embryo sexing lamp

2. Dr. Dilawar Zia
M.Phil. student at University
of Agriculture Faisalabad,
Pakistan
2012-ag-2702

3. Today's topic is
Embryo Sexing
Presented to:
Dr. Huma Jamil

4. History
Biotechnology has been practiced in animal husbandry since
the Beginning of human history.
In 1919, Karl Ereky, a Hungarian engineer coined the term
‘biotechnology
First successful embryo sexing done by Gardner,
1968 in rabbits by cytological method (Barr body
observation)

5. Introduction
• Pre implantation determination of sex of embryo at compact morula
or early blastocyst stage
• sexing of embryos before transfer and implanting has great potential
for the livestock industry.
• Manipulating the sex of offspring has been a dream of the cattle
industry for decades
• Pre selection of sex
• Modern techniques make it possible now
• Super ovulation and embryo collection

6. Methods of embryo sexing
• A. Invasive method:
• It does not maintain the Integrity of embryo and likely to damage
the potential of successful embryo transfer by effecting the viability
therefore this method is called invasive method.
• 1. Cytological method or Karyotyping
• 2. Identification of sex chromatin
• 3. Y- chromosome specific DNA probes
• 4. Polymerase chain reaction (PCR)

7. B. Non –Invasive method:
• Sexing is considered best because it maintains the integrity of embryo
and so is less likely to damage the potential of successful transfer of
embryo. The embryo is not subjected to any harm throughout the
procedure leaving it intact and viable, therefore this method is known
as Non-Invasive method of embryo sexing
• 1. Detection of X- linked enzymes
• 2. Detection H-Y antigens

8. A. Invasive method
1. Cytological methods or Karyotyping.
• karyotyping is done using cells at metaphase stage. Some cells are removed from
embryo and cultured with colchicines that causes cells to stop dividing at the
metaphase stage of mitosis. After some hours, cells are lysed osmotically and the
preparation is fixed and stained so that metaphase chromosomes can be
examined microscopically for two X in female or one Y chromosome in male.
(Seidel and Seidel,)
• Advantages:
• it is inexpensive and easy to perform
• it requires no sophisticated equipment
• it can identify chromosomal abnormalities before the embryos are
transferred.
• Accuracy of the karyotyping method is high,
• reagents are inexpensive and easy to obtain. (Kitiyanant et al., 2000)
Cont'd

9. Disadvantages
• These techniques may cause accidental harm to the survived embryo.
Reduce the viability and conception rate of embryos.
• Low percentage of embryos that can be sexed because the
analyzable metaphase plates that can be prepared are in relatively
low Limited numbers
• time consuming procedure
• required well trained personnel

10. 2. Identification of sex chromatin
• The sex chromatin method of sex selection depends on the identification of a
dark staining body, adjacent to the nuclear membrane in fixed cell referred to as a
Barr body. The cells from female embryo are expected to have Barr bodies in
them where as the male embryo will not have Barr bodies.
Advantage
• Simple and rapid technique
Disadvantage
• The granular natures of cytoplasm prevent the detection of Barr bodies
• All cells may not have Barr bodies.
• A large number of cells are required for making the smear which may damage the
embryo (White, 1989).

11. 3. Y-chromosome specific DNA probes
• The Y-specific probe technique involves the biopsy of a small number of cells
from the embryo with proteinases to expose the DNA and then hybridized with
radioactively labelled Y-chromosome specific probe. Positive hybridization results
indicate the presence of Y- chromosome and thus the male sex of
chromosome.(Leonard et al., 1987).
Advantages
• Fetal sex can be predicted with DNA samples of as little as 20 ng.
• Highly accurate and a higher percentage of embryos can be sexed.
Disadvantage
• Technique is quite complicated, expensive and time consuming.

12. 4. Polymerase chain reaction (PCR) method
• Amplification of Y chromosome-specific DNA by means of the PCR technique is the most
reliable and practical method of sexing bovine embryos.
Advantages
• Sensitive, accurate, reliable and efficient procedure and pregnancy rates have not been
affected.
• The removal of a few cells caused very little trauma to the embryos.
• It did not alter developmental potential in vitro.
• High percentage of embryos being sexed.
Disadvantages
• It requires technical skill and is time consuming.
• PCR has the risk of false positives because of DNA contamination during handling of the
PCR products in PCR procedures
Cont'd

13. ➢ Collection of embryos produced in vitro or in vivo
➢ Selection of grade one or grade two embryos
➢ Embryo washed with PBS & placed in a drop containing 200 mM sucrose under
micromanipulator
➢ Zona pellucida cut open with fine micro blade
➢ Few blastomere sucked with fine aspiration pipette
➢ Washed in KCl & transferred to Eppendorf tube
Cont'd

14. Biopsy in 0.5 ml Eppendorf tube + Proteinase-K + 9 μL of lysis buffer
Overlaid with 25 μL of mineral oil
Incubated at 37° C for 10- 60 min
Inactivation of proteinase-K at 98 ° C for 10 min.
Cooled at 4 ° C
Cont'd

15. Amplification of DNA
•15 μL of PCR reaction mixture(PCR reaction buffer, primers, 1.5 μL of Taq
DNA polymerase & 125 μg of Ethidium bromide) is added to the tube
Subjected to PCR cycling
•3 min. denaturation at 94 °C
•10 cycles of denaturation at 92 ° C
•Annealing at 50 ° C (80 seconds)
•Extension at 72 ° C for 20 seconds
Further 40 cycles at 60 ° C of annealing temperature Final extension
achieved by 5 min. incubation at 72 ° C
Cont'd

18. Loop-mediated Isothermal Amplification
• Loop-mediated isothermal amplification (LAMP) is a novel DNA amplification method that
amplifies a target sequence specifically under isothermal conditions.
• In contrast with PCR in which a target DNA sequence is amplified by a temperature change
between about 50 and 95 C, isothermal DNA amplification methods have been recently
developed. Loop-mediated isothermal amplification (LAMP) is a DNA amplification method that
can amplify a specific DNA sequence within the range of 60 to 65 C
• Detection of amplification product can be determined via turbidity caused by an increasing quantity of
magnesium pyrophosphate precipitate in solution as a byproduct of amplification. This allows easy
visualization by the naked eye

19. What advantages does LAMP offer over PCR?
• isothermal approach
• portable or less expensive instruments
• LAMP generally produces more DNA than PCR in a more rapid incubation time
• it can start the detection with small number of DNA
• easy-to-perform method

20. B) Non-Invasive method of embryo sexing
1. Detection of X – linked enzymes
• The female has two X chromosomes whereas the male has only one X chromosomes in somatic
cells and hence the enzymes associates with X chromosomes produced in female are almost
double than that produced in the males. These enzymes are Glucose-6phosphate dehydrogenase
(G6PD), Hypoxanthine guanine phosphoribosyl transferase (HPRT), Phosphoglycerate kinase,
Agalactosidase. These enzymes are measured in embryos. The high concentration of enzyme
usually denotes two X chromosomes or female embryo. While low concentration denotes one X
chromosomes or male embryo. (Monk and Handyside, 1988).
Advantages
• Allowing all embryos to be sexed.
Disadvantages
• Embryo viability was reduced, particularly for embryos with very high or very low enzyme activity.
Embryo has to be kept outside for longer duration in order to estimate the enzyme. Variations
may exist leading to false diagnosis. The assay may be toxic to the embryos. (Pratheesh et al.,
2011).

21. 2. Detection H-Y antigens
• Detection of a male-specific factor (H-Y antigen) by antibody. Embryos are incubated for 30–60 minutes with
antibodies and then for an additional 30–60 minutes with first antibody containing a fluorescent dye.
Embryos are then briefly examined with a fluorescence microscope. Male embryos fluorescent (H-Y positive)
and female embryos non-fluorescent (H-Y negative). H-Y antigen is expressed as early as the eight-cell stage
and is readily detectable on morulae. Detection becomes more difficult at the blastocyst stage. The assays
for H-Y antigen are approximately 85% accurate in identification of embryonic sex (Anderson, 1987).
Advantages
• Simple to perform and rapid enough to allow field use (Bredbacka, 1998). Although the accuracy with cattle
embryos is at best 80-90 % (Anderson, 1987). Speed and lack of need to biopsy embryos. Procedure is non-
invasive and requires no specific skills.
Disadvantages
• Need for a fluorescence microscope, commercial unavailability of reagents

22. Applications of Embryo Sexing
• Altering the male & female sex ratio in farm animals
• Increase in milk & meat production
• Control of incidence of freemartinism
• reducing the cost of MOET(Multiple Ovulation Embryo Transfer)
• In Wildlife management as a strategy for the conservation of an
endangered species and as breeding schemes in zoos

23. Constraints
• Poor infrastructure facilities
• Low level of education & training
• High cost of the technology
• Difference between research & field conditions
• Less availability of indigenous technology & materials
• Lack of 3 D’s

24. We shall overcome..
• Great progress in scientific fields in spite of constraints
• As time advances technology becomes more affordable

25. Thankyou