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Materials and methods including for sex selection

A male, optional technology, applied in the direction of chemical apparatus and methods, botany apparatus and methods, biochemical apparatus and methods, etc., which can solve the problem of suboptimal success rate, lack of reproducibility, lack of consistency of IVP protocol, etc.

Pending Publication Date: 2020-08-21
克罗莫依安私人有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Efforts have been made to reduce the discrepancy in in vitro fertilization (IVF) results between fresh semen and frozen-thawed semen, but in most IVF laboratories, IVF efficiency remains at 40-50% (Li et al., 2003; Alminana et al. People, 2005), and the optimal dose of semen is still unknown
Despite attempts to improve the IVP system using different types of media supplements and media conditions, the success rate is still not optimal
Conventional IVP systems use several media from oocyte collection to embryo culture, which can lead to system variability and lack of reproducibility
[0009] An optimal IVP system may be far from being achieved due to lack of consistency between IVP protocols used by different laboratories

Method used

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  • Materials and methods including for sex selection
  • Materials and methods including for sex selection
  • Materials and methods including for sex selection

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0080] Example 1 - Simplified IVP method that essentially uses a single medium to increase yield of porcine embryos

[0081] This example describes a simplified method for pre-implantation in vitro porcine embryo development suitable for high-throughput IVP systems.

[0082] Materials and methods

[0083] All chemicals were purchased from Thermo Fisher Scientific unless otherwise stated. All in vitro cultures were prepared by overlaying 400 μl of mineral oil in each culture well. The basal medium used in this example was M-199. In 1950, Morgan first proposed a chemically defined source of nutrients for cell culture without the use of animal products and / or tissue extracts. The advantage of using this medium is that it has a wide variety of components to support the development of oocytes and embryos at different developmental stages.

[0084] Oocyte recovery and in vitro maturation

[0085]Ovaries were collected from slaughtered prepubertal gilts and sows from a nearby sla...

specific Embodiment approach

[0209] Preferred features, embodiments and modifications of the invention can be seen from the following detailed description, which provides those skilled in the art with sufficient information to practice the invention. The detailed description should not be considered as limiting the scope of the foregoing summary in any way. The detailed description will refer to the following several drawings:

[0210] Figure 1B : Amplification profile of the Y chromosome, indicating thresholds and wells with Y chromosome amplicons.

[0211] Figure 2B : Amplification profile of the 12S chromosome, indicating thresholds and wells with 12S chromosome amplicons.

[0212] Figure 3B : Amplification profile of 12S chromosome and Y chromosome (Chr Y) combination, indicating threshold and wells with 12S chromosome and Y chromosome amplicons.

specific Embodiment approach 2

[0214] Example 1 - Differential lysis of embryos or eggs in the presence of sperm or semen for polymerase amplification.

[0215] This example describes the use of a novel lysis buffer / solution for differentially lysing embryos or eggs in the presence of sperm or semen for PCR amplification. DNA from individually lysed embryos or eggs can then be pre-amplified by PCR to generate sufficient whole-genome DNA for a variety of downstream applications.

[0216] The high-throughput method described here is a rapid, 25-minute, single-tube process that is delicate enough to selectively lyse oocytes in the presence of spermatozoa, yet sensitive enough to detect ovum from a single unfertilized egg and those that have developed. Embryos of 2, 4, 8, 16 or more cells are lysed and DNA preamplified. Furthermore, no substances were introduced that might inhibit preamplification or downstream applications. The method is also compatible with the Single Cell REPLIg Kit as well as other qPCR a...

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Abstract

A method comprising the steps of: 1. optionally subjecting spermatozoa to a treatment step; 2. subjecting the spermatozoa of step 1 to a sex selection step so as to select for either female or male spermatozoa of interest; 3. carrying out a fertilisation step using the spermatazoa of interest of step 2 to produce at least one oocyte, blastocyst, ovum, embryonic cell or embryo; 4. selectively lysing the at least one oocyte, blastocyst, ovum, embryonic cell or embryo of step 3 in the presence of spermatozoa so as to selectively release cellular material from the at least one lysed oocyte, blastocyst, ovum, embryonic cell or embryo; and 5. using the released cellular material in at least one downstream application.

Description

[0001] related application [0002] This application claims priority to U.S. Patent Application Nos. 62 / 594,124 and 62 / 594,153, both filed December 4, 2017, each of which is hereby incorporated by cross-reference in its entirety. [0003] title 1 [0004] Breeding method technical field [0005] The present invention generally relates to methods of assisted reproduction. In one aspect, the invention relates to a method of producing at least one fertilized oocyte, preimplanted embryo or blastocyst, preferably a method of producing a preimplanted fertilized oocyte, preimplanted embryo or blastocyst in a pig. [0006] Background technology 1 [0007] Over the past three decades, there has been intense interest in enhancing in vitro production (IVP) of pigs, not only to study reproductive physiology, but also as part of other biotechnological and biomedical research such as the production of transgenic pigs (Gil et al. et al., 2010; Vajta and Callesen, 2012; Liu et al., 2011; ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/073C12N5/076C07K16/40G01N33/577G01N33/569C12N5/20C12N15/13
CPCA01K67/02C12N5/0604C12N5/0609C12N5/061C12N5/0081C12Q1/6879G01N33/5005C07K16/28A61K39/39541A61K35/52A01K2227/108G01N2333/46A61K2039/505G01N33/689G01N2800/367C07K2317/33C07K2317/34C12N2509/10C12N2501/999A01K2227/10C07K2317/565
Inventor S·C·德韦特M·S·侯赛因
Owner 克罗莫依安私人有限公司
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