Animal Production management

Module 10 – Preventive Veterinary Medicine

Reproductive biotechnology
The sperm
(spermatogenesis, evaluation of sperm quality and viability, CASA, sperm sexing)

15/16-4-2024
prof. Dorota Cieslak
dorota.cieslak@up.poznan.pl
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 Date Hours / room Subject

15.04.2024
Monday 9.00AM-11.30AM Lecture : The sperm (prof Dorota Cieslak 3h)
Room 011 (basement)

16.04.2024
8.00-11.00AM Lab class : The sperm (prof. Dorota Cieslak 5h)
Room 08 (basement)

17.04.2024 9.00-10.30AM Lecture: The oocyte (dr hab. Piotr Pawlak 2h)
Wednesday 11.00-1.30PM Lecture : The embryo (dr hab. Zofia Madeja 3h)
18.04.2024 8.00AM-11.00 AM Lab class : The oocyte (dr hab. Piotr Pawlak 5h)
Room 08 (basement)

19.04.2024 2.30-5.30 PM Lab class: The embryo (dr hab. Zofia Madeja 5h)
Friday Room 08 basement

2
 The oocyte The sperm

100 µm

The embryo

50m 3
 Gametogenesis

gametes – products of the meiotic division:

one oogonium = 1 functional secondary oocyte and 3 polar bodies
one spermatogonium = 4 functional sperm cells

number of gametes
oocytes - limited by the initial mitotic divisions
(eg 4 in cattle) during fetal life
sperm cells: theoretically unlimited number of cells continuously
produced during adult life

4
 The male gonad

• Production of sperm cells

• Production of hormones (mainly androgens)

5
seminiferous tubules

6
 Cross section

The total length of the seminiferous tubules

– 8.7 kilometers (boar) and 4.9 kilometers
(bull)
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A Sertoli cell in bovine testis
Lumen of the
(cross section) seminiferous tubulus

Mature sperm cells

(microtubular lumen )
seminiferous microtubules

A round spermatide
(cytoplasmic briges)

Primary and secondary

spermatocytes

A diploid
spermatogonium
8
 Late spermatides /early sperm cells
before being released to the lumen (bull, scanning microscope) 9
Spermatogonial reservoir

1 2

Pluripotent spermatogonia (stem cell) – undergo mitotic divisions

one daughter cell differenciate into a spermatocyte (1) the second daughter cell
remains undifferentiated (2) and stays in the reservoir 10
 Spermatogoniogenesis
Mitotic divisions

Spermatocytogenesis
Meiotic division

Spermiogenesis

The numbers refers to mouse

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 Spermiogenesis

The final process of differentiation = spermiogenesis –

functional sperm cells

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 1,4. centrioles 2,3. Golgi aparat 5.mitochondrium
6. nucleus 7. miedpiece 9. tail (flagellum) 10. acrosome 13
A – head (1 – acrosome, 2 – nucleus, 3 – centriole), B – neck, C –
midpiece, D,E – tail,

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Morphology of the bovine sperm cell

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sheep cattle pig mouse dog cat human

Morphology of the sperm cells of

selected mammalian species
100m
 Sperm preparation for fertilization
capacitation

Epididymis – sperm membrane is covered by stabilizing/protecting

molecules (cholesterol, proteins) ; reduced motility and activity

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18
 Bull spermatozoa
„incubated” in the
oviduct
(note the tight contact
with the cilia of the
oviductal cells)

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Sperm journey in the female reproductive
tract

6 billion - average sperm content of a bovine ejaculate

1 thousand – estimated number of capacitated sperm in the
oviduct ampulla 8-12h later

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 Sperm capacitation in vivo vs in vitro

In vivo In vitro
Several hours of 18-20h co-incubation of
interaction sperm oocytes and sperm cells
cells with female In medium supplemented
reproductive trackt with heparin

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Acrosomal reaction

Initiated when sperm cell

reaches the cumulus cells of
a COC

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 Acrosomal reaction

• Fusion of sperm membrane with external acrosome membrane

• Acrosomal enzymes are released (mainly proteinases)

• Enzymes digest intercelullar bridges between cumulus cells

• the proces dependent on pH, temperature, ion concentrations etc

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24
Plemniki chomika penetrujące osłonkę przejrzysta oocytu 3 h po inseminacji

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 Interakcja plemników z mikrokosmkami (microvilli) na powierzchni
oolemmy oocytu chomika złocistego (po usunięciu osłonki przejrzystej)

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ICSI – intracytoplasmic sperm injection (human IVF)
 Intracytoplasmic sperm injection (ICSI)

First pregnancy reported (humans) - 1992

Efficiency - about 30%

Advise to apply ICSI

1) Low sperm cell number
2) Desabled motitily
3) Low number of morphologically normal sperm cells
https://www.youtube.com/watch?v=GTiKFCkPaUE
https://www.youtube.com/watch?v=w_OZynGBo4o

ICSI – intracytoplasmic sperm injection

The sperm cell
 ICSI is a standard
procedure in equine
IVF

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Sperm sorting („sexing”) in flow cytometer

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50% ? 50%
Sex of the offspring – impotance to dairy and
beef industies

• Dairy cattle – female offspring

• Beef industry – male offspring (better capacity of
meat growth)
DNA content – the only sperm parameter
succesfully applied to sperm sorting

The X bearing sperm contains more DNA

The difference is species related

(2.8% - human, 7.5% chinchilla)
The X and Y chromosomes differ in size and DNA content

Y
cattle 3.7% - 4.2%
Jersey 4.2%
Angus 4.07%
HF 4.01%
Hereford 3.98%

Chinchilla 7.5%
Human 2.8%

X
 Sorting in flow cytometer

• The only procedure to date with application to cattle industry

• Published in 1989 (rabbit sperm)

• Technology patented in 1991 (dr Lawrence Johnson, US Department of

Agriculture) the patent has expired

• First calves born: 1993 - IVF embryo; 1997 – AI embryo

• The first commercial license – 2002, Congent XY Inc. UK

• Based on differences in DNA content (cattle 3.8% ±0.5)

• Sorting accuracy - >90% (50% higher efficiency at 75% accuracy)

Biological background of sperm sorting
 The share of sperm bearing the X chromosome may vary
in a big range (cattle, pig)

1. Ejaculates of different males

26.5% - 96.5%
1. Ejaculates of the same male

24% - 84%
3. Calves born from different ejaculates of the same male
16.1% - 72.3%
Chandler et al. 1998 J Dairy Sci 81
How the sperm sorting proceeds?
The shape and size of the sperm head plays an important role
(Garner 2006 Theriogenology 65)

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 Garner 2006 Theriogenology 65

Shape and size of sperm heads

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 Sorting index
(area of the sperm head x difference in the DNA content %)

bull 34.5 m2 x 0.38 = 131 (easiest)

Boar 37.5 m2 x 0.36 = 115
stallion 15.2 m2 x 0.39 = 59
humans 10.8 m2 x 0.28 = 31
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Garner 2006 Theriogenology 65
 Sperm sorting - bases

• Before sorting – sperm sample dilution and staining with

fluorescent dye Hoechst 33342 (for living cells)

• The amount of the dye intercalating into DNA double strands

represents the total DNA content within a sperm cell

• After exiting the stain by the laser the fluorescence is measured by

detectors

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44
Sperm sorting - bases

The nozzle – the central part of the

system
Fluid pressure (droplets containing
single sperm cells; speed of cells)
High vibration rate
Sperm orientation

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46
Sperm sorting - bases

Sperm cells are resuspended in a

medium transformed into series of
droplets when leaving the nozzle

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 Detector 90°

Detector 0°

Niemann H. and Wrenzycki C. (ed.) (2018). Animal Biotechnology 1 – Reproductive

Biotechnologies. SPRINGER chapter 4 Technique and Application of Sex-Sorted Sperm in
Domestic Farm Animals
48
 Fluorescence detection

1. Detector 0 - front plane of the sperm head

(DNA content)

2. Detector 90 – side plane of the sperm head

(position-orietnation)

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 The orientating nozzle
(CytonozzleR SXMoFloR ,
DakoCytomation, Fort Collins,
USA)

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Discarded Sperm cells – no electric charge

Wrong head orientation

Morphologically abnormal
two or none sperm cells in a droplet

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 Time of sexing one ejaculate

• 2 sorters (each with 2 nozzles)

• Aliquots of raw semen – 50-100 million sperm/ml
• Sorting speed – 30 000 sperm/s per nozzle

• 7 hour sorting - 3 000 million sperm/4 nozzles (750 mln/nozzle)

• 1 ejaculate comprises on average 6 000 million sperm cells

Seidel GE (2014) Animal 8: 160

1 ejaculate = 7 hour sorting by 4 sorters/8 nozzles

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 Yield

1. 50% of sperm cells initially subjected to sorting will be

successfully sorted (25% of each sex)
2. The AI dose of sorted sperm - 2 x 106
(a standard dose 8-12 x 106 )
1. The yield – 14 AI doses of sperm of required „sex” / h

Seidel GE (2014) Animal 8: 160

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 Yield – an example
2 sorters with 2 nozzles

Number of sperm cells Comments

32 000 Sorted sample

- 3 000 Discarded sperm (dead, dying)
- 1 000 Discarded sperm (technical issues eg two cells per droplet, to
close in the stream)
- 8 000 Discarded sperm (not evaluated – wrong orientation)
- 4 000 Discarded sperm (biological and procedural variability)
16 000 Successfully sorted (8000 cells of each sex)
50% of the initial sperm cells
25% of each sex (X or Y)
Seidel GE (2014) Animal 8: 160

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 Factors affecting the sorting efficiency and sperm survival

Sorting negatively affects DNA integrity

1. Pressure in the nozzle – 50 psi (3.5154 kg/cm2) reduction of the pressure

to 40psi has a positive effect on sperm survival but increases the rate of
non-oriented sperm cells
2. Sperm speed by leaving the nozzle - 90km/h
3. 30 000 sperm can be evaluated per second/ nozzle
4. High extent of sperm dilution
5. The dye and laser light

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Factors affecting sperm viability during sorting

Incidence of DNA damage (double strain integrity)

1.8% control (not subjected to sorting)

3.2% sorted without staining and UV exposure

3.4% sorted only by UV exposure
3.1% sorted only by staining
3.0% complete sorting

Garner i wsp 2001

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Recent advances in Biotechnology - Animal Biotechnology
Reproductive biotechnology
The sperm – lab class
26-3-2024
sperm quality evaluation

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 Classification of sperm defects

Primary Secondary

originating during originating during

spermatogenesis and sperm maturation
reflect pathological
and processing
processes in the
(after ejaculation)
testis,

More significant to less significant to

male fertility male fertility

A maximum of 15% of the sperm cells of impaired morphology (bull)

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 Primary defects

2 double head sperm cel

3 acrosome defect
5-9 head defects
6 pyriform head
11-14 cytoplasmic droplets
15 Dag`s defect (coiled tail)
 Secondary defects

19 loose head
20 Separated acrosome membrane
21 odaxial tail position
23, 24 tail loop
Eozin-nigrozin (EN) staining

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 Eozin + nigrozine staining

1. Thaw the straw in a water bath 37C

2. Place 3µl of sperm sample on a slide
3. Mix the sperm with 3µl of eosin (by a tips) and leave for 30 seconds
4. Add 9µl of nigrozin stain and mix
5. Spread the mix on the slide with another slide
6. Let the slide dry at room temperature
7. Analyze 100 sperm cells (count live and dead cells)
Eozin + nigrozine staining
1) live to dead sperm ratio
2) % of morphologically abnormal sperm

Nigrozin – violet background

eozin – penetrates the sperm head in case of membrane damage
 1
2

live (1) and dead (2) bovine sperm cells

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stallion
66
Carell et al. 2013 Spermatogenesis. Methods and protocols 67
Cattle (bull)

68
 distal

proximal

Cytoplasmic droplets (the bar represents 10 µm)

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 2

10 µm 10 µm

Midpiece defect (1) bent tail (2)

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 10 µm

Double head sperm cell (double midpiece)

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72
73
74
 bull

14 sperm cells:
4 stained=dead
% of live perm 71%
Abnormal 0%

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 Eosine + nigrozin staining

Picture 1 Picture 2

Total numer of sperm cells in the picture

Number of sperm cells with stained head
The rate (%) of live sperm cells

Number of morphologically abnormal sperm cells

Type of abnormality
The rate of morphologically abnormal sperm cells

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1

77
6

78
4

79
5

80
6

81
Horse (stallion)

82
Dead (red) and live (white) equine spermatozoa
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Double head sperm cell
84
 2

Normal (1) and coiled (2) sperm cells

85
 Hypoosmotic swelling test (HOS)

1. Thaw a straw in a water bath 37C

2. Incubate 200 µl of sperm in 1ml of HOS medium
(4,5g NaCl, 2,2g trisodium citrate, add water up to 500ml, osmolarity
100-150 mOsm/kg, pH 7) at 37°C 60 minutes
3. Place 10 µl incubation mixture on a warm slide and cover with cover slip
4. Evaluate 100 sperm cells under light microscope:
HOS positive cells (coiled or bent tail)
HOS negative cells (with straight tail )

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 HOS
Hypoosmotic swelling test

Morphology of the sperm tail

the number of peripheral

filaments decreases and reaches
its minimum at the end of tail
 Hypoosmotic swelling – biological background

Water migrates from the HOS medium (hypoosmotic) of a lower pressure into the living
sperm cell with higher pressure
The main condition: intact and functional sperm membrane
 Hypoosmotic swelling – biological background

Active transport of water into the cell till reaching the isoosmotic conditions
Cell udergoes swelling

Functional=live sperm Non-functional = Dead sperm

Morphology of sperm tail after HOS test

A – no changes (HOS negative)

B – coiled tail (HOS positive)

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Cattle (bull)

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92
HOS test

Picture 1 Picture 2

Total numer of sperm cells in the picture

Number of the HOS+ sperm cells

The rate of the HOS+ sperm cells

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94
1

95
2

96
3

97
4

98
5

99
6

100
Pig (boar)

101
Boar sperm cells: A – HOS+

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European bison

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104
 HOS
E+N Membrane functionality

Live to dead ratio at the Live to dead ratio at the

moment of sperm staining moment of slide evaluation

% od morphologically
abnormal sperm