Flow Cytometry

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Selection of sex at conception the most sought
after reproductive biotechnology of all time
Dr. Sushil Sood Veterinary Officer (Wildlife)
RRC/MSC Tutikandi PhD (Australia), M.Sc.
(UK), B.V. Sc A.H (India)
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• Introduction Myself and Sperm Sexing
• Why , WHERE, WHEN HOW ? - sperm SORTING
• Factors -- sorting efficiency
• Sperm Insults
• Problems of Sperm Sexing
• Pregnancy Rates
• Challenges and future possibilities
• summary

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Who am I ?
Ph.D. in Reproductive technology and
physiology Master of Science in Wildlife
Management Conservation and Control Bachelor of
Veterinary Sciences and Animal Husbandry
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Who am I ?
Improvement of Sperm Functions of frozen
thawed/liquid sperm for better fertility
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• GERMANY
• Leibniz Institute for Zoo and Wildlife Research

• AUSTRALIA
• Perth Zoo (Australia)
• The Pheasant and Waterfowl Society of Australia
• Endocrine Reproductive Society of WA

NEW ZEALAND Department of Conservation, New
Zealand
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Introduction

• The latest breakthrough in animal reproduction
  is sexing of semen.

• Predetermination of sex in livestock offspring
  is in great demand and is of great importance
  for profitable dairy industry. gt50,000
  documented calves of the desired sex (Seidel,
  2009).

• With the changes that have taken place in
  animal husbandry over the past generation the
  application of sex pre-selection to production
  systems becomes increasingly important. ? Food
  Security and Competetion for similar food.

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WHY/WhERE sperm sexing?

• To produce calves of desired sex in both dairy
  and beef cattle

• Herd replacement and herd extension can be done
  very quickly

• To ensure birth of heifers when progeny testing
  of young bulls is desired

• Combination of super-ovulation and insemination
  with sexed semen further increases the desired
  calf crop

• In in-vitro fertilisation programmes, one dose
  of sexed sperms can be used to produce many
  embryos of desired sex.

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Countries Producing Sexed Semen Commercially
Argentina Brazil Canada China Japan Mexico Switze
rland United Kingdom United States
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Historical Perspective

• Gledhill 1976 First attempted to separate X
  and Y sperm by analytical flow cytometry.
• Pinkel et al. 1982 1st successful seperation of
  mammalian sperm.
• Initial methods were found to be destructive
  because the tails were removed by sonication
  leaving sperm biologically unusable (seperation
  of living sperms not possible)
• Modified cell sorter produced by beveling the
  sample injection tip within the nozzle of flow
  cytometer (Johnson Pinkel 1986) developed
  commercial cell sorter

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BOVINE sperm chromosome
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Difference between X and Y spermatozoa
Parameter Difference
DNA content Less in Y sperm
Size X sperm is larger
Motility Y sperm faster
Surface charge X sperm negative
Cell surface antigen H-Y antigen on Y sperm
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FLOW CYTOMETRY PRINCIPLE

• This technique is based on difference in DNA
  content between X and Y spermatozoa
• X spermatozoa contains more DNA
• Difference in DNA content for most mammals are in
  the range of 3-4.2
• 
  (johnson et al 2000)

X
Y
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How to measure DNA SPERM SEXING

• The DNA content of the sperm is determined using
  a DNA specific fluorescent dye
• Hoechst -33342 ( a DNA binding fluorochrome
  2-(4-ethoxyphenyl)-5-(4-methyl-1-piperazinyl)-2,5
  -bis-1H-benzimadazole-trihydrochloride)
• The stain penetrates the sperm membrane and
  binds to A-T region of nucleic acids
• The dye fluoresces when exposed to low
  wave-length laser beam. (5-watt water cooled
  argon laser)
• The fluorescence is measured by a detector and
  analysed by a computer
• Since X-sperm has more DNA ,it ends up with more
  dye than Y-sperm and it givesoff more
  fluorescence

Parameter Difference
DNA content Less in Y sperm
Size X sperm is larger
Motility Y sperm faster
Surface charge X sperm negative
Cell surface antigen H-Y antigen on Y sperm
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FLOW SROTING SPERM
Two basic Sperm Populations
Live (stain with Hoechst 33342)
Dead (Stain with Red Food dye)
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BELTSVILLE SPERM SEXING TECHNOLOGY

• Non-human sperm exclusive license to XY, Inc.
• Human sperm exclusive license to Genetics and
  IVF

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Cell sorting
Waste

• Non-viable sperm
• Drops that contain X and Y sperm
• Poorly-oriented sperm
• Sperm that overlap in DNA fluorescence

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Factors affecting the sorting efficiency of flow
cytometer

• Orientation of sperm head
• Angle of sperm presentation towards the
  excitation source, 450 is adequate
• Orientation of fluid in the nozzle
• Even with the correct orientation of cell and
  fluid 20-40 of live sperms are not measurable,
  passes directly to the waste tube
• Speed of computer processor
• Optical techniques

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Speed of cell sorting

• Standard speed system 35000 cells/h
• High speed cell sorters 15 million cells/h
• Accuracy of sorting is 85-95 (Seidel et al
  1999)

Sperm INSULTS

• Waiting to be sorted
• High concentration H33342
• High laser power
• Exit at 80 km/hour
• Very high dilution
• Centrifuge to concentrate
• Cryopreservation

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Problem of sperm sexing

• High cost of equipment
• High cost of maintenance
• Skilled manpower
• About half of the sperm sample unsexable
• Sorting efficiency
• Low pregnancy rates
• Speed

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Utility of sexed sperm in Indian context

• Production of unwanted diary cattle males can be
  eliminated/minimized as they can not be
  slaughtered in India
• It can facilitate production of required number
  of daughters for progeny testing programme in
  shortest time, thus increased genetic gain
• Production of breeding bulls as country has
  limited elite cattle and buffaloes(lt 0.1 of
  total)

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Concerns of sexed sperm in Indian context

• Costly 25 USD Imported Germplasm.
• Affordability
• Low Conception Rate ? Does not give desired
  results if used in Repeat Breeders.
• Skilled Manpower Shortage ? Cost
• May interferes with Breeding Policy of the states
  

• ACCURATE
• RAPID
• Inexpensive
• Non Damaging
• All sperm

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Pregnancy rate with sexed semen

• Offspring from seven mammalian species primarily
  cattle have been produced after H33342- staining
  and flow sorting of fertilizing spermatozoa, but
  no gross abnormalities have been produced (Seidel
  et al 1999)
• Higher pregnancy rate in heifers for sexed
  cryopreserved semen with 7-20 times more sperm
  per dose when deposited in uterine body (Seidel
  et al 1999)
• Pregnancy rate in heifers for 2106 sexed sperm
  per insemination was 56 and in control with
  10106 unsexed sperm was 61 (Garner and seidel
  2003)
• Pregnancy rate for sexed sperm was 60-80 of
  those found with unsorted control sperm when
  sperm per dose were same for both (Doyle SP
  2005)
• The use of sexed semen resulted in 45 conception
  in heifers and 28 in cows (Vries et al 2009)

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• Use of sexed semen has reduced dystocia related
  losses in heifers as compared to cows (Fetrow et
  al 2007)
• Conception rate of 69.7(30/43) for sexed sperm
  and 66.5(1545/2325) for unsexed sperm following
  AI was reported in China (Lu et al 2010)
• A review of the literature indicates that
  conception rates (CR) to 1st service with
  frozen-thawed sexed semen are 75 to 80 of
  those achieved with conventional frozen-thawed
  semen.
• Preliminary results from a large-scale field
  trial carried out in Ireland in 2013 suggest that
  significant improvements in the performance of
  sexed semen have been made, with CR of 87 of
  those achieved with conventional semen (Butler et
  al 2014)

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challenges and possibilities
Parameter Current
Fertility 75 of unsexed semen
Sex accuracy 90
Speed 6,000 cells per second
Yield 30 input correct-sex cells
Product recommendations Heifers only
Available genetics Uneconomical in high value proven sires
Arrived recently
90 of unsexed semen
90
50,000 cells per second
90 input correct-sex cells
Heifers and repeat breeders
Wide range of sires
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Future
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Conclusion

• Rapid improvements have occurred in flow
  cytometry technique which is the most reliable
  and fully validated method at present for sperm
  sexing
• Sexed semen although has been advocated to be
  used on heifers will soon be used in both heifers
  and repeat breeders
• Sexed semen can be routinely used for embryo
  transfer and IVF to produce calves of desired sex
• Calves appear normal
• No excess abortions

• ACCURATE
• RAPID
• Inexpensive
• Non Damaging
• All sperm

Sexed semen conception rates average 25 lower
than with conventional semen, but field trial
results have varied widely. A 25 reduction is
approximately 10 to 15 percentage points in
heifers
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Possibilities

• Costly 25 USD Imported Germplasm.
• Establish our own Sexed Semen Lab
• Buying of Patent Rights ??
• Low Conception Rate ? Not Anymore Can be
  compensated by increasing the sperrm
  concentration in the straw.
• Skilled Manpower Available
• May interfere with Breeding Policy of the states
  Not if we raise our own bulls ? Supplements
  Progeny Testing

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Associations and Affiliations
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THANK YOU