Appendix 7.1 261 APPENDIX 7.1 DATA SHEET USED TO COLLECT REPRODUCTIVE SPECIMENS AND DATA TORRES STRAIT DUGONGITURTLE PROJECT DUGONG DATA AND SPECIMEN COLLECTION CHECKLIST .................. Recorderls ............................ Date Time .................. Location Mabuiag Boigu Badu ........... Specimen Number Tag colour Blue Green Red White 0 ...................................................... .............. Name of hunter N o of fishers Date caught .................... Time caught .................. Place caught ...................... Number days offishing trip > 1 day days 1 2 3 Time spent lookingfor dugongs >1 hour all day I all day & night Fishing preference 1 morning/aftemoon/night all night 0 Please tick Dugong 0 Turtle Crayfish 0 Fish Other catch: Turtle (no. caught) ......... Please tick data sheet yes no MEASUREMENTS 1.Body length (O.Ocm) ................... 2.Facial disc to anus distance (0.Ocm) .................
Transcript
Appendix 7.1 261
APPENDIX 7.1
DATA SHEET USED TO COLLECT REPRODUCTIVE SPECIMENS AND DATA
TORRES STRAIT DUGONGITURTLE PROJECT
DUGONG DATA AND SPECIMEN COLLECTION CHECKLIST
.................. Recorderls ............................ Date Time ..................
Location Mabuiag Boigu Badu
........... Specimen Number
Tag colour Blue Green Red White 0
...................................................... .............. Name of hunter N o of fishers
Date caught .................... Time caught .................. Place caught ......................
Number days offishing trip
> 1 day days 1 2 3
Time spent looking for dugongs
>1 hour
all day
I all day & night
Fishing preference
1 morning/aftemoon/night
all night 0
Please tick Dugong 0 Turtle Crayfish 0 Fish
Other catch: Turtle (no. caught) .........
Please tick data sheet yes no
MEASUREMENTS
1.Body length (O.Ocm) ................... 2.Facial disc to anus distance (0.Ocm) .................
Appendix 7.1 262
SPECIMEN COLLECTION
Please tick box if specimen collected
Skin sample put sample in plastic via1 with DMSO
Blubber
1. Collect specimens and take measurements as indicated on Dugong Fat Collection Data Sheet.
2.1 0cm square left ventral side 13 put sample in a labelled clipseal bag. To be frozen
3.10 cm square left ventral side wrap in foil and put in a labelled clipseal bag. To be frozen
Kidney tissue 1. Take samples as required in Dugong Fat Collection Data Sheet G
put sample in labelled clipseal bags. To be frozen
2. right side wrap sample in foil and put in labelled clipseal bags. To be frozen
3. right side put sample in labelled clipseal bags. To be frozen
Liver tissue 1. Take samples as required in Dugong Fat Collection Data Sheet
2. lOcm tissue wrap sample in foil and put in labelled clipseal bags. To be frozen
3. 5cm tissue 13 put in labelled clipseal bags. To be frozen
Head O put in labelled onion bag. Teeth U put in labelled seived bag.
Tusk Not erupted I? put in labelled seived bag
Erupted put in labelled seived bag
Stomach Content put in 10% SWF jar.
Eyelens Right 0 Left Put in labelled glass jar with IO%SWF
FEMALES
.......... Reproductive tract Photograph id.# slit open bursa. Record frame # of film
............ Ovaries Photograph id.# Right Left
Put each ovary in separate labelled onion bags. To be put in 10%SWF drum.
Endometrium at most recent scar midlength of horn Put each sample in labelled histology cassette. Put in 10% SWF jar.
Uterus Photograph id.# ............ cut and check horns for embryolfetus. Count and measure scars.
max. width ........... cm max. length ......... cm
Embryo 13 put in labelled sieved bag. To be put in 10%SWF drum.
Foetus 13 put in labelled oniodsieved bag. Record information on separate data sheet. To be put in 10%SWF drum.
Mammary gland lactating specimen 0 not lactating O
put in labelled histology cassette. Put in 10% SWF jar.
Milk 1. collect in a labelled glass vial. To be frozen.
2. O collect in a labelled glass vial. To be frozen
MALES Testes Right O. Left
Put in labelled histology cassette. Put in 10% SWF jar
Epididymis Right 0. Left
put in labelled histology cassette. Put in 10% SWF jar
Appendix 7.2 263
APPENDIX 7.2
DATASHEET USED TO COLLECT BODY CONDITION DATA
DUGONG FAT COLLECTION DATA SHEET
....... Recorderls .................. Date .............. Time
Specimen No. ............ Tag colour ..............
1
Antericr M~ddle Posterior
Axis
(a) Dorsal *
@) Lateral
(c) V e n d
A Muscle/tissue sampling sites
MEASUREMENTS
................ 1. Facial disc to tail notch cm
................ 2. Girth behind flipper cm
................. 3. Girth at umbilicus cm
4. Girth at anus ................ .cm
5. Girth at tail ............... ..cm
SPECIMEN SAMPLED Put each sample in labcllcd clipseal bag
MuscleIBlubber Ala M1a [7 M2a PIP P2a
Liver Fat
Mlb M2b P l b [1 P2b Kidney Fat
Alc M l c M2c P l c P2c Ovary Fat
Liver [7
! Kidney BLUBBER DESCRIPTION
Firm white [7 firm yellow Jelly like [7 Watery
Thick Thin
BACKBONE Obvious Not Obvious
OTHER NOTES .............................................................................................................................
Appendix 7.3 264
APPENDIX 7.3
METHOD USED FOR AGE DETERMINATION
Tusks were prepared forage determination based on methods described by Marsh (1980). Large tusks
(>5 cm long) were cut longitudinally in the mesiodistal plan with a 20 cm carbon steel blade. They were
secured in place during bisection using a custom-made mount. Small tusks (<2 cm long) were set in
plastic containers with Wests System epoxy resin and left to dry for 4-8 hours. They were then glued to
microscope slides with crystal balm before being cut with a 10 cm diamond saw.
The cut surfaces of both large and small tusks were polished using progressively finer grades (180, 240,
800 and 1200 grit) of wet and dry sandpaper. Tusks were etched in 5% HCL in 70% ethanol (sensu
Marsh 1980; see below), however, I required a longer etching period of 8-12 hours rather than the three
hours suggested by Marsh (1980) in order to produce satisfactory results. After the extended period in
etching reagent, individual tusks were rinsed thoroughly in water for at least five minutes before being
left to soak in a tray under gently running water for a further4-6 hours, Insufficient rinsing in water,
resulted in 'blistering' on the cut surface which made it very difficult to score the growth layer groups
(GLGs) accurately. When this occurred, re-polishing to obtain a smooth surface and re-etching
invariably provided satisfactory results providing the tusk was thoroughly rinsed as described above.
Etching Solution
Method to make up 2 litres of etching solution:
300 ml32% HCL
1400 ml high grade ethanol
300 ml distilled water.
Appendix 7.4 265
APPENDIX 7.4
MACROSCOPIC AND HISTOLOGICAL FEATURES OF PLACENTAL SCARS
In fresh uterine specimens, the macroscopic appearance of placental scars was generally darker than
the surrounding endometrium. As noted by Marsh eta/. (1984a), the appearance and colour of placental
scars varied considerably between different dugongs. Scars ranged from dark purple patches to a faint
grey in colour. However, a dark coloured scar in one dugong could not be assumed to be more recent
than a fainter scar in another individual. In individual dugongs with multiple scars, one that was dark
coloured was presumed to be more recent than a fainter coloured scar. The most conspicuous scars
had well defined edges that were easily identified in macroscopic examinations (see Figure 1).
Histologic assessment of placental scars (which had been confirmed by macroscopic examination)
indicated that implantation sites are characterised by a number of features. Firstly, generally extensive
amounts of haemosiderin are present either as clumps of golden brown coloured granules or as
macrophages that have pale brown cytoplasm containing flecks of haemosiderin. The walls of arteries in
the endometrium and stroma are considerably thickened with its connective tissue appearing
conspicuously spiralled. Active inflammation was evident by one or more foci of inflammatory cells
(lymphocytes) in the stroma. In some animals, mineral deposits presumably of calcium and evidence of
oedema were also present. In addition to information from ovarian activity, these features were used to
verify the presence of placental scars in some female dugongs.
In a several females, it was necessary to confirm the presence of scars by histological examination of
endometrium tissue because either the edges of scars were very faint or the scar tissue appeared red
rather than purple coloured in fresh specimens or dark grey in preserved uteri (Figure 1, Table 1). In the
uteri of fifteen females (Table I ) , the endometrium presented as a diffuse area with illdefined edges of
homogenous dark red (in fresh) or dark grey (in fixed uterine specimens) coloured tissue that appeared
hyperaemic or haemorrhagic (Figure 1). These areas occurred either as discrete patches on the uterine
cornua or were appeared to be 'overlaid' or adjuncting a placental scar (with defined edges). Some of
these areas also appeared as though they were extensions of an adjacent scar. The length of discrete
areas of red endometrial tissue along the uterine horn was between 4.3 to 13.5 cm. In contrast,
confined placental scars were generally smaller being 2.4 to 8.1 cm in length.
Appendix 7.4 266
Figure I. Hyperaemic areas on the uterus of MD41 showing there are two hyperaemic areas present on the left uterine horn; one hyperaemic area is lying adjacent to a placental scar with conspicuous dark edge on the right uterine horn.
Histological examination of these areas indicated hyperaemia (i.e., presence of excess blood vessels)
rather than haemorrhage (i.e, external bleeding from blood vessels) to be the likely cause of the red
appearance of endometrial tissue in all animals examined. However, the presence of haemosiderin
particules or haemosiderin macrophages in all such areas, are indicative of haemorrhage rather than
hyperaemia. Other histological features consistent with placental scars such as thickened arterial walls,
active inflammation and mineralisation were found in nine animals. However, on the basis of the
available histological data of the uterus and information on ovarian activity, it was not possible to
determine whether these features resulted from residual effects of adjacent confirmed scars on the
same horn, leakage from haemorrhaging during oestrus or were actual sites of implantation (Table 1 ) .
Appendix 7.4 267
Table 1. Histological characteristics of discrete areas with no adjacent scars on uterine horn.
Possibly associated with RH PS or ?site of implantation
Possibly associated with RH PS
Possibly associated with LH PS
Probably associated with RH PS
Probably associated with RH PSs
Probably associated with LH PSs
Probably associated with RH PS
Very thick in endometrium and muscle layer
Moderately thickened (7.5 um)
Moderately thickened (1 1.5 urn)
Moderately thickened (1 1.5 urn)
Very think in endometrium and muscle layer
Very think in endometrium and muscle layer Very thick in endometrium and muscle layer
A few granules and macrophages
Moderate number macrophages
Moderate amounts of granules & a few macrophages Moderate amounts of granules & a few macrophages
Many granules and a few macrophages
Many granules and a few macrophages
Many granules and macrophages
MD23 16 years 2 RH PS RO- 22 CL, 18 C A LO-LFG MD96 16 years 1 RH PS I LH PS No ovaries collected MD97 I 1 years 1 RH PS RO-20 CL, 14 C A LO-SGF MD98 32 years 2 RH PS 2 LH PS
MD102 27 years 3 RH PS 2 LH PS RO-47 CL LO-44 CA MD108 I I years 1 RH foetus, 1 LH PS RO-20 CL LO-19 CL, 5 C A MD122 10 years 1 RH PS RO-27 CL, 26 C A Part LO only MD124 32 years 3 RH PS 3 LH PS RO-13 CL, 36 C A LO-22 CL, 10CA MD168 9 years 1 RH PS RO-27 CL,
1 7 C A
Possibly associated with RH PS or ?site of implantation
Probably associated with LH PS
Possibly associated with LH PS or ?site of implantation
Probably associated with RH PS
Possibly associated with RH PS or ?site of implantation
Proabably associated with RH PS
Moderate amount granules
A few dark stained granules
Moderate amount of granules and macrophages
Moderate amount of granules and macrophages
A few granules
A few granules and macrophages
Moderate amount of granules
RH
LH 1
LH2
RHllRH2
RH
RH 1
RH2
Very thick in endometrium and muscle layer
Not thick
Moderately thickened (1 1.5um)
Moderate-very thickened
A few with very thickened walls endometrium and muscle layer
Moderately thickened
Slightly thckened
A few foci of lymphocytes
A few foci of lymphocytes
A few foci of lymphocytes
A few foci of lymphocytes
A few foci of lymphocytes
A few foci of lymphocytes
A few foci of lymphocytes
Present
No
No
No
No
Present
No
Appendix 7.5 272
APPENDIX 7.5
HISTOLOGICAL FEATURES OF LACTATION
Proliferating mammary glands
In the early proliferating mammary gland in the dugong, the ducts are enlarged with often numerous
epithelial sprouts. In the latter stage proliferating gland, alveoli buds are evident, and form distinct
alveoli consist of single layers of cuboidal epithelial cells, which are separated by conspicuous dense
connective tissue.
Lactating mammary gland
Lobules composed of numerous oval or spherical alveoli are the most distinguishing histological feature
of active lactating mammary glands in dugongs. Alveoli are lined with a single layer of cubiodal and
myoepithelial cells. Fatty secretory products are also commonly seen in the lumen of alveoli. The
interlobular connective tissue is reduced, loose and thin particularly in the immediate vicinity of the ducts
and lobules.
Inactive mammary gland
The inactive mammary gland is distinguishable from the lactating gland by a predominance of loose
connective tissue and absence of alveoli. The epithelial cells and ducts are contained in loose
connective tissue, which is readily distinguishable from the surrounding dense connective tissue. The
epithelial cells are of stratified cuboidal form and are typically two cells thick. Alveoli are not present,
however, its precursors are represented as cellular thickening of the duct walls.
Appendix 7.6 273
APPENDIX 7.6
MEASUREMENTS OF THE UTERUS AND OVARIES OF FEMALE DUGONGS IN VARIOUS
REPRODUCTIVE STAGES.
Stage Corpus uterus Right horn Left horn Right ovary Left ovary fat diameter diameter diameter fat depth depth (+ s.e) (f s.e) (f s.e.) (f s.e) (f s.e)
Left Left Left Right Right Right Left Right Right Right Right
Left Left Left No data Right Right Right Right Right Right Left Right Right Left Right Left Right
74.2 20 57.2 26 50.08 22 111.8 20 81.2 18 71.2 22 50.6 0 52.4 No data
No data No data 1 33 0 38 18 6 13 1 20 14 0 10 29 6 27 26 7 2 27 17
15.5 0 85.3 16 61 4 No data 23 105.2 46 104.2 12 103.2 No data 4.1 21 118.6 4 76.6 44 130.02 20 2.1 28 85.4 35 44.8 39 30.08 20 61.2 0 106.2 30
8 0 0 0 10 6 34 No data
No data 10 30 I 0 0 19 1 No data 7 1
37 29 14 14 0 0 0 0 8 0 0 No data No data 0 19 39 15
11 4 0 0 0 0 0 82 0 3 1 0 0 20 6 No data No data
No data No data 34 15 0 0 0 10 0 0 45 0
- BL = body length; ROCL = corpora lutea in right ovary; LOCL = corpora lutea in left ovary; ROCA = corpora albicantia in right ovary; LOCA = corpora albicantia in left ovary. a age determined from lens weight bsimultaneously lactating and pregnant Cpregnant with proliferating mammary glands
Appendix 8.1 275
APPENDIX 8.1
HISTOLOGICAL CHARACTERISTICS USED IN PHASE ANALYSES OF MALE DUGONGS
Phase Cellular organisation and features in seminiferous tubules Phase -4 Essentially solid tubules lined with Sertoli cell nuclei and types A and B
spermatocytes. Occasional Sertoli cell nuclei and vacuolated Sertoli cell cytoplasm in the centre.
Phase -3
Phase -2
Phase -1
Like Phase -4 with small numbers of primary spermatocytes and more Sertoli cell nuclei in central area of tubules.
Like above phases but with numerous primary spermatocytes. Tubule lumens may be opened or closed.
Lumen of tubules usually evident. Primary and secondary spermatocytes present and organised in layers.
Phase 0 Like Phase -1 but with a few rounded spermatids or spermatozoa present.
Phase 1 Sertoli cell nuclei and spermatogonia line basement membrane. Primary and secondary spermatocytes organised in layers. Lumen surrounded by round spermatids.
Phase 2 Spermatids elongating and becoming associated with Sertoli cells.
Phase 3
Phase 4
Spermatid bundles well organised associated with Sertoli. Maturation divisions occurring among primary and secondary spermatocytes.
Two generations of spermatids present. Elongate spermatids have tails and are more central to lumen.
Phase 5 Sperm line the lumen tubule. Round spermatids also present.
Phase 6 Spent tubule at the end of period of sexual activity. Lumen of tubule very wide and surrounded by an incomplete assemblage of cell layers which may contain a subset of any of the cell types;
Appendix 8.2 276
APPENDIX 8.2
DETAILS OF MALE DU GONGS ASSESSED FOR REPRODUCTIVE STATUS ON THE BASIS OF PHASE ANALYSES (sensu Marsh et a/. 1984c; Hernandez eta/. 1995)
Tubule Diameter (um) 49.59 f 5.6 43.47 f 4.62 51.33 + 8.69 46.35 i- 5.88 50.72 f 5.95 51.07 f 4.68 37.5 f 4.56 49.69 f. 5.92 47.4 f 6.70 63.83 + 8.01 61.07 7.07 51.79 f 4.92 64.03 f 0.78 56.67 f 8.65 53.93 f 5.25 Total Mean 51.71 f 6.85
Mean Tubule state -3.71 -4 -4 -4 -4 -4 -4 4 -4 -4 -3.8 -4 -4 -4 -4
State of tubule (percentage at each stage) -4 -3 -2 -1 0 1 2 3 4 5 6
Mean Tubule state -2.63 -2.82 -2.82 -3.59 -1 -08 -2.02 -2.92 -3 -3.3 -3 -3.14
State of tubule (percentage at each stage) -4 -3 -2 -1 0 1 2 3 4 5 6
Density of sperm in epididymis 0 0 0 0 0 0 0 0 0 0 0
Density of spermatozoa in'epididymis: O=no spermatozoa; l=a few spermatozoa; 2=moderate amount of spermatozoa; 3= tubules densely packed with spermatozoa
State of tubule (percentage at each stage) Density of sperm in epididymis
3 2 N A N A 3 2 3 3 3 2 2 3 2 3
Density of spermatozoa in epididymis: O=no spermatozoa; l =a few spermatozoa; 2=moderate amount of spermatozoa; 3= tubules densely packed with spermatozoa
Appendix 8.2 279
Resting male adults (n = 3)
Dugong ID Age (yr) Body Tubule Mean State of tubule (percentage at each stage) Length (m) Diameter Tubule
(urn) state
715198 MD29 NA 2.88 48.42 + 7.28 -3.33 32.7 67.3 3130199 MD104 23+ 2.43 95.61 + 12.57 -3 100 411199 MD109 16+ NA 8 5 f 17.51 -3 100
Density of spermatozoa in epididymis: O=no spermatozoa; l=a few spermatozoa; 2=moderate amount of spermatozoa; 3= tubules densely packed with spermatozoa
Appendix 8.3 280
APPENDIX 8.3
CHARACTERISTICS OF SEMINIFEROUS TUBU LE USED IN PHASE ANALYSES
The mean diameter of seminiferous tubules was used to make comparisons among animals. As reported
for manatees (Hernandez et a/. 1995), a strong correlation was detected between testicular stage and
tubule diameter in dugongs in Torres Strait (Figure 2, n = 49; Spearman's rank correlation test; r = 0.842,
pc0.01). Strong correlations were also evident for tubule diameter and age of dugong (based on GLGs)
(Figure 3; n = 36; Spearman's rank correlation test; r 0.626, p<0.01) and testicular stage and age of
dugong (based on GLGs), (Figure 4, n 36; Spearman's rank correlation test; r = 0.696, p<0.01).
The seminiferous tubules of immature prepubescent dugongs were narrow and ranged in mean diameter
from 38 to 64 um, a result very similar to that previously reported for dugongs (Marsh et a/. 1984~) and
manatees (Hernandez eta/. 1995) (Appendix 8.2).
Animals classified as immature maturing/recmdescent dugongs had most tubules in phase -3 or 4 with
mean tubule diameters of between 44.29 to 65.09 um (see Appendix 8.2).
Males with more active stages of spermatogenesis (stages -2 to 0) had larger mean tubule diameters than
those at stages -3 to 4 (Appendix 8.2). One male (JCOI) with a mean tubule diameter of 97 um had 39%
of his tubules at phase -2 and 61 % at phase -3 and 4 (Appendix 8.2).
Two dugongs (MD59 and KD19) had testes at more advanced stage of spermatogenesis with most
tubules approaching stage -2 to 0 with mean tubule diameters of 112 - 120 um (see Appendix 8.2). None
of the eleven males from Mabuiag reported in this category had erupted tusks (Appendix 8.2).
Fully spermatogenic males were characterised by very large tubules with mean diameters ranging
between 135 to 206 um (Appendix 8.2).
Appendix 8.3 28 I
! Age (years)
I
Figure 2. Relationship between testicular stage and age (n 28)
0 10 20 30
Age (years)
Figure 3. Relationship between seminiferous tubule diameter and age (n = 36).
I I
Tubule diameter (um)
Figure 4. Relationship between testicular stage and tubule diameter (n = 49).
List of Acronyms
LIST OF ACRONYMS
AFMA AQUlS C A CDEP CITES CL CPUE CSlRO DlMA ENS0 G AM GBRMPA GLG GLM HRSCATSIA
ICC IlHED ILUA IMCRA I PA IUCN LGF LH LO MaSTERS
NPA NO0 NW PBR PNG PS PZJA QFS RF RH RO SE SGF Sol SPRITS
TSRA TSPZ WCED
Australian Fisheries Management Authority Australian Quarantine and Inspection Service Corpora albicantia Community Development Employment Program Convention on International Trade of Endangered Species Corpora lutea Catch per unit effort Commonwealth Scientific Industrial Research Organisation Department of lmmig ration and Multicultural Affairs El Nino Southem Oscillation Generalised additive model Great Barrier Reef Marine Park Authority Growth layer group Generalised linear model House of Representative Standing Committee on Aboriginal and Torres Strait Islander Affairs (Torres Strait) Island Coordinating Council International Institute for Environment and Development lndigenous Land Use Agreement Interim Mari ne and Coastal Regionalisation for Australia lndigenous Protected Areas The World Conservation Union Large Graafian follicle Left uterine horn Left ovary Marine Study of the Torres Strait Environment Resource Strategy (Marine Strategy) Northem Peninsula Area (Cape York Peninsula) National Oceans Office North-West monsoon season (November to April) Potential Biological Removal Papua New Guinea Placental scar Protected Zone Joint Authority Queensland Fisheries Service Recovery factor Right uterine horn Right ovary South-East season (May to October) Small Graafian follicle Southern Oscillation Index Strategy for the Planning of Resource Integration in the Torres Strait Torres Strait Regional Authority Torres Strait Protected Zone .
