WO2011072354A1

WO2011072354A1 - Circuit for collecting goat and sheep embryos transcervically

Info

Publication number: WO2011072354A1
Application number: PCT/BR2010/000415
Authority: WO
Inventors: Jeferson Ferreira Da Fonseca; José Henrique BRUSCHI; Paulo Mangus Cabral De Barros
Original assignee: Empresa Brasileira De Pesquisa Agropecuaria - Embrapa
Priority date: 2009-12-17
Filing date: 2010-12-15
Publication date: 2011-06-23
Also published as: BRPI0905581B1; BRPI0905581A2; WO2011072354A4

Abstract

The present invention relates to a system for collecting embryos transcervically, developed specifically for small ruminants. More particularly, said system is provided with devices that prevent contact with the outside while the method is carried out, protecting the embryos and the (embryo donor) animals from outside contamination, and with a device having a double flow pathway which allows the direction of uterine infusion to be reversed with a simple touch. Additionally, the present invention also relates to a method for collecting embryos.

Description

"TRANSCERVICAL EMBRYO COLLECTION CIRCUIT IN GOATS AND EGGS".

FIELD OF INVENTION

The present invention relates to a transcervical embryo collection system developed specifically for small ruminant animals. More particularly, said system is provided with devices that prevent contact with the external environment during process manipulation, protecting embryos and animals (embryo donors) from environmental contamination. Additionally, the present invention further relates to an embryo collection process.

BACKGROUND OF THE INVENTION

Currently, the transfer of embryos in small ruminants mostly uses materials developed for other species, especially cattle and humans. Goat / Sheep agribusiness has been growing worldwide. To meet increasing demand, the exchange of superior genetic material has been warming and reaching surprisingly high and rising numbers year after year. The collection, preservation and exchange of male genetic material (i.e. semen) is relatively efficient and dominated in small ruminants. However, in females, this reality is quite different.

The bovine species has been the subject of intense research in recent decades. During this period, among other advances, embryo collection and transfer techniques underwent intense changes from totally surgical to totally non-surgical. The advantages of non-surgical procedures are obvious, including animal welfare, decreased life-threatening and surgical sequelae such as hernias and adhesions, among others.

Embryo collection in goats and sheep is predominantly performed surgically and laparoscopically with the animals undergoing general anesthesia. Despite being safe and precise techniques, both the surgical and the laparoscopic form present all the risks and sequelae inherent to the surgical processes involving the exploration of organs. abdominal organs, mainly adhesions. However, the surgical procedure is not routinely repeated more than three times, since this seems to be the limit of surgical collections, when surgical sequelae, such as adhesions, make new collections unfeasible.

In the past decade, the non-surgical transcervical collection procedure has been proven to be effective in goats and sheep and is currently the most widely used method in Brazil. Recently, data from the literature showed that by using misoprostol 200pg (PGET) for embryonic collection by transcervical surgical technique, 95% success was achieved in Dorper ewes and 63% in Santa Inês ewes. Other data show that administration of cloprostenol 50pg 12 hours prior to embryonic collection resulted in a 59% success. The procedure can be performed with the animal in season. Epidural and local anesthesia (cervix), as well as mild sedation, minimize animal discomfort, facilitating the process.

Salles, H. (Rev. Ruralnet; Technical Articles CNPC (http: // www. Cnpc. Zembrapa.br/artigo11.htm, 2001) describes the collection of embryos in goats by transcervical route through a circuit. The document does not mention how the liquid was injected into the uterus.In small ruminants this procedure can only be done under controlled pressure.In this way, to inject the volume of 100 ml per uterine horn, as mentioned in the document. There is undoubtedly a need to disconnect the injector device (syringe) both in a horn and in the exchange of horns, which characterizes the use of a system with a risk of contamination of the material by the external environment.

Other data revealed in the technique show the use of the non-surgical collection technique in Boer goats through the introduction of a transcervical catheter. For this, the non-surgical collection methodology was used as a viable alternative to replace the commonly used surgical procedures. However, there is no mention in the technique about the use of closed circuits for embryo collection. Additionally, a fundamental aspect for the proper functioning of the transcervical catheter is the need for an external connection. mentioned by documents that currently comprise the state of the art.

WO0152767 describes a device that includes a tube or catheter that is introduced into the animal's vagina into the cervical canal, a flexible tube covered with a coating of also flexible material thus allowing the tube to advance along the cervical canal. and then along the uterine horn after reaching the distal end of the tube. However, the device shown prevents the absorption of reflux when it is transported out.

FR2607011 discloses a device that can be used for transcervical or peritoneal transfer of embryos. However, the device presented consists of a piston syringe, a device already known for use in artificial insemination, and not susceptible for use in embryo collection, especially goats and sheep, due to its morphological peculiarities. In this case, the term embryo transfer refers to the act of innovation. Innovation is the act of depositing in the female uterus (recipient) an embryo previously collected from another female (donor).

DE3730347 describes an embryo collection instrument for collecting embryos from small ruminants such as goats and sheep. The device has holes in the end and uses fluids to promote embryo suspension. However, the embryo collection described is made through the laparoscopy method.

The related technique further comprises several other patent documents, as well as scientific articles describing methods of embryo collection via the transcervical approach. However, the described methods are surgical methods that present the need for improvement to optimize the process and to promote better conditions of its accomplishment.

The only similar device available and currently used to promote a completely closed circuit is that used for cattle. However, in all of the above references there is a need to open up the system at least once, which makes it incorrect to use the term closed loop for methods currently known in the art.

However, for smaller species, the circuit now used for cattle is inadequate due to its dimensions to the reproductive anatomy of these species. The similar bovine, due to the high caliber and size, hinders the efficient flow of injected and recovered fluid in the womb. The coupling of similar bovine to the collection probe requires extra connection, due to the high caliber.

More specifically, in goats and sheep, surgical techniques are most commonly used because of the difficulty in manipulating the female genitals and because there is no proper equipment to allow the use of other techniques for embryo collection.

In the transcervical collections described in the state of the art, human probes and bovine circuits were used which, due to size and caliber differences, cause losses and injuries.

In Brazil and worldwide, techniques involving the whole area of Goat and Sheep Embryo Technology are still under development. There is a clear search for more efficient protocols for increasing the number of viable donor-collected embryos, as well as for simpler, less invasive procedures that allow recovery rates close to those obtained with traditional (i.e. surgical) methods. Brazil is one of the pioneer countries in non-surgical transcervical embryo collection in goats and sheep and one of the few countries that use this route as the preferred and commercially implemented goat route. However, the materials used are improvised, unsuitable and reused - not designed for this purpose.

The development of specific equipment for embryo transfer in goats and sheep, which takes into account the anatomical peculiarities of these species and the necessary flexibility, precision, diameter and size, may consolidate embryo collection by non-surgical transcervical approach.

Such aspects may reflect in improved welfare conditions. during the process, as well as reducing risks and surgical sequelae, allowing the use of animals for a relatively longer period. In addition, in addition to the prospect of using the technique in other countries that use surgical collection, it may provide additional consumption and export of the product.

To solve the problems still in the art, the present invention has been developed in order to provide the technique of a system comprising devices with measures suitable for goats and sheep in particular, by double flow and closed loop.

SUMMARY OF THE INVENTION

The present invention relates to a transcervical embryo collection system developed specifically for small ruminant animals. More particularly, said system is provided with devices that prevent contact with the external environment during process manipulation, protecting the embryos from environmental contamination, as well as a device provided with a double flow path, to allow the change of direction of the embryo. Uterine infusion of the culture medium with a simple touch. Specifically, the function of the developed technology is to sequentially and sequentially carry the culture medium flush fluid into the female's uterus, allowing the uterine content of females subjected to superovulation and embryo collection to be completely enclosed. . Additionally, the present invention further relates to a process for collecting embryos.

DESCRIPTION OF THE FIGURES

Figure 1 shows the embryo collection system object of the present embodiment.

Figure 2 shows the connecting device (1) and flow restrictors (2).

Figure 3 shows the filter system of the invention.

Figure 4 shows the flow restricting device assembly.

Figure 5 shows the system connecting system with the uterine lavage pouch used in the system of the present invention. DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a transcervical embryo collection system developed specifically for small ruminant animals such as goats and sheep, taking into account the anatomical peculiarities of the species. The present invention is not limited to the use of said system in goats and sheep and said system can be used on any animal of similar size.

More particularly, said system has great efficiency in embryo collection and is provided with devices that prevent contact with the external environment during process manipulation, protecting the embryos from environmental contamination.

A second particular aspect of the present invention is that said system comprises a device provided with a double flow path, to allow the uterine infusion of the culture medium to be changed with a single touch. This feature represents a great facilitation in the embryo collection process.

The embryo collection system is also referred to by experts in the art as the embryo collection circuit, and especially the present system will be known by such specialists as embryo collection loop.

The elements that make up the present system are made of a material with specificity suitable for the small volume used for the type of animal for which it is intended, for example, for goats and sheep, maximizing the return flow of fluid from inside the womb. Said material may be sterilized by cobalt 60 gamma radiation and / or by ethylene oxide gas, being a non-toxic and pyrogen-free material.

For the present invention, the entire system is comprised of plastics material, preferably but not limited to, for example, vinyl polychloride - PVC - ATP-109 CRISTAL. The characteristic of the system related to the use of a smaller amount of plastic material makes the developed system less polluting to the environment. The function of the developed technology is to carry the flushing fluid with culture medium in the bag, to the female's uterus, sequentially and successively, allowing the uterine content of the females submitted to superovulation and embryo collection. For the present embodiment, the washing liquid used as culture medium was a phosphate buffered saline solution - PBS. However, any other solution that has the same characteristics as the PBS solution may be used as a wash solution for the system now developed, provided they meet the requirements for embryo collection.

In order to facilitate understanding of the present invention, but without limiting the scope of protection to this preferred embodiment, the present system will be described and detailed by the accompanying Figures.

The embryo collection system (1) now developed is fully shown in Figure 1 and comprises:

- an embryo collection device (1)

- restrictive devices or flow controllers (2);

- an embryo collecting filter (3) and the filter seal (4);

- a flow control device (5) and a seal for the collecting device (6)

a system connector device with the two-part uterine lavage bag; the internally coupling connector (7) and connector seal (8); and

- tubes (9)

Figure 1 shows the complete system with all its elements.

All ends of the system, as well as the other devices that make up the system (Figure 1) of the present invention, are protected by protective devices (seals) to prevent contamination with the external environment and to ensure a fully closed collection procedure.

The collection device (1) of the embryo collection system is shown in Figure 2. The connector device for the collection probe (1) and the flow control devices (2) can be seen. Such a device has a Y-shape and has different elements connected to each of the 3 system outputs.

At one end is the collecting device (1) formed by a connector for the collection probe. Figure 2 shows this outlet at which the flow restricting device (5) is coupled to the connector of the embryo collection probe (1) that passes through the cervix and is positioned within the uterus. Said flow restricting device (5) has a Y-shaped structural configuration, allowing both the flow of liquid into the uterine interior and the flow of liquid to the filter. For its sequential total locking a pulley is turned inside. The preferred distance of the location of said device (5) between the connector (1) and the embryo collection filter (3) is approximately 80cm.

At the other end of the system is attached the filter device for embryo collection (3) and its corresponding seal (4), housed in a ventral manner. About 50-80 cm, preferably 65 cm, from this connection, the flow restricting device (2) is positioned. This set constitutes the second output end of the system. The filter, which is coupled to the flow restricting system (2), allows the passage of liquid and the retention of embryos. As the volume inside the filter increases, the filter device allows controlled disposal while maintaining a safe amount of liquid inside, preventing overfilling or drying. The filters of the invention are shown in detail in Figure 3.

At the other end and about 5-30 cm, preferably 15 cm from the connector (1) is the device that constitutes the third output end of the system and consists of the flow control device (5), which now passes through to be referred to throughout this report by the usual nomenclature of those skilled in the art, 3 (three) way tap (5). This 3-way faucet or flow control system and its seal (6) are detailed in Figure 4.

In said 3 (three) way tap (5) is coupled to a sterile dosing device (not shown in the figures), such as a syringe, which allows a perfect dosage of the amount of liquid to be used in the embryo collection procedure without the need to open the system (Figure 1) external medium. For the present embodiment, such a metering device holds approximately 60 mL. However, devices of other capacities may be easily adapted to the system according to its dimensions.

After the flow controller is the connector device with the uterine lavage bag. Said device consists of a connector (7) and a seal (8), which internally engage forming the third end of the system (Figure 1) which is connected to a bag containing the wash solution, not shown in the accompanying Figures. present report. Figure 5 shows details of the connection system to the uterine lavage bag.

The embryo collection process developed by the present invention basically comprises: (i) aspirating the culture medium into the syringe; (ii) reversing a tap; (iii) injecting the culture medium into the female's uterus in volumes of approximately 10 to 30 mL, more preferably 15 mL sequentially, making 240 mL per uterine horn.

After each injection of fluid into the uterine interior, the flow restricting device pathway that carries the fluid into the uterus is closed, allowing gravity to return the uterine content through the other pathway of the uterine content device to the uterus. filter. Subsequently, it is again opened for a new injection procedure and so on.

Since the whole process is done through gravity, there is no need for any other type of instrument.

The distance measurements between the devices comprising the present system developed herein are exemplary values of the present invention, and are not limited thereto to such values, and said system can be sized according to the characteristics of the present invention. female submitted to superovulation and embryo collection, referred distances adjusted to the new dimensioning.

The present invention provides a suitable embryo collection system for small ruminant animals, such as goats and sheep, with dual loop and closed loop, reducing contact with the external environment and avoiding contamination.

Due to these previously unpublished characteristics, the developed system presents great efficiency in the collection.

As a closed system, collected embryos are protected from environmental contamination. Because it is a system provided with a double flow path, it is possible to change the direction of uterine infusion of the culture medium with a simple touch, which facilitates the collection process.

The present system is a product that adds functionality to anatomical concepts specific to sheep and goats. Additionally, the product is cheaper than similar bovine because it uses less fluid to perform uterine lavage, is more efficient because it allows greater retrograde flow of this fluid and is more aseptic because it allows collection in a completely enclosed environment.

In a specific embodiment, the process for embryo collection, sometimes called closed loop, comprises:

1) Fit the ends of a connector to a solution bag, embryo collection filter and dosing syringe;

2) Turn the tap of a 3 (three) way tap clockwise to maintain free access;

3) Aspirating a volume of saline, for example 60 ml of PBS-buffered saline into the syringe;

4) Turn the 3 (three) tap tap counterclockwise;

5) Inject half of the aspirated saline into 3, for example about 30 ml PBS all the way to the filter;

6) Close the ventral seal of the filter; 7) Inject the other half of the aspirated saline into 3, for example the remaining 30 ml that will be retained in the filter;

8) Close both flow restrictors;

9) Couple the equipment in an embryo collection probe;

10) Aspirating new volume of PBS buffered saline, for example 60 ml of PBS buffered solution;

11) Open the PBS buffered saline restrictor and inject a volume, for example, between 10 ((initially) to 30 ml PBS at a time into the uterus;

12) Close the PBS restrictor and open the draining stem restrictor to the filter to allow backflow of fluid from the uterus into the filter;

13) Repeat PBS injection and drainage procedures until the uterus has been properly flushed.

The amount of PBS buffered saline may vary depending on the size of the ruminant to be used and is well known to those skilled in the art.

Claims

1- Circuit for embryo collection by small animal transcervical pathway characterized by the fact that it comprises devices that prevent contact with the external environment during the process manipulation and device provided with a double flow path.

2. Circuit for embryo collection by transcervical approach according to claim 1 characterized by the fact that it comprises:

- embryo collection device (1);

- flow restricting devices (2);

- embryo collection filter (3) and filter seal (4);

- flow control device (5);

- seal for the collecting device (6);

- system connector device with the uterine lavage bag (7),

- seal of the connector (8); and

- tubes (9)

Embryo collection circuit according to Claim 1, characterized in that it is suitable for goats and sheep.

Embryo collection circuit according to Claim 1, characterized in that the elements are made of a plastic material.

Embryo collection circuit according to claim 4, characterized in that said plastic material is polyvinyl chloride - PVC - ATP-109 CRISTAL.

Embryo collection circuit according to claim 2, characterized in that the connector comprises two distinct units (7 and 8) which internally engage to form an end connected to a pocket containing the wash solution.

Embryo collection circuit according to Claim 6, characterized in that the wash solution used as the culture medium is a phosphate buffered saline solution - PBS.

Embryo collection circuit according to Claim 1, characterized in that it allows the conduction of the washing liquid with culture medium packed in the pouch to the female's uterus, sequentially and successively.

Embryo collection circuit according to Claim 1, characterized in that all ends of the collector are protected by protective devices.

Embryo collection circuit according to Claim 1, characterized in that it is connected to an embryo collection probe.

Embryo collection circuit according to claim 2, characterized in that the connector (1) is 5 to 30 cm apart from the flow control device (5).

Embryo collection circuit according to claim 11, characterized in that the connector (1) is preferably 15 cm apart from the flow control device (5).

Embryo collection circuit according to claim 2, characterized in that the flow control device (5) is a 3-way tap.

Embryo collection circuit according to claim 13, characterized in that the 3 (three) way tap (4) is coupled to a sterile dosing device, the second end of the 3 (three) way tap (5) being connected to the connector (1).

Embryo collection circuit according to Claim 14, characterized in that the sterile dosing device is a syringe.

Embryo collection circuit according to claim 14, characterized in that the sterile dosing device has a volume between 30 and 120 ml.

Embryo collection circuit according to claim 16, characterized in that the sterile dosing device has a preferred volume of 60 ml.

Embryo collection circuit according to Claim 2, characterized in that the flow restricting device (2) has a Y-shaped structural configuration and is coupled to the embryo collection probe connector (1). Embryo collection circuit according to claim 18, characterized in that said flow restricting device (2) permits both the flow of liquid into the uterine interior and the flow of liquid to the filter.

Embryo collection circuit according to Claim 2, characterized in that it has an embryo collector filter (3) connected to one end of the system (1).

Embryo collection circuit according to claim 2, characterized in that the embryo collection filter (3) is provided with a ventrally housed seal (4) and allows the passage of fluid and the retention of the embryos.

Embryo collection circuit according to Claim 2, characterized in that the embryo collector filter (3) is 50 to 80 cm from the connector (1).

Embryo collection circuit according to claim 20, characterized in that the distance between the filter (3) and the connector (1) is preferably 65 cm.

24. The embryo collection process as defined from 1 to 23, characterized by comprising:

Fit the ends of a connector to a solution bag, embryo collection filter and dosing syringe;

Turn the flow controller register clockwise to maintain free access;

Aspirate an appropriate volume of PBS-buffered saline into the dosing system;

Turn the flow controller register counterclockwise;

Inject half of the aspirated saline into 3, for example, across the circuit until it reaches the filter

Close the ventral seal of the filter;

Inject the other half of the aspirated saline into 3 to be retained in the filter;

Close both flow restrictors; Attach the equipment to an embryo collection probe; Respirate adequate volume of PBS-buffered saline,

Open the PBS buffered saline restrictor and inject an appropriate volume into the uterus;

Close the PBS restrictor and open the draining stem restrictor to the filter to allow backflow of fluid from the uterus into the filter;

Repeat PBS injection and drainage procedures until you are sure the uterus has been properly flushed.

Process according to Claim 24, characterized in that all liquid flow occurs through gravity.

Process according to Claim 24, characterized in that the culture medium is injected into the uterus of the female in volumes of approximately 10 to 30 ml.

Method according to Claim 24, characterized in that the culture medium is injected into the uterus of the female, preferably of 15mL.

Process according to Claim 24, characterized in that the injection of the culture medium into the female's uterus is carried out sequentially.