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OF PASTURE-FED LAl'<1BS (BIRTH TO 8 WEEKS)
A thesis presented in partial f'.llfilment o:f the requirements
for tha degree of
Master of-Philosophy in Veterinary Science at
Massey University New Zealand
KE!'."NETH YERRO ILIO 1983
ABSTRACT
The histology, mitotic index, ultrastructure and Na+-K+-ATPase cytochemistry of lambs reared on pasture
w~re studied during the period of weaning.
Two embalmed mature non-pregnant Romney cross-brGd ewes and thirty Romney-cross lambs reared on i:vlassey University sheep fa.rms pastures were used. Five lambs
{three in 1981 and tvTO in 1982) were taken from their dams on pasture at each of the following respective ages: within 24 hours of birth, and at 12, 23, 34, 45 and 56 days. Stomach-tissue sareples from 1 adult and from the lambs reared during the 1981. lambing season
were prepared for histology using Haenatoxylin and Eosin, Nasson 's green t.richrome, Feriodic-acid-Schiff and
Toluidine Blue stains, and for conventional transmission electron microscopy. Tissue samples from the rumens of lambs reared duri:r:.<; the 1982 season were 12sed for
strontium-capture technique Na+-K+-1\TPase cytochemistry . Gross dissection o£ the stomach of one-day-old lambs
.
confirmed that th2 'largest compartllient at birth is the abomasum, follo·wed, in decreasing order of size; by the rumen, re~iculum and omasum. Progressive development resulted in the forestomach compartments assuming their adult proportions by 56 c.:.ys of age.
Preliminary histological studies of epithelium taken from the rumen, reticulum, omasum and reticular groove of the adult sheep c0nfirmed it to be a stratified
keratinizing epithelium. Five general cell layers ~e£e
clearly seen: s t.r atu.:;:;1 basa1e, :; trct tu;.~;. splnc~uiD.,
stratum granulosum, stratus transiticnale and str~tum
corneum. (In previous stu~ies, the stratum granul0sun and the stratum transitionale have been considered as one cellular spaces in the str~tum cornGum.
&xamination of the forestomach mucosa
revealed a numbe.r o:f changes between birth and 56 days of age: (1) an ~ncrease ~n pap~~~ary • . . . . 1 length;
(2) starting from 23 days, the _deveL)pment of extensive papillary process-epithelial bulb interactions accompanied by proliferation of blood vessels in the papillary
processes; (3) a decrease in epithelial thickness for
•
the first 45 days of age; (4) the appearance of a
com~,=~~ :~y=r of transitional cells by 45 days; {5) the disappearance of glycogen £rum the epithelium; (6) an increase in the amc:.unt of mucopolysaccha.d.de in the intercellular spaces in the stratum corneumj (7) the increase i:1 the number of non-ke.ratinocytes in the basal layer; and (8) the appearance of apoptotic bodies due to single cell death in the basal layer of the epithelium from 45 days of age.
The mitotic index of the epithelium in developing lambs decreased from birth until 23 days cf age, had increased at 34 days, but decreased again between 45 and
56 days.
E-'<amination of the ultrastructure of the a.d'...llt epithelium provided general results consistent with previous studies. . HoVJever, gap junctions ·w·ere found in the stratum basale, stratum spinosum and were extensive in the stratum granulosum. Tight junctions (zonulae occludentes) were seen between the cells· of the stratum corneum.
Langerhans cells and mast cells/globule leul-~ocytes
were classified as non-keratinizing cells. Other non- keratinocytes recognised were the 'indeterminate cells',
lymphocyte-like cells and cells similar to i.,1erkel cells.
Completely keratinized cells appeared in the epith~lium
at about 12 days of age, in association with the increased endoplasmic reticulum protein ~nd merebrane-coatins granules.
Proliferation o£ IDitochon~ria in th2 basal l&y2rs s~artcd
at about 12 days of age, and glycogen deposits in the intermediate layers were not found after this age.
iv Increased folding of the basolateral membrane surfaces of basal cells and progressive thinning of the endothelium of sub-epithelial blood vessels were·also observed. Gap
Ju._~tions in the stratum granulosum became · progressively more obviouti between birth and 56 days of age.
gap junctions were also found.
Annular
Na+-K+-ATPase enzymatic sites were identifit:d fran 12 days of age, on the cytoplasmic membranes of lower granular, spinous and basal cells: the reaction products being localised on both the cytoplasmic and inter-
cellular-space 3urfaces of the plasma mem~ranes.
inhibited the formation of deposits only on the
Cuab.:J..in cytoplasmic side. Alkaline phosphatase activity was
localised in the stratum corneum. ;-.:g ++..:.ATPase was demonstrated in the stratu:;n corneum, the intercellular
spaces in the stratum granulosum and stratum transitionale,.
and in mitochcindria.
It is conclu~ed that the ultrastructural £eatures and Na +--K+-AT.Fase cytochemistry o:f th2 epi~helium a+
days of age appeared to be similar to those found in older animals. However, structural (and presumably
functional) maturity did not ap;?ear -to be complx?te untiJ.
after 45 days of a9e at i.\·hich stage the stratum transi- tionale had become cohlpl2te and the mitotic index and the thickness o£ the epithelium had become stable.
The increase of non-keratinocytes suggests the increasing immunoco'Jlpetence of the epithelium.
Tight junctions and extruded contents of membrane- coating granules in the intercellular spaces of the stratum corneum could provi~e a barrler to th? diffusion of
solutes across the epithelium. The development of gap j1..1ncti ons c:"nd the presence of Xa +-K +--':,TPase e:-1::::yma tic sites in the membranes are consistent with the absorptive
and transport f~J.nctions of the epithelium, particularly the active transepithelial movement of sodium ions.
Future studies could well show hormones, hormone-like substances and antibiotics. to be important in th2
development of the £oresto~ach epithelium in ruminants.
to thank the govern~ents o£ New Zealand and the Republic of the Philippines for giving me the
oppurtunity to study for this degree.
>·lv si.ncer.::> grati tud<? is also extended to Professor R. E. ~~nford for providing the opportunity and fa~ilities
to undertake the study and to my supervisor, Dr. E. J.
Kirk for his advice and assistance during this study.
I would also like to thank ~~Ir. P. Whitehead for his willing help with the supply of ani~als, as well as :vu. Dill :.;orris, :.'·1r. Doug Stewart and .::.1r. :'·1attheY:
;(a tur.1ai tavul~i fer assis-ting in the collection o£ the la!"lbs.
T1·'J.a:1l:s are also ex·ten.dcC to >lrs. Fa::n Sla.clc and ~>lr.
Doug Hopcroft for their assistance and advice in the electron microscope ~·:ork; ·to :'·lr. r.;ervyn 2i:r:tles, :'-irs.
Irene Hall and r~Ir. Roy Sparkman for t·heir help in the preparation of sliqes for histology; to .-1r. Neil ~·lard
for the supply of materials; to >Ir. Toi11 Law for allowing the use of his darkroom and to i'vlr. \\'al t Abel for the use o£ his compute.c.
Thanl·.:.s are also given to T. C. Balassu and P. D.
Buckley for their support during the preparation of this thesis.
CONTENTS
Abstract
Acknowledgements List of Contents List of Figures List o£ Tables INTRODUCTION
CH.t\PTER I - REVIEW OF LIT2IU\TCRE
1 .1 Early Studie_s on the Ruminant Stomach
1. 2 Func1:ional Anatomy of the Ru01inant Sto<nach 1.2.1 Gross anatomy
1.2.1.1 The rumen and reticulum 1.2.1.2 The omasum
1.2.1.3 The abomasun
1.2.2 For·estomach mucosal for:u and architecture
1.2.2.1 Ru:nen.
1.2.2.2 Reticulum 1 . 2 • 2 • 3 G'masum
1.2.3 Blood ~upply and drainage, ly~phatics
and innervation.
1.2.3.1 Arterial suppy 1.2.3.2 Venous drainage 1.2.3.3 Lymphatics
1.2.3.4 Innervation 1.3 Histology
1.3.1 Rumen
1.3.2 Reticulum, reticular groove and omasum
1 . 3 . 3 A~o:nast:~.:11
1.4 Embryology
1.4.1 Orgarwgen2sis
1 A ?
-...""'r.,_, l-lis tog enesis
vi
Paqe No.
· i i v vi
X
i
1.
6 6 8 10 10 12 14
14 15 1?
17 18 18 19 19 19 20 20 22 23 23
')'?
'~ .J
25
Paae No.
1.5 Functional Organisation of the Forestomach
epithelium 27
1.5.1 The ultrastructure of the forestomach
epithelium 28
1.5.2 Proposed model for transport pathways in the epithelium 1.5.3 Histochemistry
1.5.4 3pithelial cell differentiation 1.5.4.1 Keratinization
1. 5. 4. 2 i,Ii totic index
1.6 Establishment of the Ru~inant State 1.6.1 Anato3ical development
1.6.2 Histological development 1.6.3 Physiological development
2.1 Animals
1.6.3.1 Absorption and transport of solutes
1.6.3.2 Metabolism
2.2 Gross Dissection 2.3 Tissue Sampling
2.4 Histological Techniques
2. 5 Electron )'1icroscopic Techniques
2.5.1 Conventional electron microscopy 2.5.2 Scanning electron microscopy 2.6 Mitotic Index
2.7 Na+-K+-ATPase Cytochemistry 2.8 Illustrations
2.9 Statistical ~ethods
CHAPTER l i i - ReSULTS 3.1 Gross Dissection 3.2 Histology
3.2.1 General observations
3.2.2 Histological appearance at dif£er.::mt ages
3.2.2.1 Birth to 24 hours 3.2.2.2 12 days
3.2.2.3 23 days
31 33 34
34 35 37 39 40 42
43 44
47 47 48 49 50 50 52 53 53 55 55
56 56 SB
61 61 62 63
3.2.2.4 34 days 3.2.2.5 45 days 3.2.2.6 56 days
3.2.3 Changes in the number of layers of the epithelium in different ages 3.3 ~itotic Index
3.4 Electron )tlcroscopy
3.4.1 General obseLvations
3.4.2 Observations on the cytoL)gy of epithelial cells
3.4.2.1 Cells of the stratum bas ale 3.4.2.2 Cells of the stratum spinosum 3.4.2.3 Cells of the stratum
gra.nulosum
3.4.2.4 Cells of the stratum -transitio:1ale
3.4.2.5 Cells of the stratt..lm corneum 3.4.3 Observations on the ultrastructure
the epithelium in different ages 3.4.3.1 3irth to 24 -f1ours
3.4.3.2 12 cays
3.4.3.3 23 and ::; "Li ~ days 3.4.3.4 45 and 56 days 3. 5 Na +-K+-ATPase Cytochemistry
3.5.1 Light niicroscopy 3.5.2 Elect£on microscopy CHAPTER IV - DISCJSSION
4.1 Classification and Nomenclature
4.2 Organisation of the Forestor:"a.ch ~·lucosa
and Epithelium as Related to their Functions 4.3 Keratinization
4.4 Epithelial Non-keratinocytes 4.4.1 Lang~rhans cells
4.4.2 Indeterminate cells
4.4.3 Intraepithclial lym?hocytes
4. 4. 4 ;:,~as t cells and .globule leukocytes 4.4.5 Other non-keratinocytes
viii
Page No.
63 64 64 65 65 66 66 67 67 70 71 72 7'3
74 74 74 77 78 78 78 79 81 81 37 91 94 94 97 97 98 99
Paoe No.
4.5 Structural Changes from Birth to 56 days
of Age 102
4.5.1 Gross anatomical development of the forestomach
4.5.2 Gross and histological development of the 'forestomach mucosa
4. 6 ivli totic Index
4.7 Ultrastructural Features o£ the Zpithelium during Development
4.8 Na+-K+-ATPase Cytochemistry CP.APTER FIVE - CONCWSIONS .
APPENDIX .._ T APPENDIX II APPENDIX III APPENDIX IV
~;PPE?'-IDIX
v
R3FE.i:(ENCES
102 102 110 113 118 124 128 129 132 135 137 139
X
LIST OF FIGUI~ES
Figure No. TITLE Between Pa9es
1.1 Diagrammatic representation o£ the stomach of the sheep
2.1 Tissue sampling sites
3 .. 1 Diagrammatic representation of the stomach c£ the adult she:ep; right side 3.2 Reticula-rumen of sheep to show position
of internal structures
3.3 Diagrammatic representation of the stomach of a one-day-old lamb (actual size).
3.4 Scanning electron micrograph of the
abnormal structure found in the reticulum of a one-day-old lamb killed in 1982
3.5 Light micrographs of papillae taken from different-locations in the forestomach of the adult
3.6 Light micrographs of epithe1ia taken .from the forestorr.ach of the adult
3.7 Light micrographs o£ the forestomach 3.8
3.9 3.10 3.11
mucosa taken £rom one-day-o1d lambs Light
taken Light taken Light taken
T "' '\... •
L~9ll 1:
micrographs of the rum.inai mucosa from 12-day-old lambs
micrographs o£ the ruminal mucosa
£rom 23-day-old lambs
micrographs of the ruminal mucosa from 34-day-old lambs
r:1icrographs of the ru;:ninal mucosa
10 - 11 49 - 50 56 - 57
57 - 58
58 - 59
58 - 59
59 - 60 60 - 61 61 62 62 - 63 63 - 64
63 - 64
taken froB 45-day-old lambs 64 - 65 3.12 Light
taken from 56-day-old lambs 64 - 65 3.13 v~an ~itotic indic~s in the ruminal
epitheliu~ in different ages
3.14 Semi-diagrammatic representation of the.ruminant forestomach epitheliun 3.15 Electron ~icrograph of the epithelium
taken from the forest~ach sf the adult
66 - 67
6"'• - 67
sheep, stratum ~~sale. 67 - 68
3.16 Electron m~crographs of the furestomach epithelium taken from the ~euJt. Stratum
Basal0 68 - 60
3.17 .:ilectron microQrapl1s o£ the epithelium tak.;;;n fruru ~he £0restomach of the adu 1 t sheep. Stratum Bc,sale.
3.18 E~~2+ron micrographs of the epithelium taken from the forestomach of the adult sheep. Stratum Spinosum.
3.19 Electron micrographs of the epithelium taken fro,n1 the forestomach of the adult sheep. Stratum Granulosum.
3.20 Electron micrographs o£ the epithelium taken from the forestomach of the adult sheep. Str-atum Transi tionale.
3.21 Electron micrographs of the epithelium taken from the forestomach of th2 adult sheep. Stratum Corneum.
3.22 Electro~ micrographs of the epithelium taken from one-day-old lambs
3.23 3lectron micrographs o£ the epithelium taken from the forestomachs of 12-day- old lambs
3.24 Electron micrographs of the epithelium·
taken· from the forestomachs of 23- and 34-day-old l~~bs.
3.25 Electron micrographs of the epithelium taken from the forestornachs of 45- and
Between Faqes
69 - 70
70 - 71
71 - 72
72 - '7? ' - '
73 - 74
74 - 75
76.- 77
77 - 78
56-day-old lambs. 78 - 79
3.26 Na+-K+-ATPase cytochemistry of the ruminal epithelium. Complete ~·ledium.
3.27 Na+-K+-ATPase cytochemistry o£ the ruminal epithelium. Controls.
4.1 Schematic representation of three
models for transepithelial volume flow.
4.2 Schematic repr2sentation of the
transport model for Na+ based on the res•.1lts of the present study and in accord with the model proposed by ,\Iills, Ernst and DiBona ( 1977)
78 - 79 78 - 79 87 - 88
122 - 123
I
II.·
III
IV
v
LIST OF TABLES TITLE
Normal values of mitotic index of the ovine ruminal epithelium Proportions of the compartments of the stomach of grazing lambs as percentages of w~ight of the whole stomach
Number o£ light-staining cells in the forestomach epithelium in selected ages expressed as a percentage against the number of
xii
Between Pages
36 - 37
40 - 41
basal colls 65 ·· 66.
Nu;:uber of individual cell layers in t-he ruminal epithelium in
different ages 65 - 66
Mitotic Indices
(%)
of the ruminal epithelium taken frompairs of lambs at different ages 65 ·- 66
