Payette for artificial insemination of cattle with capacitation activator

Abstract

This utility model relates to veterinary reproductive medicine. The method involves using a straw containing sperm and a heparin solution, separated by an air bubble. Heparin at a concentration of 50 μg / ml activates sperm capacitation immediately at the moment of insemination. Insemination is performed 18-21 hours after detection of estrus. The method expands the time window available for insemination.

Description

[0001] The utility model relates to the field of veterinary biotechnology and reproductive science, namely to devices for increasing the efficiency of artificial insemination of cattle, and can be used on a permanent basis in breeding farms.

[0002] It is known that standard artificial insemination protocols for cattle involve semen injection 10-12 hours after detection of heat. However, individual differences in the timing of ovulation, as well as decreased sperm viability after thawing, and human factors lead to insemination timing not matching the optimal fertile window, resulting in lower conception rates.

[0003] Scientific and patent sources indicate that one of the key factors determining the fertilizing ability of bovine sperm is the process of capacitation. Heparin has been established as one of the most effective and reproducible agents for inducing capacitation of bull sperm. Fundamental studies (Parrish JJ et al. Capacitation of Bovine Sperm by Heparin1 / / Biology of Reproduction. 1988. Vol. 38. No. no. 5. Pp. pp. 1171-1180.) have shown that the addition of heparin to bull sperm in vitro leads to a sharp increase in the proportion of sperm capable of fertilizing oocytes.

[0004] The use of heparin as a standard capacitating agent is reflected in a number of foreign patents. For example, patent US8,211,629B2 uses FCDM (a "defined fertilization and capacitation medium") containing 0.02% heparin to prepare bull sperm for fertilization in vitro, which is explicitly stated as a capacitation induction step prior to fertilization. A similar approach is described in patent US8,486,618 B2, where heparin is used to prepare sperm for fertilization in heterogeneous insemination systems. Earlier patents (US6,524,860B1; US6,149,867) consider heparin to be a recognized standard for capacitating bull sperm, with the applicants explicitly citing experimental data confirming its effectiveness.

[0005] Russian patent sources also support this concept. Patent RU2620004C1 ("Method for Increasing the Survival Rate of Bull Sperm During Cryopreservation") describes a method for increasing the survival rate of bull sperm during cryopreservation, in which the sperm are maintained in a medium containing capacitation inducers, including heparin, before freezing. Patent RU2639268C2 ("Method for Capacitating Cryopreserved Bovine Sperm for In Vitro Fertilization of Eggs") lists heparin as the primary agent used for capacitating cryopreserved bovine sperm in vitro. In more recent patents devoted to the production of bovine embryos in vitro (RU2818068C1), heparin is included in the composition of the media used at the stages of sperm preparation, which further confirms its widespread and recognized use in reproductive biotechnology.

[0006] Thus, the prior art confirms that heparin is an effective and widely accepted agent for capacitating bovine spermatozoa. However, in these examples, heparin is typically added to the medium beforehand, leading to premature sperm activation and limiting the window of time for their effective use.

[0007] The technical result of the claimed utility model is an increase in the efficiency of insemination due to the controlled, delayed activation of spermatozoa directly in the female's reproductive tract, as well as an extension of the permissible time interval between the detection of sexual heat and the moment of insemination, which is due to the design features of the straw.

[0008] Diagram of a straw (in Fig. 1) with separation of sperm and heparin solution by an air bubble to prevent premature mixing (1 - straw body; 2 - cotton wad; 3 - heparin solution; 4 - air bubble between the heparin solution and the bull sperm suspension; 5 - bull sperm suspension; 6 - air bubble between the bull sperm suspension and the sealed edge of the straw; 7 - sealed edge of the straw).

[0009] The technical result is achieved by using the above-mentioned straw for artificial insemination of cattle (Fig. 1). The body (1) is made of polypropylene, a synthetic material with transparency for monitoring the contents inside the straw. Volume: 0.25 ml, length: ~133 mm, external diameter: 2.83 mm. The straw body is sealed with a cotton wad (2), occupying 33 mm. Unlike a standard straw, which is filled only with a suspension of bull sperm, in the proposed utility model, a heparin solution (3) at a concentration of 50 μg / ml is added first. To compensate for compression during freezing, when filling the straw, it is separated from the bull sperm suspension (5) by an air bubble (4) (complies with GOST 26030-2015 dated 01.07.2016 "Reproduction means: frozen bull sperm"). An air bubble (6) is placed after the sperm sample (5). After filling, the straw is sealed (7).Separating the sperm and heparin solution into separate segments prevents their premature mixing during freezing and thawing. The dose contains at least 15 million sperm with linear forward motility. The straw, frozen in liquid nitrogen, is thawed in a water bath at 36-37°C for at least 40 seconds and inserted into the uterine cavity. When the straw contents are expelled into the female's reproductive tract, the sperm and heparin solution mix, ensuring early activation of the sperm, allowing insemination to be performed closer to ovulation. Insemination can be performed 18-21 hours after detection of estrus, allowing for later insemination if the standard insemination time is missed without significantly reducing the effectiveness of the process.

[0010] Example of the method implementation

[0011] To determine the feasibility of the proposed method for improving artificial insemination efficiency, we studied a herd of 30 Holstein cows in their third lactation in the Leningrad Region. For the experiment, the herd was divided into two equal groups. The first half was inseminated using the standard technique—10-12 hours after the onset of estrus with thawed semen. The second half was inseminated 20 hours after the onset of estrus using the proposed straw with added heparin. The 0.25 ml straws contained 15 million active sperm and a heparin solution with a concentration of 50 μg / ml; the solutions were separated by an air bubble. The straws were thawed at 36-37°C without shaking.

[0012] The method resulted in a slight increase in pregnancy rates in the experimental group compared to standard insemination, as well as the preservation of sperm motility and morphological integrity after thawing. Furthermore, delayed insemination times did not affect the effectiveness of heparin, which was separated from the semen until insemination of cattle.

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