RU2297197C2 - Method for increasing fertilizing capacity of ram's sperm - Google Patents

Abstract

FIELD: animal science.

SUBSTANCE: the medium for sperm cryoconservation should be supplemented with FLPG preparation - the complex of synthetic analogs of prostaglandins F_2α, E, E₁, E₂ deposited in vegetable oil at concentration being 125 mcg/ml diluted sperm. The innovation enables to increase fertilizing capacity of ram's sperm, stimulates reproductive function in ewes due to positive impact onto ovicells and spermium movement to the site of fertilization.

EFFECT: higher efficiency.

2 ex, 3 tbl

Description

The invention relates to the field of animal husbandry and can be used for artificial insemination of sheep to improve their reproductive function - to increase coagulation and multiple fertility.

Currently, for artificial insemination of ewes, freshly harvested (native), chilled or cryopreserved sperm of sheep is used. In the last two cases, in the process of processing the native sperm is diluted 3-4 times with cryoprotective media. Consequently, the content of biologically active components, for example, such as prostaglandins (PG), which regulate the reproduction processes in females, in one sperm dose (0.2 ml) will become 15-20 times less than in one ram ejaculate.

In total, 20 species of prostaglandins were found, of which 13 species were found in the seminal fluid of rams (about 100 μg / ml.)

From the above it follows that one of the reasons for the lower efficiency of artificial insemination of ewes with cryopreserved sperm is the low content of prostaglandins (5-6 μg) in one sperm dose. This has a negative effect on the progress of sperm to the place of fertilization.

To increase the effectiveness of artificial insemination of ewes with cryopreserved sperm, the drug Enzaprost-25 is used (4). Enzaprost-25 is a synthetic luteolytic drug, an analogue of prostaglandin F _2α , in which dinoprost is the active principle:

(as sodium salt) 25.0 mg in 5 ml of solution (1). The drug is approved by the Main Veterinary Directorate for use in veterinary practice to induce estrus and calving in cows (heifers), to synchronize estrus in ewes, as well as to induce and synchronize farrowing in sows.

However, enzaprost cannot contribute to the full realization of the reproductive ability of ewes when inseminated with cryopreserved sperm, since it contains only one type of prostaglandin - F _2α . In addition, the active principle of PG F _{2α is} rapidly dissolved and excreted from the animal.

The aim of the invention is to increase the safety of the biological usefulness of ram sperm outside the body after dilution, cryopreservation and defrosting, which ensures an increase in the percentage of coagulation and multiple pregnancy of ewes during artificial insemination.

The goal is achieved by enriching the synthetic medium with a biologically active substance of prolonged action containing several types of prostaglandins.

The essence of the method consists in the use of a biologically active preparation FLPG, which contains several types of synthetic analogues of prostaglandins:

The advantage of the present invention, in comparison with the prototype, is that the preparation FLPG more effectively increases the fertilizing ability of cryopreserved sperm of sheep and stimulates the reproductive function of ewes. Due to the fact that the proposed drug includes not only one PGF _2α , but also PGE, PGE ₂ and their derivatives, it has a wider spectrum of physiological effects not only on sperm, but also on the body of ewes, having a positive effect on the promotion of the egg and sperm to the place of fertilization.

The method is as follows. For cryopreservation of sperm, GLC-Tris-buffer medium with an oil solution of FLPG preparation with a calculated activity of 125 μg per 1 ml of diluted semen was used. We prepare the medium according to the “Manual on the use of GZHUK-Tris-buffer medium for dilution, freezing, storage and use of sperm of ram-producers” (3).

According to the recipe, lactose, dextrin, xylitol, tris- (oxymethyl) aminomethane, trilon-B are added to a clean chemical flask. The components are thoroughly mixed and poured with boiled distilled water (80-90 ° C) in the required volume. To dissolve the components, the flask is tightly closed with foil or plastic film, placed in a water bath and at a temperature of 100 ° C, constantly shaken until the components are completely dissolved, then sterilized by boiling for 30 minutes. Then the solution is cooled to 40 ° C. Considering the fact that vegetable (refined) oil strongly emulsifies, the FLPG preparation is not administered into the finished medium, but into the yolk (one of its components), which, after thorough mixing, is introduced into the solution. After dilution, a sanitizing preparation and glycerin are introduced, as indicated in the instructions for use of GHUK-Tris buffer medium.

The prepared medium is used to dilute sperm within 4 hours from the time of preparation. Store at a temperature of 4 ° C, heated to 28-30 ° C before use. The optimal concentration of the experimental preparation FLPG in the composition of GHUK-Tris-buffer medium was established in laboratory experiments conducted at the All-Russian Research Institute of Breeding. Scientific and production experiments to study the effectiveness of the FLPG preparation on the fertilizing ability of cryopreserved sperm were carried out in the sheep-breeding special farm "Country of Soviets" in the Belgorod region, Belgorod region.

Example 1. Sperm for research was obtained from rams of the breed Prekos using an artificial vagina. After taking each ejaculate, its volume and a number of qualitative indicators were determined (sperm activity in points, concentration in billion / ml, total number of sperm in the ejaculate in billion). Sperm dilution was carried out according to instructions 3-4 times. In the experiments we used ejaculates that met the requirements of instruction.

To study the effect of the drug FLPG (a complex of prostaglandins F _2α , E, E ₁ and E ₂ ) on the preservation of the biological usefulness of ram spermatozoa outside the body after its cryopreservation, the following substance concentrations were used: 250 μg, 125 μg and 60 μg per 1 ml diluted sperm. To obtain such concentrations in the first experimental group, 0.75 ml of medium with 250 μg of the drug was added to 0.25 ml of native sperm, in the second group 0.75 ml of medium with 125 μg of the drug was added to 0.25 ml of sperm, in the third group - to 0.25 ml of semen was also added 0.75 ml of medium with 60 μg of the drug. The fourth group, used as a prototype, contained 0.25 ml of sperm and 0.75 ml of medium with 125 μg F _2α PG (enzaprost). In the control group, native ram sperm was diluted with GHUK-Tris buffer medium without PG preparations.

The diluted sperm of the rams was packaged in numbered vials and, together with the control sample, was equilibrated (cooled) for 2 hours at a temperature of 2-4 ° C. Next, sperm were frozen on a fluoroplastic plate in pairs of liquid nitrogen, in 0.2 ml wells, according to the method of VNIIplem (2). For biological control, experimental samples of frozen sperm were thawed in a 3% solution of sodium citrate at 42 ° C, then sperm motility was determined every hour until they died. The absolute survival rate (Sa of spermatozoa) in the experimental groups was determined at an incubation temperature of 38 ° C. The experiments were carried out in triplicate.

The optimal concentration of prostaglandins in diluted ram sperm was determined after defrosting by the best indicators of sperm motility and survival (Table 1).

Table 1
The effect of FLPG on sperm motility after cryopreservation and sperm defrosting Experience number Sperm motility in points at a concentration of GHG FLPG in μg / ml (experiment) Enzaprost (prototype) 250 125 60 0 (control) 125 I 4.5 ± 0.10 4,5 ± 0,17 4.5 ± 0.10 4.5 ± 0.10 4.5 ± 0.10 II 4.0 ± 0.13 4.4 ± 0.08 4.4 ± 0.07 4.4 ± 0.07 4.3 ± 0.08 III 4.2 ± 0.08 4.3 ± 0.08 4.1 ± 0.07 4.0 ± 0.08 4.0 ± 0.10 On average 4.2 ± 0.15 4.4 ± 0.06 4.3 ± 0.12 4.3 ± 0.15 4.2 ± 0.12

Studies have shown that with the inclusion of exogenous prostaglandins in the medium, sperm activity did not change significantly. Higher results after defrosting of cryopreserved sperm were obtained in the second group, with a content of 125 μg of FLPG preparation in 1 ml of diluted semen - mobility 4.4 points. This is 2.3% higher than in the control group, and 4.8% higher than the similar indicator of the prototype.

The absolute indicator of sperm survivability in experimental samples of defrosted sperm is shown in Table 2.

table 2
The effect of FLPG on the survivability of ram sperm after defrosting No. of experience Sperm vitality (in conventional units) at a concentration of PG FLPG in μg / ml (experiment) Enzaprost (prototype) 250 125 60 0 (control) 125 one 14.3 ± 2.10 24.7 ± 0.58 12.6 ± 2.06 15.4 ± 1.92 16.0 ± 2.03 II 17.2 ± 2.15 19.6 ± 1.94 18.9 ± 1.04 18.8 ± 2.40 19.2 ± 1.70 III 18.3 ± 1.58 14.4 ± 1.47 21.1 ± 1.50 17.4 ± 1.83 17.6 ± 1.60 On average 16.6 ± 1.19 19.6 ± 2.80 17.5 ± 1.50 17.2 ± 0.99 17.5 ± 1.80

From table 2 it follows that at a concentration of FLPG 125 μg / ml in the medium, sperm survivability was 14% higher compared with the control, and 10% higher than the prototype (125 μg / ml enzaprost). The obtained difference in the survivability indices of the experimental and control groups (14% and 10%) is statistically significant in the first and second cases. When using other concentrations of the drug (250 μg / ml and 60 μg / ml), the sperm vitality of the experimental and control groups did not differ significantly.

Thus, the studies showed that the prostaglandin complex (F _2α , E, E ₁ , E ₂ ) as part of the FLPG preparation proved to be better in GZHUK-Tris buffer medium than enzaprost, having a positive effect on the preservation of the biological usefulness of ram spermatozoa organism after cryopreservation and defrostation: by mobility by 4.8%, and by absolute survival rate - by 14%.

Example 2. The effect of FLPG on the percentage of sheep ejaculation and multiple pregnancy was studied in a scientific production experiment only at a concentration of 125 μg / ml. The reason for this decision was the fact that at this concentration of the drug in the composition of GLC-Tris-buffer medium, the highest quality indicators of defrosted ram sperm were obtained. This has been identified as the best way to increase the fertilizing ability of ram cryopreserved sperm and sheep eutrophy.

The results of these studies are presented in table.3.

Table 3
The effect of the introduction of FLPG in diluted semen of sheep before its cryopreservation on the results of artificial insemination of ewes Dose of the drug Artificial Insemination Results Inseminated
ewes Lounged Received lambs per 100 queens goals % Goal % Without GHG (control) 185 85 46.2 ± 3.6 122 100 ± 0.7 FLPG, 125 (experience) 150 88 58.8 ± 4.0 138 113 ± 2.9 Enzaprost 125 (prototype) 145 73 50.7 ± 4.1 127 104 ± 1.4

The data obtained (Table 3) show that the fertility of ewes in the experimental group, where the optimal dose of FLPG (125 μg / ml) was used, was higher than in the control group by 12.6%, and multiple pregnancy - by 16 animals (138 lambs per 100 ewes versus 122 goals in the control group). The difference obtained in favor of experience is statistically significant at a probability level of P <0.02.

Compared with the results obtained using enzaprost (prototype), in the experimental group, the fertility of ewes was also higher by 8.1%, and fertility by 11 goals (per 100 queens). The difference in favor of experience is statistically significant at a probability of P = 0.1.

Thus, the introduction of FLPG (a complex of prostaglandins F _2α , E, E ₁ , E ₂ and their derivatives) into a Gluc-Tris buffer medium at a concentration of 120 μg / ml immediately before sperm cryopreservation can significantly improve the biological usefulness of sperm after defrostation (absolute survival rate Sa + 14%), as well as increase the fertility of ewes by 12.6%, their multiple fertility - by 16 goals (for every 100 queens) compared with the control (medium without PG). Compared with the prototype (enzaprost medium, 125 μg / ml), the efficiency of use was higher by 8.1% and 11 goals for every 100 queens, respectively. The difference obtained in favor of experience is statistically significant in both cases.

Statistical processing of research results to determine the nature of the dependence of the fertility of ewes on the number of prostaglandins in the used sperm dose revealed a direct reliable relationship between these indicators, the correlation coefficient was r = 0.854.

Information sources

1. Manual on the use of enzaprost. Quinoin, Pharmaceutical and Chemical Enterprise LTD. Budapest, Hungary.

2. Prostaglandins. Big medical encyclopedia. Moscow, 1985.

3. Guidance on the use of GZHUK-Tris-buffer medium for dilution, freezing, storage and use of sperm of ram-producers. VNII tribe. Forest Glades, 1992.

4. Klinsky Yu.D., Pavlov V.A., Bulynin V.A. The use of prostaglandins to regulate reproductive function in sheep. Livestock. 1980. No4.

Claims (1)

A method of increasing the fertilizing ability of ram sperm, including the introduction of a biologically active substance (BAS) into the environment for cryopreservation of ram sperm, characterized in that FLPG (a complex of synthetic analogues of prostaglandins F _2α , E, E ₁ , E ₂ ) is deposited in vegetable oil, at a concentration of 125 μg per 1 ml of diluted semen of sheep.