CS265943B1 - Method of obtaining eggs of grayling (Thymallus thymallus L.) - Google Patents

Abstract

The method of obtaining eggs during artificial spawning of grayling (Thymallus thymallus L.) is carried out in such a way that at the time of pre-spawning maturity, the eggs are administered intramuscularly with a synthetically prepared peptide - an analogue of salmon LH-RH p-Glu-His- -Trp-Ser-Tyr-D-Tle-Trp-Leu-Pro-GlyNH2 in doses of 50 to 150 µg/kg or an analogue of porcine LH-RH pGlu-His-Trp-Ser-Tyr-D-Ala-Leu-Arg- -ProNHEt in doses of 10 to 40 µg/kg of live weight of fish. The peptide is administered to egg-laying fish kept in unheated water, under natural conditions, at a temperature of 2 to 12 °C. The administration of these substances not only increases the percentage of successful ovulation, but at lower temperatures significantly reduces the time necessary to achieve ovulation. Compared to carp pituitary, very small doses of an almost pure substance are used, and therefore the incidence of complications, unlike pituitary, where administration often ends in death or reproductive disorders, is minimal.

Description

The invention relates to a process for obtaining the eggs of the grayling (Thymallus thymallus L.) using synthetic analogues of natural gonadoliberins.

While there are already a number of communications on the use of certain synthetic analogues in economically interesting fish species, either alone or in combination with justified carp pituitary glands,. obtaining eggs of sport fish, wild and caught by electric generators, is not easy and is successful only with a small part of the fish caught. Beneficial performance of artificial spawning of linseed and some other economically interesting fish species by administration of luteinizing hormone release factor analogue (hereinafter LH RH) (AO 214 366, AO 244 397), as well as preparation of salmon derivatives LH-RH (Czech author's certificate no. 254,850) led to a project in which the conditions for successful spawning of grayling grayling were found, under conditions close to natural, i.e. low temperatures and little sunshine (short day).

The essence of the method for obtaining the eggs of the grayling (Thymallus thymallus L.) according to the invention is that at the time of pre-eradic maturity the intramuscular synthetic peptide luteinizing hormone releasing factor (LH RH) analogue of the general formula p-Glu-His-Trp-Ser-Tyr- x (I), where X is bud

D-Tle-Trp-Leu-Pro-GlyNH ₂ or

D-Ala-Leu-Arg-ProNHE _t .

The method for obtaining eggs is carried out by administering the peptides of the general formula I to submarine grayling eggs reared in unheated water at a temperature of 2 to 12 ° C, for analogues of the formula p-Glu-His-Trp-Ser-Tyr-D-Tle- Trp-Leu-Pro-GlyNHj (II) in doses from 50 to 150 ug / kg body weight of fish and in analogues of the formula p-Glu-His-Trp-Ser-Tyr-D-Ala-Leu-Arg-ProNHEt (III) in doses from 10 to 40 μg / kg body weight of fish.

Administration of these substances not only increases the percentage of successful ovulation, but also at lower temperatures significantly reduces the time required to achieve ovulation.

The following are examples of the process for obtaining grayling eggs according to the invention.

In the individual examples, eggs harvested by an electric generator in the autumn of the previous year in rivers and chambered on hatchery buildings in ponds were used (Examples 1, 2, 4 to 5). In two cases (Examples 3, 6), grayling eelgrasses drained by an electric aggregate were used in spawning grounds in the spring before spawning.

In all cases, the weight was determined individually for each fish before injection with an accuracy of +5 g.

The peptide of formula II derived from salmon LH-RH was injected in saline. The peptide concentration was always chosen at each dose to inject the fish with a volume of 1.0 ml / kg body weight. In two experiments, the dose range of the analog was 1.0 to 125 μg / kg (1, 5, 25 and 125 μg). In selected cases, a combination of 25 μg, analog / kg and 5 mg pituitary (extract / kg) and pituitary alone 5 mg / kg was injected. In Example 3, peptide doses in the narrower range, i.e. between 25 and 125 .mu.g, were used.

Peptide III was administered (Example 4 and 5) at a dose of 25 _/ ug / kg, respectively. 10 to 10 .mu.g / kg (Example 6). The detail of the design is then evident from the description in individual cases. The results are shown in Tables 1 to 6.

He added.

Eggs of grayling (Hynčice farm - April) weighing about 200 to 250 g were divided into groups, which were placed in laminate troughs (water temperature during the experiment 4 to 8 ° C) with a volume of 400 l, separately fed with surface water. The water temperature was monitored 2-3 times a day, the peptide was injected intramuscularly. During the period of expected ovulation, the fish were inspected and, in the case of ovulation achieved, artificially wiped. The length of time from injection to swab is given in both days and daily degrees (d ° C). The results are summarized in Table 1, which presents the results of the application of 4 different doses of the peptide of formula II, the peptide in combination with pituitary carp and pituitary carp alone and saline (control).

The results show that 100% successful swabs at 4-8 ° C were achieved with the administration of 125 .mu.g, the compound of formula II, as well as the dose of the pituitary gland alone, respectively. pituitary gland in combination with a peptide. In the case of administration of the peptide alone or the peptide with the pituitary gland, there was also a significant reduction in the time required for a successful swab, from 9 days to 7.2, respectively. 5.2 days.

Example 2

Technically, the experiments were performed as described in Example 1. The eggs were weighed 180 to 220 g, the experiment was performed on the Príbovce farm at a water temperature of between 2 and 9 ° C. The results are shown in Table 2, where success is captured in both daily degrees and relative fertility.

The results show that more than 50% (57%) of the swab was achieved when 125 [mu] g of peptide alone was applied. in combination with the pituitary gland (64%). The highest pituitary swabs were obtained on this farm, but autopsy of the untreated fish showed that the juvenile fish were included in the groups tested for peptide effects, which did not achieve the expected result. The application of the peptide itself leads, as in the case, to a significant reduction in the time required for a successful swab, from 11 days to 8 days.

Example 3

The experiments were carried out analogously to Example 1. The fish ranged from 130 to 320 g, the water temperature was between 6 and 9 ° C. The fish were placed in metal halters, submerged at the bottom of a concrete pond with a strong flow of water. The results are summarized in Table III.

As in Examples 1 and 2, the application of the peptide (already 25 .mu.g / kg) of the pituitary peptide and the pituitary itself was successful. In comparison with the control, resp. application of the pituitary gland alone did not significantly reduce the time required for swabbing after peptide administration at this temperature.

Example 4

The experiments were performed as in Example 1 (Hynčice farm). The eggs received a single dose of porcine LH-RH analog (25 .mu.g / kg) and were kept at 4-8 ° C. The pituitary gland was infected either alone or in combination with the peptide. The results are shown in Table 4.

In the given example, the applied peptide was already successful at a dose of 25 μg / kg. Even in this case, administration alone or in combination with the pituitary gland significantly shortened the expected swab time, compared to the pituitary gland alone in a single administration by up to two days. (One eggworm included in the control group spontaneously ovulated after 5 days).

Example 5

Experiments were performed on the effects of the peptide alone, the pituitary gland alone, and a combination of both. The results are summarized in Table 5.

The results of the donor rui f 61 <»Γ-όιγιο r. success (comparable to pituitary administration), thus shortening the time required to achieve ovulation.

Example 6

The experiments were performed on a farm in Kaplice. The weight of the eggs ranged from 150 g to 260 g. In this experimental setup, the effects of different four doses of peptide were tested.

The water temperature ranged from 11.2 to 1.3 ° C.

The results are summarized in Table 6.

A dose-response study of the peptide showed a dose efficacy of 5 to 40 [mu] g of peptide (temperature 11.2 to 13 [deg.] C.), the swab success being increased by about 100% compared with the control.

At this temperature, compared to the control, the time required to achieve ovulation was not reduced.

Overview of tables 1 to 6

Table 1- Results of an experiment with induced artificial spawning of grayling eggs at a fish hatchery in Hynčice using substance II and pituitary carp at a water temperature of 4 to 8 ° C.

Table 2 - Results of an experiment with induced artificial spawning of grayling hatcheries in a fish hatchery in Príbovce using substance II and pituitary carp at a water temperature of 2 to 9 ° C.

Table 3- Results of an experiment with induced artificial spawning of grayling eggs at a fish hatchery in Hynčice using substance II and pituitary carp at a water temperature of 6 to 9 ° C.

Table 4 - Results of an experiment with induced artificial spawning of grayling eggs at a fish hatchery in Hynčice using substance III and pituitary carp at a water temperature of 4 to 8 ° C.

Table 5- Results of the experiment with induced artificial spawning of grayling in Příbovce using substance III and pituitary carp at a temperature of 2.3 to 9.2 ° C.

Table 6- Results of an experiment with induced artificial spawning of grayling eggs in a fish hatchery in Kaplice using substance III at a water temperature of 11.2 to 13 ° C.

Results of an experiment with induced artificial spawning of grayling eggs at the fish hatchery in Hynčice using a substance of formula II of the pituitary gland at a water temperature of 4 to 8 ° C

Weight Number of eggs _____ Length of time interval eggs in the experiment wiped from injection to swab - + s pcs pcs% - + s - + s __d _____________ d ° ________ 208 - 89 5 ________ 2 ______ 40 ______ 8.0 - 4.2 _______ 44.7 - 20, S 230 - 74 5 2 40 9.0 - 0.0 46.7 ± 0.0 232 - 72 5 2 40 9.0 ± 0.0 46.7 t 0.0 224 -87 55 100 7.2 - 2.5 43.5 ± 7.4

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OBJECT OF THE INVENTION

Claims (2)

11 OBJECT OF THE INVENTION A method for obtaining eggs in the case of artificial spawning of the grayling (Thymallus thymallus L.), characterized in that at the time of pre-spawning maturity, an intramuscular synthetic peptide - luteinizing hormone releasing factor (LHRH) analogue of the general formula is administered to the eggs. P ~ <; 1 u-Hl n-Trp-Sor-Tyr-x (I), where X is bud. D-Tle-Trp-Leu-Pro-GlyNH ₂ , or D-Ala-Leu-Arg-ProNH.Et. 2. A process for obtaining eggs in the artificial spawning of grayling according to claim 1, characterized in that the peptides of general formula I are administered to grayling eggs grown in unheated water at a temperature of 2 to 12 ° C for analogs of formula 'p-Glu-His -Trp-Ser-Tyr-D-Tle-Trp-Leu-Pro-GlyNH ₂ (II) in doses from 50 to 159 ug / kg body weight of fish and in analogs of the formula p-Glu-His-Trp-Ser-Tyr- D-Ala-Leu-Arg-ProNHEt (III) at doses from 10 to 40 ug / kg body weight of fish.