Effects of freezing on ram and boar sperm

Y

EFFEC1S OF FREEZING ON R.AM A¡TD BOAR SPERM

a thesÍs

submitted by

Betty Y.

M. ^CHENG

to the University of

Adelaide towards ^*,he degree

cf

^Master

of

Âgricultural

^Science

Department

of

Anj-mal ÞhysÍo1.ogy

Waite

Agricul.tural

Research

Irrstitute

Universíi.y

of

Àclelaide

JuIy

1976

I

Ta-ble

of

Conten'cs

I.

^Table

of

Contents

2.

Declaration

3.

Acknowlegement

4.

^Summary

5. fntroduction

fhesis

Thle advantage

of frozen

^semen Chapter One

Literature

^Review

Storage

of

sheep spermatozoa Ð<periments

with pig

spermat-ozoa

Freezing, storage and thawing j-njury The biochemical

function of

^GoT

The release

of

GOT aJ¡d GE{l from ram and

boar

spe:rmatozoa

Protein

leakage from

frozen

^sernen

Electron micrograph

description of

ram and

boar

frozen

sperm

General

characteristics of

ram and

boar

^semen

A.

Boar semen

Constituents

of

^semen

Gelatinous

material

Chemical com¡ronents

a.

Choline

b.

Ergothioneine

c. Lipids in the

seminal plasma

d.

^Fructose

e. Inositol

and

sorbitol f. Citric

^acid

g.

Proteins

B.

Ram semen

(1)

Grading ram semen

Q)

Quantity

of

^{semem and number}

of

s¡:ermatozoa Chemical composition and metalcoU sm

I t

2 3

3 5

7

! ó

I

9

9 10 10

I1

L2

13

L4

I5

1s

(3) Protein constituents of

ram semen

Sperm

motility

^and

evaluation of

^{semen qualiEy}

(A)

Evaluation

of

^semen

quality

(i)

General methods

(ii) Histol.ogical

^methods

(iii)

Chemical and biochetnical methods

(B)

Factors which

affect

^sperrn

motility it ¡¡;!p (i)

Management

(ii)

Temperature and lighÈ

(iii) Nutrition

(C) Sperrn

motility in

vitro_

(1)

_PH

(2)

Díluents

(3)

Temperature and storage (,4) osmotic pressure

(5) Electrolytes

(6)

Female

genital- tract fluid (7)

_Toxin

(8) Nutrients

Material

and methods

L. Animal

Training

2.

Semen

collection

3.

Semen

evaluation

steps

4. Live

and dead

staining 5. Motility tests

a)

I¡lave motion

b)

Oxygen consumption

6.

Freezing and bhawing procedures and media

¿)

Rarn semen

diluents b)

^{Boar semen}

diluents

7.

^pH,

osmolality

and

ionic conductivity

measurements

8.

Glutamic-oxaloacetic-transaminase an.elysis

15

I5

16

\7

20

20-2L

22

23-24

25 25 26 L8 19

)a

26

27

2B

Removal

of

spermatozoa from seminal plasma Sample treatment

Colorimetric reaction for

determination

of

^GOT

9.

Polyacrl'larnide ^geJ- electrophoresis

Separation

of

seminal plasma

proteins

Stain

10. Electron

rnicroscopy

of

spermatozoa Procedure

of

washing sPermatozoa

Fixation

Dehydration -- ^Embeilding

.Sectioning Staíning Results

I.

O>q¡gen consumption

of

^{ram and}

boar

spermatozoa

Effects of diluents

Compariaion

with solution of different osmolality 2.

^CÐT

release of

ram and

boar

spermatozoa

Effects of different diluents

^{on CÐT release}

Ran spermatozoa Boar spermatozoa

3.

Disc electrophoresis Èoar spermatozoa

Ram spernatozoa

29 29

30

31 31-32

34 35-36 33

37-39

40 40 4L

4. Electron

microscopy

of

ram ^and. bOar spermatozoa before

and

after freezing

⁴²

Ram spermatozoa Boar spermatozoa

Discussion

Section I.

Frozen semen

evaluation by

GOT enzyme nethod

43

i

t ì

I ì

I

Section 2. Protein

leakage a¡rd

role of reaction

and low

temperature

44

Section 3. Ultrastructure of frozen spermatozoa

45

Seqtion

4. Protein

leaJcage and polyacrylamide ge1

eleetro-

phoresis

₄₆

Conclusion

TLre

best diluent for

ram

spermatozoa

47

flre best diluent

and temperature

for

storage

boar

spermatozoa 48 Appendix

References

Acknowledgements

I

hereby wísh

to

thank ^{a1-1 persons} connected.

with

the production

of this tfiesis. In particular I wish to

acknowledge

the

^guidance

and

supervision of

Professor W.

V.

Macfarlane

during the

experimental work and

wríting of

tJ1is

tåesis.

Thanks

are

^due

also to Dr.

^D.E-

Brooks

for his inspiration.

Further I wish to

thank

Dr.

R.

Bais, Mr. R. tr[iles

and

Mr. B. Palk for technical assistance, Mr. P.

Ifeap and

l4r. ts.

^Stone

of the pig

_Research

unit,

Department

of Agricultur:e for theír

cooperation

in the boar

^semen

collection

and. many

others.

To ^nV husband Charl-es C"K. Ctreng

with

my

sincere

^thanlcs

for

encouragenrent and company throughout

the

experiment.

Declaration

I declare that thiS thesis

does

not incorporate without

acknowtedgement any

material previousty

submitted

for

^a

degree

or

^diploma

in

^any

university;

^arrd

that to the

^best

of

^{my knowledge}

it

does

not contain

any

material previously

published

or written by

^another

person

^{except where} due reference

is

^made

in the text.

Bet Y.M.Cheng (nee Ju)

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Summary

sperm sr-rrvival

in frozen

^{semen eppears}

to

be determined

largely by the

^anount

of intracel-'l-ular

enz)¡rfles

during freezing

and thawing.Data

are

^presented

to

shorv

the effect-s of different diluents,

tetnperature and

gtycerol

as

well as

^egg

yolk

concentrations

I

^on glutamic-oxaloacetic-transaminase ^(GOT) release.

The

influence of clifferent diluents

^{has been}

related to their osmolality, ionic conductivity

^{and pH.} The imporf,ance

of

^the

enzyme GOT

is that it indicates the

^degree

of cell

damage, and

also efficiently

^shows

the quality of ftozen

^semen

in a

30 rninute

test.

polyacrylamide

gel electrophoresis

has been used

to detect extra dense protein

^{bands which appear}

in frozen

^samples

of

semen.

lt

seems

that the protein leaks

^{from damaged}

celIs.

Electron

microscopy

of

boar and ram spermatozoa

yields a picture of

severe

disruption of the frozen cell

^membrane'

It is

concluded

that the optirnal conditions for freezirtg

^ram and

boar

spermatozoa are:

AnimaI Boar

Ram

Temperature 37C@

5 c

-1964c

Thawing

40c

¿l

37C

-196 o^C o

pH

7.4 7-4

7.4

Diluents

Tris buffer

^fructose

Tes

buffer with

egg

YoIk

^and

glycerol

Tes

buffer with

egg

Yolk

^and

glycerol or

^{BF3 extender}

Tris buffer with

fructose

Sperm Ringer phosPhate

with

fructose

Sperm Ringer _PhosPhate

with

fructose

7.4 7.4

7.4

Thawing

4ooc 7.4

Citrate

^glucose

These

diluents

^{which minimize GoT}

release'