A study of the transport of spermatozoa in the reproductive tract of the ewe after artificial insemination : a thesis presented in partial fulfilment of the requirements for the degree of Master of Agricultural Science at Massey University

Copyright is owned by the Author of the thesis. Permission is given for

a copy to be downloaded by an individual for the purpose of research and

private study only. The thesis may not be reproduced elsewhere without

the permission of the Author.

REF1CDUCTIVE 'I'RACT OF TriE K.'.'E AFTER A '!tTIFICIAL IK SEI.:InATI OF

A thesis presented in partial �ul�ilment o� the require�ents

�or the degree o� 1-:aster o� Agricultural 2cience in Animal Science

at

Massey university

�:tiN HAThE GILES ^.

1975

A Qillion willion spermatozoa, All of the= alive:

Out of their cataclysm but one poor r�oah, Dare hcpe to survive.

---- ALDOUS LECTA 'W EUXIBY

This investige.tion was conducted at the Sheep Production Centre of the Sheep P.usbandry Department, !.:assey University.

The experimental work was conducted in July, 1974- and from February till June 1975, and represents original rese2rch

by the author under the supervision of Dr. L::C. i.IcDonald, Reader, Sheep Husbandry Departrr,ent, �.�assey University.

A

C K }; C VT L E D G E E F. N T S

I am e sp ecially indebted to Dr . I.:. F . r.:cDonald for h i s superv ision of the nork, his invaluable a s si s tance i n the plar�ing and c onduct of the expe rimental work, and helpful critici sm of

the text.

Appreciation is extended to �.:r . Co l in r-.;uir fo r technical a s si stance ; i.:r

.

!.:urray Ford for routine management o f the animal s , and to I.:r . Ivan \.'alker f'o r acting as c hief slaughte rman .

Gratitude to Dr .

L L.

racmillan of t..he Awahuri Artificial Breeding Centre is extended f o r provision of several p ie c e s of equipment and for hi s intere st in the s tud�r throughout the year.

Spec ial thar�s are due to Profe s so r

A. ^L.

tae fo r advice on statis tic s and to all o thers with vrhom the author had helpful

discus sion. 1;:embers of the 1.�as sey J,ibrary s taff de serve thar.ks for their a s s i s tance in research of the literatur e .

I thank 1.:rs.E. Lynch for typ ing thi s manuscrip t . Finally,

^I

exp re s s my deepest apprecia tion to my wife N"oeline for he r pat ience and p art icipation in my p o st-graduate

studie s , and to all other �emb ers of my_family who have contributed in

many ways

to enable the pre sentation of this thes i s .

Financ ial ass i stance from the Helen E. Ake rs Scholarship i s acknowledged .

ABSTRACT

Two prelimin�J experiments were conducted with arti�icially inseminated ewes to e.'Car::ine the considerable variation in counts o�

spermatozoa recovered in �lushings �rom the reproductive tract. In the second trial, road transport stress immediately after

insemination caused a reduction in the number of spermatozoa reaching the Fallopian tubes.

In the third trial, sperm transport after insemination with fresh, undiluted semen at two sites in the tract at predetermined times relative to S)rnchronized ovulation

(

progestagen-HCG treatment

)

was investigated. �eposition o� semen at the uterine end of the cervix or within the uterus using a specially adapted inseminating pippette, resultec in the recovery of higher numbers of spermatozoa from the uterus and Fallopian tubes 4 hours after insemination, than did insemination at the external cervical os. Twenty-four hours a�ter insemination similar numbers of spermatozoa were found in all segments for both sites o� insemination. Deposition of sen:en be�ore or after the expected time 0� ovulaticn, or at both times, had little effect on the numbers o� spermatozoa recovered. i.:ore ova vlith

accessory spermatozoa were recovered from oviducts containing high numbers of sperm.

The transport o� spermatozoa after deposition of �resh or frozen semen into the uterus by surgical and non-surgical methods, was studied in a fourth experiment. The number of spermatozoa recovered from the uterus and isthmus was similar for both types of semen, but only 'fresh' spermatozoa d eposited at surgery, reached the ampulla in significant numbers. 'Frozen' sperm

deposited by both methods, and fresh sperm deposited non-surgically,

did not reach the ampulla within � hours of insemination.

Impaired transport of 'frozen' spermatozoa to the s ite of fertilization appeared to be a result of reduced longevi� of these cells within the female trac t, and may involve factors s uch as bre��age of sperffiatozoa in the uterus, and delayed

transport in the oviducts.

The i�rovement of sperm transport in the ewe through t he develop ment of non-surgical, intrauterine inseminations and

using either fresr�y exte�ded or frozen senen, are discussed.

vi

Chapter

I

II

Ill

P'RE'?ACE

AClTOIDEDGEr.�rTS :ABSTR-<\CT

IJIST

OF TA.BDS LIST

_OF

FIGt.T?..ES

LIST

OF PLA'ISS

n:T?.c

DUG

^TI _or

snrc::�C�IZA':'IC 0? OVT.)I.A':'ICr AFD

:?B"?.TII"ITY

TO JT�::IrATIC·I·:S AT P1:1.ED::Tf.cu�::-!:"S"J TH:SS

S�rt:::A'l'OZOA Ir THE "TWPRCT)liCTJ:V"':

':"'�ACT SPE?J.:

l"LJ1.:3"S'1S ^At� FE'?.TILIZATIOE

FACTC1S ccr: '?JBUTn:G TC FAILL�E OF 'SFB"l::

FACTC'?.S

AF'57.CTJTG ��'U.:A?OZOA

�J:SPORT IN V

P'JRPC� ArD ^SCCFE OF STUDY

�-':A'.!.SRLt...LS AF:l �TIEC·DS '?.XPE?JJ.:Et�TAL A�;Ji,:ALS

SEJ.:E?·; COLIECTIOE

.Al�D

EVALUATIGr� -./

iii iv V X

xii xii

1 2

2

4 6 18

19

20 23

25 25 25 27

SLAUGHTER ^OF

E.''ES 28

POST-SLACGH':'EP.. T'?..EATI.':ET�T OF REPRODUCTIVE Tn.AC'l'S 28

EVALUAT:::cr:

C"'

�CUJ'"':-u�S

EXPETtH:SFT

1

^-

-

_THE

DISTUBUTIGr

OF SFBry�:A

T

_CZ^OA

IN

T�

GEI"ITAL T"_4.CT AFTS1. A-q,TD'ICIAL Il'TSEI�U.:ATIO�

�A'!'-LY

_0:1. ^LATE _H�

CESTRUS

EXPSUI��T

2

^{· --} TJ-:S 'F:£1' :<'l'.:C':' OF ".CAD T?_4.I:SPC? ^..

T D:J·�DIATSLY

_AFT?'!{ ARTI:.'ICIAL IrSE:.:nrATIC.Y, O'fiT TEE

DISTIUBUTICN

_011' �.�i.FATOZCA IN THE

REP1.0DU�TIVE �CT

OF � �·�

CO'fiTCLUSICNS

31

31

33 36 IV EXPE1:1.JJ:-r.1-:T 3 ^-

-

�p�nJ.:..�TCZOA D� THS n_?,P'!lODUCTIVE Tu_A..CT

AFTSR

A'I'J.TIFICIAT:

n:�;.·n·ATIO:i" '-':'IT.r "?'i.,-r.:SH,

UJi'DIIU�D

37 37

C_ha_pte_r

:RESULTS

I II

II^I

IV

(a) General

(b) :Sxpe_rimen_tal Design (c) Stat_isti

c

al �nalyses

TH:E

CF CVTJLA TI ^CN

l.:.;CO'\Tl';�Y CF

SP'"'l'ATC·ZOA (a) ?rom the Vagina (b) ?rom the Cervix (c) From the Uterus

(d) From the Fallopian

Tubes

(e) Total Sperm �ecovered

THE DI�T'ftiBUTIC!T OF ::�'t?J: IF THS ·:merE l?.SPRCDUCTIVE TRACT

(a) Vaginal Insemination (b) Uterine Inserr.iration

ovm; REeov;;:Jty .AJ-:-D "CS�TII.JI ZATI CN DISCUSSIOr

(a) Synchronization of' Cvulation

(b)

Sperrratozoa Transport

SURGICAL A!':::l YCE-SU�GICAL D�?CSITICT: CF :<'"1"8�:-: c-q

EATEUALS AriD l.ETI-:CDS RESULTS

I 'Y.Ll::E CF ^{OVU"T ..} ATICN

II :?..ECCVE�Y OF sp:;:n.� 'A'XZOA (a) From the Cervix (b) Fro� the Uterus (c) From the Isthmus (d) From the Ampulla (e) Total 8perm Recovered III EP.EAKAGE OF �PE�l.:.ATO�OA

IV IEUKOCYTES HT TJ-� 1tEP�ODUCTIVE T'O.ACT V OVUM ?.ECO"'J'£-q_y AFD :<'E�TILI7ATION

DISCUSSION

( a) Time _of' Ovulation (b) Spermatozoa Transport

37 37 38 39 39v 40 l+O 40 4 1

43 43 44 44 45 45 45

48 48 1!-9 49 ^v' 50 50 5 1 5 1 5 1 52 53 53 54 54 54 54

Chapter

VI

THE VAR.IABILITY CF SPERr COID;TS INTRA-UTE!tH:E IYSEkiFATIO�,: ^OF

E1.'!ES SFEm::ATOZCA TRJ�:-SI'ORT

_AF�'1 ARTIFICIAL

INSE::IFATI or;·

THE �?FECTS OF STP.ESS 01':: SP"SR1.: T1ATSFC::tT IN THE AllTIFICIALLY IFSEI.IH'A'T.'ED E':.::S

CONCL'JSI ^m;s

Pace

58 5'8 59 61

66 69 72 89

L'�' or ..,-(.l.lSL-ES

Table Page

3.1

3.2

3.4-

4-. 1

4-.3

4-.5

4-.8

4-.9 4.10 4.11

Estimations of th e time taken for spermatozoa to travel from the anterior vagina to the Fallopian Tubes in the ewe

Treatment group

s -- Experiment 1

�Turubers of spermatozoa recovered from the genital tracts of enes in Experi ment 1 Estimated numbers of spermatozoa recove

r

^ed

from the rep ro .i^{u c}tive tracts of erres slaughtered 4- hours after inse:r.ir..ation

Numbers of spercatozoa recovered from within the reproductive tracts of trucked and

non

-

trucked e

·.

^:es

Incidence of ovulation at intervals after HCG

--

'Expe rirr_en t 3

�ffect of interva

l

^{from PCG} to slaughter on the number of e;.-e s ovulating

!�um1)ers ( transforr.:-,ed.) of sperr...atozoa recovered from the anterior ^vagina of vaginally inserrinated ewes

�ffect of

s

^ite of insemination on the number of erres yielding sperm from the ce

rv

ⁱ

x

Numbers (transformed.) of spermatozoa recovered.

f rom the cervix of vaginally inser.�ina tea ev;es numbers (transferred) of sp er

s

at^c

z

oa recovered from the uterus (Y.xperiment

3)

The interaction of site of inse�ination and time of slaughter on the numbers of sperm recovered f ron the uterus (Experiment 3) The interaction of site of insemir..ation and.

time of slaughter

with

_{time of} insemination Numbers (transfor^med

)

of spermatozoa recovered.

from t he ovid.ucts (Experiment

3)

The interaction of site of insemination ard time of slaughter on numbers of sperm in the oviducts (Experiment 3)

The distribution of ewes classified. accord.ing to the number of spermatozoa recovered. from the Fallopian tubes

f 6 32 f 32

f 35

f 35

f 39

f 39

f 40

4 1 f 4 1

f

4-1

f 41 f 4-2

f 4-2

f

43

f 43

Table

4.13 5 .1 5 .2

5 .3

5 .4

5 .5 5 .6

5 .7 5 .8

5 .9

5 .10

5 .11 5 .12 5 .13

Treatment e�fects on the percent recovery of sperrrGtozoa from the genital tract Incidence of the recovery of ova with and without sperm on or in the zona pellucida Incidence of ovulations at intervals after HCG

(

Experiment 4

)

Treatment effects on follicular development and the number of corpora lutea on ovaries at slaughter

:rumbers

(

trar:sfor:r:ed

)

of sperr:,atozoa recovered from the cervix of ewes after intra-uterine

• • � 4

�nse:'!J.na v�Ons

!�umbers

(

transforDed

)

of spem.atozoa recovered from the uterus after intra-uterine deposition of semen

Numbers

(

transforced

)

of sperrr�tozoa recovered from the istrzus

Interaction of I!:ethod of insemination and time of s laughter on numbers of sperc in the istr.2us

Numbers

(

^transfo�ed

)

of spermatozoa recovered from the aBpulla

Analysis of variance of numbers of suerm recovered fro� the acpulla

24

ar3 48-hours after ir>.s emir:ation

Mean percent recovery of spermatozoa from the tract after inserr:ination with fresh or frozen sel!!en

1:ean percent broken spermatozoa in the flushings of the uterus

Presence of leukocytes in the uterus

Incidence of the recovery of fertilized ova Summary of some mechanisms of sperm transport in the reproductive tract

f Refers to f ollowing page.

f

43

50

f 50

f

50

f

51

f 51

f

51

f

51

f

52

f

52

53

f

5 3

f

54

f

5 7

LIST OF FIGUP.ES

Figure

4.1 Plan of Experiment 3 (Block 1) 4.2 Sequence of procedures relative to

aclrninistration of HCG -- Experiment 3 4.3 Distribution of sperm in the

reproductive tract (Experiment 3) 4.4 �elationship betrreen number of tubal

speroatozoa

e.r..d

proportion of ova with on or in zona pellucida

5.1 Plan of Experio.ent 4

5.2

^·

Sperrratozoa populations in the isthmus 5.3 Sperm populations in the ampulla after

deposition o� fresh or frozen semen

Plate 2 .1 2 .2 2 .3

LI�T OF PLATES

Components of the artificial vagir�

Photomicrogr�ph of spermatozoa after live-dead stainin; (eGsi�nigrosin)

Inseminating instruments Insemination in progress

sperm

5.1 Exteriorised uterus with inserted pippette at laparotomy

5.2 5.3

5.4 5.5 5.6

Photomicrograph of phagocytosis of a spermatozoan in uterine flushings

Single-cell, unfertilized ovum recovered 24 hours after non-surgical insemination with frozen semen

2 -cell ovum recovered 24 hours after surgical insemination with fresh semen 6-cell ovum recovered 43 hours after

non-surgical insemination with fresh semen 8 -cell ovum recovered 48 hours after

surgical insemination with frozen semen

f �efers to following page.

Page f 37 f 37 f 43

f44

f 48 f 51 f 52

f

30

f 30 f 30 f 30 f 54 f 54

f 57

f 57

f 57

f 57