herein by reference, a present invention also includes Salmon GnRH analogue as active ingredient of the composition.

One of the treatments for spawning was the application of Ovaprim®, a stable two-component solution: OvaRH®, which is a composition in aqueous saline solution of the analogous hormone of sGNRHa, and domperidone an antidopaminergic agent that allows the hormone to function properly . The hydrophobic nature of the latter made the final composition of Ovaprim® an emulsion, due to the oil component necessary to solubilize domperidone. However, the high viscosity of the resulting emulsion has caused difficulties in the fish injection process. Ovaprim works as a potent ovulant / sperm agent that promotes and facilitates the reproduction of many species of fish, whose ovulation time is highly predictive with high fertility and viability of the eggs. As a result of its application, a peak of maturation hormones is reached from the pituitary that induces the final maturation of gametes through endogenous gonadal steroidal hormones. ■ These steroidal hormones are essential for the final maturation of the gamete, which means that this product does not act interfering with the spawning component or viability of the gamete. Finally, to verify the effects of its application, the fish should be checked 4 to 10 days after treatment. The composition of the invention can be administered as an injection where it preferably comprises between 10 to 100 μg of active ingredient per suspension unit. When the composition according to the invention is administered, it is expected to administer a dose between 10 to 40 μg of sGnRHa per kg of fish weight.

The invention also provides a process for the management of reproduction in fish which comprises administering a sGnRHa composition in an oily mixture of two or more oils, and pharmaceutically acceptable excipients. For each administration of the composition the fish will be anesthetized slightly before injection, according to standard protocols known to the art expert.

In the injection the hormone is found in an oily mixture vehicle, which will be administered in the fish's dorsal sinus, such as salmon.

After administration the fish are kept in properly oxygenated ponds and in controlled environmental conditions, conducive to spawning. The process of administering the composition of the invention obtained results that improved spawning synchrony by 38% for the first 6 days with respect to the group that received the implant (Ovaplant®). This result is surprising and is linked to the type of release of the analogous hormone of the composition of the invention. For the aquaculture productive sector, especially in the salmon breeding industry, this invention will be useful for improving productivity rates. With the same physical space of the fish farming ponds, the number of breeding females in the spawning stage can be increased, as well as their production process.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1. Bar graph showing for Groups A (negative control), Group B (sGnRHa implant), Group C (injectable sGnRHa / sesame oil: ethyl oleate) and Group D (without GnRHa / sesame oil: oleate ethyl) the percentages of survival, post-shocking mortality, and infeasibility of salmon eggs in the females of the study. Figure 2.- Bar graph where it is shown for Groups A

(negative control), Group B (sGnRHa implant), Group C

(injectable sGnRHa / sesame oil: ethyl oleate) and Group D (without GnRHa / sesame oil: ethyl oleate). The parameters measured are: number of abortions, number of deaths without cause, number of deformed and number of edemas of the sack, measured in the fry planted in a fish farm in the South of Chile, coming from each group of the study.

Figure 3. Bar graph showing for Groups A (negative control), Group B (sGnRHa implant), Group C (injectable sGnRHa / sesame oil: ethyl oleate) and Group D (without GnRHa / sesame oil: oleate ethyl), the number of female salmon spawned on day I ^or , day 6 ^or , day 10 and day 13 during the study.

EXAMPLES

EXPERIMENTAL PROCEDURES:

The experimental examples and field studies in fish farming in southern Chile are described below, as an example of the development of an effective tool for the management of reproduction in fish such as salmon, through the administration of the composition of the invention.

The formulation of the invention is an oily suspension with aluminum stearate as a "gelling agent" and / or suspending agent. It is a type of formulation where hydrophilic peptides are adsorbed into hydrophobic particles such as aluminum stearate. Due to the great attraction or affinity between the dispersion medium and the dispersed phase (stearates) these suspensions form spontaneously when the liquid vehicle comes into contact with the solid phase. They are thermodynamically stable and reversible, easily reconstituted. (one)

In the prior art it is disclosed that the ideal ratio between the amount of peptide and the fatty acid salt (aluminum monostearate) as adsorbent is 1: 5 to 1:10. Thus, for example, 0.1 to 0.3 mg of peptide can be adsorbed in 1 mg. of aluminum monostearate. In the case of the invention, a formulation with an improved ratio of 0.15 mg of sGnRHa in 500 mg of aluminum monostearate was developed for each

100 ml of vehicle (sesame oil: ethyl oleate), which while in excess meets another advantageous condition: the proportion of monostearate: oil should preferably be 0.5 to 2% (weight / volume) of aluminum monostearate in oil. So being conservative with the latter we opted for 0.5% aluminum monostearate in the vehicle, making sure we have excess adsorbent and lower the viscosity. (2) We also saw that when using 2% aluminum monostearate, there were variations in the gelation with sesame oil from the same supplier but from different batches, this may have to do with the seasonality of sesame oil, as the concentration decreases From aluminum monostearate, the variation from batch to batch was minimized and the spectrum of possible oil suppliers was expanded.

In the experimental design (DOE) for the development of the composition, the need to incorporate another fatty acid, such as ethyl oleate, was observed in the preparation, this decreases the viscosity and facilitates the application and also allows it not to solidify. low temperatures. Tests of the preparation for 24 hrs at a temperature of 4 ° C it was observed that the preparations with ethyl oleate and 50:50 sesame oil were kept liquid at that temperature. The diffusion capacity of the mixture in fish serum (salmon) was measured, using standard pharmacodynamic methodology.

Preparation of the composition:

In the search for suitable oils for the formulation, sesame oil was identified as a good oily vehicle, however, due to its high viscosity it is widely used in conjunction with reducing agents such as ethyl oleate, which has a low viscosity , about ten times smaller than olive oil. This mixture allows them not to originate nodules decreasing the pain in the fish, after the injection.

Sesame oil can be replaced by other hydrophobic agents as long as the viscosity reducing agent is miscible with the selected hydrophobic agent. The mixture of filtered oil and ethyl oleate is mixed with the aluminum stearate. It is heated to 120 ° C slowly, to form the suspension, then cooled to 40 ° C. The sGnRHa in benzyl alcohol and vitamin E acetate is added, with stirring for 30 minutes. It is packaged in vials. Example 1.- Suspension of the oily mixture of salmon sGnRHa.

Determination of sGnRHa Acetate:

Syntex SA standards were used which is performed by HPLC chromatography, with Luna 5μ C18 column (2): 250 x 4.6 mm; (Phenomenex). Mobile phase: buffer A: 0.1% TFA in linear gradient acetonitrile: 20-40% buffer B in 20 minutes. Temperature 25 ° C, flow rate of 1.2 ml / min. Length at 215 nm, sample volume of 20 L (full loop). Samples are injected into the chromatograph in triplicate. For the preparation of the reference solution A of sGnRHa standard acetate, 7.5 mg is dissolved in Water (purified for

HPLC) for a final volume of 50 ml of concentration 150 mcg / ml. Reference solution B of standard sGnRHa acetate is dissolved 2.5 mg in 1 mL of 0.1M hydrochloric acid and heated in a thermostatic bath at 65 ° C for 4 hours. It is added

1 mL of 0.1M sodium hydroxide and diluted to 5 mL with HPLC quality water. The solution shown with the hormone is extracted from a 50 mL aliquot in a separating vial with 50 mL of acetone pa. It is stirred forming an emulsion, the phases are separated, centrifuged, the precipitate is returned and extracted in the same volume of Acetone and both precipitates are re-suspended in 1% v / v acetic acid. The content of sGnRHa in the samples is determined by HPLC. Field efficiency tests.

To show the efficiency of our composition in spawning control, salmon was used in fish farming, located in southern Chile. The breeders' ponds are 27 m ³ . These aquaculture farms have controlled temperature and photoperiod conditions, the variables of the external environment of the study being controlled. Efficiency in spawning control was compared with a device or implant containing the hormone in pellets. The total population of the study was 100 female Atlantic salmon (Salmo salar), 3 winters, which were in a condition close to spawning and with a similar gonadosomatic index. The fish in the study had an average weight of 12.5 kilos.

The females were marked and separated into four groups, and placed in their respective ponds. Group A: with negative control treatment, Group B: with sGnRHa pellet implant treatment (150 g) (Ovaplant®), Group C: with injectable sGnRHa / sesame oil treatment: ethyl oleate (1: 1) application 150μg / kg fish weight, and Group D: with treatment without GnRHa / sesame oil: ethyl oleate). The fish had an average weight of 12.5 kg comprising a total biomass of 1,252.8 kg of female breeding salmon.

Table 1. Detail of the study fish

The fish were acclimatized for 9 days before treatment. The water flow was supplied at a constant rate to ensure 80% oxygen saturation. The photoperiod was 8 hr light: 16 hr darkness, the fish were not fed. After the acclimatization time, the fish were treated with the hormone. For this, mechanical stress was applied to each pond and then a bath with anesthesia, supplemented with diffusion oxygen. The maturation status of females was examined manually by palpation, ensuring that there was no regression of the maturation by previous mechanical stress. The fish were heavy. Both the group marked for treatment with the Ovaplant® implant and the group for treatment with the oily sGnRHa injection were placed in the dorsal sinus behind the dorsal fin of the female salmon. Intramuscular implants were administered in the dorsal sinus behind the dorsal fin, one per fish, while the injectable composition was administered at a dose of 15μg / kg of mass.

The fish were returned to their ponds for recovery and were not disturbed. After 4 days, the fish were removed from the ponds, sedated with low doses of isoeugenol and examined by palpation to measure gamete expression by a qualified technician, who was not aware of the type of treatment received in each group. The ovulation of the fish was noted for each treatment. When fish ovulation is detected, it is expected 6 to 10 days for spawning. Egg volume was measured and weighed without ovarian fluid. The females were weighed before and after spawning. The occurrence of standard diseases was monitored. Fertilization was completed for fish of all treatments.

The eggs were fertilized and incubated according to standard culture standards and evaluated for viability according to the eye phase and the percentage of hatched.

This ovulation and spawning process was repeated every four days until 95% of the females had spawned, and the study was considered completed. In each spawning the total number of eggs was determined, which was estimated by direct counting and observation. The eggs were subjected to shocking (action of dropping the eggs with water from one container to another) at a height of between 0.5 to 1 mt in order to determine the percentage of survival, mortality and infeasibility of the post eggs shocking These determinations were made by calculating the eggs corresponding to each group, multiplying ^'value by 100 and then dividing by the total number of eggs.

The percentage of each determination is detailed in Table No. 2.

Table 2. POST INVIABILITY SAMPLING

SHOCKING

The results are shown in Figure 1, Figure 2 and Figure 3, in the annexes.

Table 3. SHEEPED SEEDS IN PSICULTURE

Table 3 shows the causes of infeasibility of the eggs for the four study groups. Table 4 shows the field results obtained for the viability of post fertilization eggs for the four study groups. The treatment with the injectable composition of the invention shows that there are no significant differences compared to the other groups, so this did not affect the functionality of the eggs.

Table 4. VIABILITY OF POST SHEEP

FERTILIZATION

Table 5. Number of females that spawned in each group.

Table 5 shows the number of females that spawned after certain days after treatment. The distance from the implant to the spawning of the fish of Group B (Ovaplant®) and Group C (injectable with sGnRHa / sesame oil: ethyl oleate) It was only 10 days, spawning in just two pulses. Groups A and D still maintained females that had not spawned yet on day 11.

The safety and efficacy of the composition of sGnRHa in an oily mixture of sesame oil: ethyl oleate, in a 1: 1 ratio, the advantages over the single-dose implant (Ovaplant®) and its industrial applicability in the control of Ovulation synchronization and spawning in salmon.

Claims

1.- An injectable veterinary composition for the synchronization of spawning in fish with the gonadotropin releasing hormone similar to CHARACTERIZED salmon, because it comprises a vehicle that is an oily mixture with one more fatty acids, vegetable oils, and pharmaceutically acceptable excipients.

2. - A composition according to claim 1 CHARACTERIZED, because the oily mixture of fatty acid ester and vegetable oil are in a ratio of 1: 3 to 3: 1.

3. - A composition according to claim 1 CHARACTERIZED, because the oily mixture of fatty acid ester and vegetable oil are in a ratio of 1: 2 to 2: 1.

4. - A composition according to claim 1 CHARACTERIZED, because the oily mixture of fatty acid ester and vegetable oil are in a ratio of 1: 1.

5. - A composition according to claim 1 CHARACTERIZED, in that the fatty acid ester mixture is selected from triglycerides, derived from triglyceride, ethyl propionate, propylene glycol monoacetate and ethyl oleate.

6. - A composition according to claim 5 CHARACTERIZED, because in the mixture the fatty acid ester is ethyl oleate.

7. - A composition according to claim 1 CHARACTERIZED, because in the oily mixture the vegetable oil is selected from cotton oil, carnauba oil, jojoba oil, marigold oil, sesame oil, soybean oil and He drew it.

8. - A composition according to claim 7 CHARACTERIZED, because in the oily mixture the vegetable oil is sesame oil.

9. - A composition according to claim 1

CHARACTERIZED, because it contains a ratio of 0.15 mg of gonadotropin-releasing hormone similar to salmon per 500 mg of aluminum monostearate per 100 ml of vehicle of the composition (sesame oil: ethyl oleate in 1: 1).

10. - A composition according to claim 1 CHARACTERIZED, because in addition the oil mixture comprises pharmaceutically acceptable excipients such as aluminum stearate, vitamin E acetate and benzyl alcohol.

11. A composition according to claim 1 CHARACTERIZED, because a dose of 15] ig of the analogous gonadotropin release hormone is applied per kg of fish weight.

12. - A process to control the synchronization of spawning in fish with a composition containing the gonadotropin releasing hormone similar to CHARACTERIZED salmon, because it comprises the steps of: a) Identifying mature females by palpation b) administering an injectable composition comprising sGnRHa, aluminum stearate, oily mixture of ethyl oleate and sesame oil, vitamin E and benzyl alcohol, c) Identify immature females by palpation for a period of 3 to 4 days until they are identified as mature d) administer the composition with hormone to those females of stage c), e) Wait 6 to 10 days for group spawning of females. f) Desorve, separate and label the batches of produced grapes.

13. - The use of the composition claimed in 1 CHARACTERIZED, because said oil mixture composition synchronizes the spawning of salmon in captivity.