Improving Australia’s Crocodile Industry Productivity — Understanding runtism and survival— RIRDC Publication No. 09/135
RIRDC
Innovation for rural Australia
Improving Australia’s Crocodile Industry Productivity — Understanding runtism and survival —
Sally Isberg, Cathy Shilton and Peter Thomson
September 2009 RIRDC Publication No 09/135 RIRDC Project No PRJ-000550
© 2009 Rural Industries Research and Development Corporation. All rights reserved.
ISBN 1 74151 934 9 ISSN 1440-6845 Improving Australia’s Crocodile Industry Productivity— Understanding runtism and survival— Publication No. 09/135 Project No. PRJ-000550 The information contained in this publication is intended for general use to assist public knowledge and discussion and to help improve the development of sustainable regions. You must not rely on any information contained in this publication without taking specialist advice relevant to your particular circumstances. While reasonable care has been taken in preparing this publication to ensure that information is true and correct, the Commonwealth of Australia gives no assurance as to the accuracy of any information in this publication. The Commonwealth of Australia, the Rural Industries Research and Development Corporation (RIRDC), the authors or contributors expressly disclaim, to the maximum extent permitted by law, all responsibility and liability to any person, arising directly or indirectly from any act or omission, or for any consequences of any such act or omission, made in reliance on the contents of this publication, whether or not caused by any negligence on the part of the Commonwealth of Australia, RIRDC, the authors or contributors. The Commonwealth of Australia does not necessarily endorse the views in this publication. This publication is copyright. Apart from any use as permitted under the Copyright Act 1968, all other rights are reserved. However, wide dissemination is encouraged. Requests and inquiries concerning reproduction and rights should be addressed to the RIRDC Publications Manager on phone 02 6271 4165.
Researcher Contact Details Dr Sally Isberg Porosus Pty Ltd, PO Box 86, Palmerston NT 0831 Phone: 08 8988 5554 Fax: 08 8988 2001 Email:
[email protected] In submitting this report, the researcher has agreed to RIRDC publishing this material in its edited form. RIRDC Contact Details Rural Industries Research and Development Corporation Level 2, 15 National Circuit BARTON ACT 2600 PO Box 4776 KINGSTON ACT 2604 Phone: Fax: Email: Web:
02 6271 4100 02 6271 4199
[email protected]. http://www.rirdc.gov.au
Electronically published by RIRDC in September 2009 Print-on-demand by Union Offset Printing, Canberra at www.rirdc.gov.au or phone 1300 634 313
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Foreword The Australian crocodile industry relies on the production of saltwater crocodile skins for the international skin trade. For the industry to continue to develop and ensure its environmental and economic sustainability, it not only needs to ensure a reliable supply of hatchlings (wild or captive egg harvests) but also to ensure maximum survival rates are achieved to meet the end product usage. The emerging status of this industry means improvements in animal husbandry and a better understanding of the underlying dynamics of production inefficiencies will ensure the industry meets this goal. In addition, understanding the dynamics of the underlying causes of production inefficiency, such as mortality rates, aids in defining research priorities. Runting causes crocodile mortalities and results reported indicate that wild collection area effects, and captive breeding genetic effects are highly significant. The initial histopathology results presented herein indicate that immunosuppression and chronic stress are the most likely cause of runting. Recommendations are given to continue addressing this area of large economic loss. This project was funded from RIRDC Core Funds which are provided by the Australian Government. This report, an addition to RIRDC’s diverse range of over 1900 research publications, forms part of our New Animal Products R&D program, which aims to accelerate the development of viable new animal industries. Most of RIRDC’s publications are available for viewing, downloading or purchasing online at www.rirdc.gov.au. Purchases can also be made by phoning 1300 634 313.
Peter O’Brien Managing Director Rural Industries Research and Development Corporation
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Acknowledgments The outcomes reported in this project arose from a collaborative effort between RIRDC, Darwin Crocodile Farm (Porosus Pty Ltd) and the University of Sydney. Without the enthusiastic support of the Porosus Pty Ltd Board of Directors, this project would not have eventuated and the authors wish to sincerely acknowledge their contribution to this project. Sincere thanks must also go to the management and staff at Darwin Crocodile Farm for their assistance with data collection over the last three years. At times, there have been interesting debates regarding which category of death to put some animals in. The conversations have, to this effect, been thought-provoking and challenging. The authors would like to thank the generous donation of in kind work by Berrimah Veterinary Laboratories and, in particular, Anton Janmaat for originally agreeing to laboratory participation in the study. The following laboratory staff performed technical aspects of the pathology portion of the study: Lynne Chambers (haematology, data entry), Sue Aumann (histology processing and serum biochemistry), Steve Davis (corticosterone assay), Suresh Benedict (bacteriology) and Lois Small (parasitology).
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Abbreviations Abbreviations used in this study are defined below (in alphabetical order), although they are also described within the text.
Age
Age of animal (days)
ALP
Alkaline phosphatase
ALT
Alanine amino-transferase
AST
Aspartate amino-transferase
BVL
Berrimah Veterinary Laboratory
Bwt
Bodyweight (g)
CBV
Crocodile breeding value
CK
Creatine kinase
CI
Confidence interval
EDTA
Ethylenediaminetetraacetic acid
GGT
Gamma glutamyl-transferase
HDays
Number of days between hatching date and 1st of January in that particular year
NoHatch
Number of live hatchlings in a particular clutch
NVA
No visible ailments
PCV
Packed-cell volume
SD
Standard deviation
SE
Standard error
TL
Total length (mm)
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Contents Foreword.................................................................................................................................................. iii Acknowledgments ................................................................................................................................... iv Abbreviations ............................................................................................................................................v Executive Summary ..................................................................................................................................x 1. Introduction...........................................................................................................................................1 Objectives .............................................................................................................................................1 2. Methods and Materials.........................................................................................................................2 2.1 Survival analysis.............................................................................................................................2 2.1.1 Crocodile resources...............................................................................................................2 2.1.2 Collection of mortality data ..................................................................................................2 2.1.3 Statistical analyses ................................................................................................................5 2.2 Histopathology ...............................................................................................................................6 2.2.1 Haematology .........................................................................................................................7 2.2.2 Serum biochemistry ..............................................................................................................7 2.2.3 Corticosterone assay .............................................................................................................7 2.2.4 Bacteriology..........................................................................................................................8 2.2.5 Parasitology ..........................................................................................................................8 2.2.6 Histology...............................................................................................................................8 3. Survival Analysis Results ...................................................................................................................10 3.1 Porosus resource...........................................................................................................................10 3.1.1 Survival analysis results......................................................................................................10 3.1.2 Kaplan-Meier survival functions ........................................................................................15 3.2 Pedigree resource..........................................................................................................................19 3.2.1 Pair model survival analysis results ....................................................................................19 3.2.2 Correlation between crocodile breeding values (CBVs).....................................................24 3.2.3 Pair model Kaplan-Meier survival functions ......................................................................25 3.2.4 Animal model survival analysis results ..............................................................................28 4. Histopathology Results .......................................................................................................................29 4.1 General findings ...........................................................................................................................29 4.2 Haematology ................................................................................................................................29 4.3 Biochemistry ................................................................................................................................29 4.4 Corticosterone assay.....................................................................................................................30 4.5 Bacteriology .................................................................................................................................30 4.6 Parasitology ..................................................................................................................................30 4.7 Histology ......................................................................................................................................31
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4.7.1 Lymphoid tissue..................................................................................................................31 4.7.2 Adrenal gland......................................................................................................................31 4.7.3 Bone ....................................................................................................................................31 4.7.4 Liver, gastrointestinal tract and pancreas............................................................................32 4.7.5 Yolk sac ..............................................................................................................................32 4.7.6 Other tissues........................................................................................................................33 5. Discussion.............................................................................................................................................34 5.1 Survival analysis...........................................................................................................................34 5.1.1 Collection area and Pair effects ..........................................................................................34 5.1.2 Heritability estimates ..........................................................................................................35 5.1.3 Hatch days (HDays)............................................................................................................36 5.1.4 Number of hatchlings (NoHatch)........................................................................................36 5.2 Histopathology .............................................................................................................................36 6. Implications .........................................................................................................................................39 7. Further research .................................................................................................................................40 8. References............................................................................................................................................41
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Tables Table 1.
Summary statistics of nests from different collection areas in the Porosus resource available for analysis....................................................................................................................................................3
Table 2.
Descriptive statistics of the runts and “normals” in each phase of the study..........................................6
Table 3.
Summary statistics of the Porosus resource data used in the survival analyses from Darwin Crocodile Farm .....................................................................................................................................10
Table 4.
Total number of deaths in the Porosus resource including a breakdown into the six defined categories. .............................................................................................................................................10
Table 5.
Significance summary for explanatory variates used in the Porosus resource analyses for the different causes of mortality .................................................................................................................11
Table 6.
Estimates (±SE) of year effects for each mortality cause, their hazard ratios and the antilog of the 95% confidence interval (CI) using the Cox proportional hazards model ...........................................12
Table 7.
Probability of a crocodile surviving to day 365 and day 1077 for each cause of mortality..................16
Table 8.
Summary statistics of Pedigree resource data used in the survival analyses from Darwin Crocodile Farm......................................................................................................................................................19
Table 9.
Total number of deaths in the Pedigree resource and a breakdown into the six defined mortality categories. .............................................................................................................................................19
Table 10.
Significance summary for explanatory variates used in the Pair model analyses for the different causes of mortality
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Table 11.
Correlation coefficients between crocodiles breeding values (CBVs) for the different causes of mortality................................................................................................................................................25
Table 12.
Probability of a crocodile surviving to day 365 and day 1002 for each cause of mortality..................25
Table 13.
Significance summary for explanatory variates used in the Animal model analyses for the different causes of mortality using the Pedigree resource ....................................................................28
Table 14.
Crocodile breeding values (CBVs) were offset by a weighted economic value using the percentage of death in each category (%) multiplied by AU$52.37 .....................................................34
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Figures Figure 1.
Examples of congenital defects. a) cleft palate; b) weak yolk scar suture, rupturing exposes internal organs; c) undershot jaw; d) crocodile born with no eyes; e) born with no tail. .................. 4
Figure 2.
Compared to the same aged “normal” crocodile (below), the runt crocodile (top) appears emaciated with wasting particularly obvious in the neck and tail areas. ........................................... 4
Figure 3.
Probability of mortality and standard errors for each collection area (Areacode) for overall survival, congenital defects and runtism ......................................................................................... 13
Figure 4.
Probability of mortality and standard errors for each collection area (Areacode) for the diseaserelated, stress-related, no visible ailments (NVA) and management categories of mortality.......... 14
Figure 5.
Kaplan-Meier estimated survival functions for crocodiles between hatch and one year of age (365 days) for each cause of mortality ............................................................................................ 17
Figure 6.
Kaplan-Meier estimated survival functions for crocodiles between hatch and day 1077 (2.95 years) for each cause of mortality. ......................................................................................... 18
Figure 7.
A) Log hazard pair estimates (±SE) of overall juvenile survival in the Pedigree resource. B) Pair CBVs (±SE) at 365 days using the Cox’s proportional hazards model. .................................. 21
Figure 8.
Pair CBVs (±SE) at 365 days for the runtism cause of death.......................................................... 23
Figure 9.
Pair CBVs (±SE) at 365 days for disease-related deaths................................................................. 23
Figure 10.
Pair CBVs (±SE) at 365 days for stress-related deaths. .................................................................. 24
Figure 11.
Pair CBVs (±SE) at 365 days for deaths occurring from no visible ailments (NVA). .................... 24
Figure 12.
Kaplan-Meier estimated survival functions for crocodiles in the Pedigree resource between hatch and one year of age (365 days) for each cause of mortality using the Pair model................. 26
Figure 13.
Kaplan-Meier estimated survival functions for crocodiles in the Pedigree resource between hatch and day 1002 (2.75 years) for each cause of mortality using the Pair model. ....................... 27
Figure 14.
Dollar deviation of crocodile breeding values as expressed as a dollar ($) deviation from the herd average for the runting and NVA causes of death................................................................... 35
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Executive Summary What the report is about and who is the report targeted at? This project assessed the incidence of different causes of juvenile saltwater crocodile deaths on an Australian crocodile farm. In addition, a pilot histopathology study was conducted to determine if there are any primary causes for runting in captive saltwater crocodiles. This information is targeted at Australian crocodile producers to enhance their production efficiency by reducing juvenile mortalities, particularly from runting. Background Industry standard mortality rates have been accepted to be 10-15% in the first year and 5% thereafter on Australian crocodile farms. This obviously has a large economic impact on the industry and decreases the overall level of production efficiency. Previous investigations into captive crocodile mortality have grouped deaths into one overall encompassing category. However, it was of interest to know the incidence and trends associated with specific causes of deaths on farms so that management regimes could be adjusted accordingly. Furthermore, the previous heritability estimate for overall juvenile survival was 0.15 (SE 0.04). It was of interest to estimate the heritability, and subsequent breeding values, for the different causes of mortality for incorporation into CrocPLAN. Anecdotal evidence suggests that runting results in the highest incidence of mortality on crocodile farms. Runting refers to extremely poor growth in young animals compared to similarly aged conspecifics. Runts are eventually lost to the industry due to culling or early natural death in a profoundly wasted state. Little research has been conducted into the reasons why this syndrome occurs. Aims/objectives There were two main objectives of this project. Firstly, to conduct a categorical risk analysis of all mortality data collected over three years at Darwin Crocodile Farm. This was done using two datasets i) combined data from both wild and captive-bred crocodiles, and ii) captive-bred crocodiles of known parentage only. This will allow mortality to be investigated within a non-genetic and genetic framework, respectively. Estimated breeding values of crocodiles from the latter will be incorporated into CrocPLAN as separate breeding values rather than the collective “overall” survival. Secondly, a histopathology study was conducted to examine the issue of runtism and attempt to observe any differences between “normal” and runt crocodiles. Similar studies have been conducted in other crocodilians but not for saltwater crocodiles. Methods used Both the survival analysis and histopathology study were conducted using animals and data collected at Darwin Crocodile Farm. Animal ethics approval was obtained from the University of Sydney (N00/9-2005/3/4204). Mortality data were collected using clutch and individual scute-cut identification which allowed clutch of origin, hatch date and parentage/wild nest area to be determined. In addition, the cause of death was recorded as one of the following categories: congenital defects, runting, disease-related, stress-related, no visible ailments (NVA: unknown) and management. Three years of data were collected for analysis using various Cox’s proportional hazards models and adjusted for various environmental, geographical and genetic effects.
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The histopathology component of the study was conducted at Berrimah Veterinary Laboratories (Berrimah, Northern Territory). The study was split into two phases: 2005 and 2007. Each phase consisted of ten “runts” and ten “normal” crocodiles of similar age. The animals were sacrificed and subjected to a thorough examination including full post-mortems, general bacterial culture, faecal parasitology, standard diagnostic haematology and serum biochemistry, histological evaluation of an extensive range of tissues, and, in the 2007 group, serum corticosterone. Results/key findings From the analyses presented herein, runtism constitutes 49% of deaths followed by deaths for no visible ailments (23%) and disease (12%). There were significant collection area and genetic effects, as well as time of hatch and number of hatchling effects. With the exception of runtism (0.71 SE 0.08), heritability was estimated to be 0.76 (SE 0.09) for all other causes of death using a Pair model due to confounding of the data. Additional data collection will rectify this situation allowing clutch to be included. The heritability estimates from the Animal model varied from 0.28 (SE 0.02) for deaths for no visible reason to 0.60 (SE 0.04) for runting. Crocodile breeding values estimated from these data show considerable variation which will allow producers to start selecting superior, and replacing inferior, animals from the higher risk mortality categories (runtism, no visible ailments and diseaserelated) to quickly ensure the economic impact of these causes of death are minimised. Many of the findings in the histopathology study were expected due to the emaciated state of the runts that characterises the condition. However, the major findings were the presence of marked lymphoid atrophy, suggesting immunosuppression, and vacuolated adrenocortical cells due to chronic stress. Implications for relevant stakeholders and recommendations Runtism should be set as the number one mortality research priority. Areas of particular research interest to reduce the incidence of this syndrome, as well as the other categories, are a thorough investigation of the crocodilian immune system, exploration of potential viral infection(s) and chronic stress, although other areas should also be explored including alternative pen designs, ethology and endocrinology. There are significant geographical effects in the incidence of each cause of death. As a result, producers should adjust their management policies appropriately for each area. The difference in crocodile breeding values for the different causes of death will allow producers to select against higher risk categories when considering the implementation of their genetic improvement programs.
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1. Introduction Juvenile crocodile deaths still remain an area of large economic loss for Australian crocodile producers. Webb (1989) commented that producers should aim for 95% survival in the first year after hatch, but in reality, survival rates are typically between 85-90% (Isberg et al. 2004). After the first year, the risk of mortality decreases and a 95% survival rate is the aim from one year old to slaughter at about 3.5 years on average (Webb, 1989). There is a large variation in this trait and Isberg et al. (2004) revealed that the probability of a crocodile surviving to day 400 is only 56%, which is extremely low. Since every animal is potentially worth in excess of AU$500 at harvest, the economic loss from mortality and space inefficiency is immense. Isberg et al. (2004) reported the heritability for the breeding objective, juvenile survival, to be 0.15 (SE 0.04). However, this heritability estimate was based only on whether an animal lived or died and not why the animal died. Crocodile deaths, as with deaths in any species, can occur for a variety of reasons and is often ignored when addressing the overall issue of survival (Southey et al. 2004). Therefore, gains obtained from implementing recommendations based on overall survival may not be as great compared to those recommendations that consider different causes of death. For this to occur, the highest risk factors need to be identified. Runts constitute a large proportion of juvenile deaths on Australian crocodile farms (Hibberd et al. 1996; personal observation) and have been an ensuing problem for producers. Runtism is described as a condition of hatchling crocodiles whereby they fail to grow in comparison to the rest of their cohort and generally appear emaciated (anorexic; Huchzermeyer 2003). Buenviaje et al. (1994) suggested that runting was a failure to adapt to a particular rearing or management environment, whilst Peucker and Mayer (1995) proposed that the condition is inherited. Mayer (1998) reported that injecting runts with vitamins or changing the type of food (live worms, tinned cat food) could be potential cures, although both strategies required further study. In contrast, Anderson et al. (1990) and Kanui et al. (1993) conducted trials to investigate the effect of human and bovine growth hormone, respectively, with varying success. Bacterial hepatitis and septicaemia, caused predominantly by gram negative bacteria, were described by many authors as a leading cause of death in hatchling saltwater crocodiles (Ladds and Sims 1990, Buenviaje et al. 1994, Hibberd et al. 1996, Ladds et al. 1996). These were described as opportunistic infections that predominated in the winter months. However, as the industry has developed management regimes (provision for heating systems, etc), deaths from bacterial septicaemias have become less frequent (Buenviaje et al. 1994). A similar situation has also been reported for parasitic and mycotic infections.
Objectives There were two major objectives of this study. They were: a) To evaluate the specific risk factors associated with juvenile mortality, in particular runtism and disease susceptibility, within both a genetic and non-genetic framework. b) To conduct a histopathology study to examine the issue of runtism and attempt to observe any differences between “normal” and “runt” crocodiles.
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2. Methods and Materials Darwin Crocodile Farm (Porosus Pty Ltd) has provided data and animal resources essential for the research reported herein. The initial part of this section, describes the data collection process and statistical methodology for the survival analysis component of this study, whilst the latter part describes the sampling strategy and methodology for the histopathology. Animal ethics approval was obtained from the University of Sydney (N00/9-2005/3/4204).
2.1 Survival analysis 2.1.1 Crocodile resources Data were collected from all animals hatched at Darwin Crocodile Farm (Noonamah, Northern Territory, Australia) between 2005 and 2007. The eggs were sourced from either the captive breeding population or during wild egg collections under the approved Northern Territory management plan (Department of Natural Resources, Environment, and the Arts). The eggs were incubated on-farm under standard industry conditions described in Isberg et al. (2004). Upon hatching, the crocodiles were identified according to their clutch number using the scute marking system described in Richardson et al. (2002) and Isberg et al. (2004). The crocodiles were then placed into raising pens and fed standard industry diets according to their size and age class similar to those described by Isberg et al. (2004). Two data-sets were created from the data allowing different analyses to be performed. The first dataset, herein referred to as the Porosus resource, included all progeny records from both wild and captive nests hatched at Darwin Crocodile Farm between 2005 and 2007 (n = 36,346). For the purposes of the analysis, all captive nests were allocated into one collective group, whilst the wild eggs were allocated into twelve separate collection areas according to the landowner (for example, private cattle station, indigenous community group, etc.; summary statistics are given in Table 1). The second dataset, herein referred to as the Pedigree resource, includes only the progeny records from 67 known-parent breeding pairs at Darwin Crocodile Farm (Pedigree resource; n = 2,721). This dataset was used to estimate genetic parameters.
2.1.2 Collection of mortality data Mortality data were collected in a similar manner described in Isberg et al. (2006) during routine feeding and cleaning procedures. The dead animal’s clutch of origin was determined from the scute cuts and used to retrospectively determine the date of hatch (used to calculate age at death) and the origin of the clutch (captive breeding pen or wild egg collection area). In addition, it was decided to allocate the cause of each death to one of six categories (described below) so the main risk factors associated with juvenile crocodile deaths on Australian crocodile farms could be identified. The categories used to allocate crocodile deaths were congenital defects, runtism, disease-related, stress-related, no visible ailments and management-related. Further descriptions of these are given below. Congenital defects- Deaths related to this category were generally seen immediately upon hatch and include defects such as unabsorbed yolk sacs, weak yolk scar sutures, jaw deformities (cleft palate, under-shot or over-shot jaws), spinal deformities, tail deformities (no tail, partial tail missing, “curly” tails), and any other gross deformity. These deaths generally occur within the first month of life. Figure 1 shows some examples of defects that would be classified as congenital.
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Table 1. Summary statistics of nests from different collection areas in the Porosus resource available for analysis. n is the total number of live hatchlings from each area in the particular year, NoHatch is the number of live hatchlings from each clutch put to farm.
2005 Area 1 2 3 4 5 6 7 8 9 10 11 12 13
n 2750 0 2868 263 12 69 1533 0 65 2313 556 1192 0
2006 Av. NoHatch ± SD 32.76 ± 11.07 36.77 ± 11.70 28.67 ± 9.61 12 ± 0 26.86 ± 7.36 38.67 ± 10.41 32.32 ± 9.05 35.87 ± 11.02 35.43 ± 8.62 38.85 ± 14.14 -
2007 Av. NoHatch ± SD 29.57 ± 10.20 35.23 ± 11.68 37.84 ± 11.14 41.08 ± 11.68 35.76 ± 16.89 41.25 ± 11.45 42.02 ± 1.00 37.17 ± 14.95 34.51 ± 10.47 35.46 ± 10.92 38.42 ± 12.65 -
n 2323 1508 2661 180 110 0 1265 84 465 1621 1199 969 0
n 2729 787 3012 469 18 0 250 170 1111 1641 1237 231 685
Av. NoHatch ± SD 27.37 ± 10.16 32.27 ± 11.61 38.35 ± 11.03 36.21 ± 11.84 11.78 ± 4.28 39.58 ± 10.45 38.49 ± 9.65 38.77 ± 12.26 35.22 ± 12.40 35.26 ± 11.33 35.38 ± 9.18 43.51 ± 15.00
Runtism was defined by an emaciated, non-thriving animal in comparison to others of a similar age (Huchzermeyer, 2003), shown in Figure 2. Disease-related was determined following a pathological investigation at Berrimah Veterinary Laboratory (BVL; Northern Territory Department of Primary Industries, Fisheries and Mines). These were considered independent to the stress-related deaths described below. When appropriate, antibiotics were administered as determined by antibiotic sensitivity testing at BVL. Stress-related is defined when deaths occurred within a short time period after a management-induced stress event. These include minimising size variation within pens (grading), moving animals between pens, hot water services failing or pens left without water. In the majority of these cases, animals sent to BVL returned positive septicaemia pathology results and antibiotic treatment followed as appropriate. No visible ailments (NVA) - Deaths were allocated to this category when neither a disease outbreak nor stress incident was noted. These deaths usually occur randomly in pens with no distinct trend in mortalities. Management is any other event that does not fit into the above categories. An example is an injury event. When a crocodile died, it was denoted a one (1) in the appropriate category. However, in each year cohort, there were crocodiles that were still in the production system when this study period concluded (31st December, 2007). These animals were included in the study as censored records (coded as 0) to account for the study period ending before mortality could be observed (Southey et al. 2001). In addition, to maintain data integrity, there were some observations that were omitted as their scute cuts corresponded to nests that had zero hatchlings, either their hatch date or their death date were not recorded so their age at death could not be calculated or their scute cuts were not recorded.
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a)
e)
d)
b)
c)
Figure 1. Examples of congenital defects. a) cleft palate; b) weak yolk scar suture, rupturing exposes internal organs; c) undershot jaw; d) crocodile born with no eyes; e) born with no tail.
Figure 2. Compared to the same aged “normal” crocodile (below), the runt crocodile (top) appears emaciated with wasting particularly obvious in the neck and tail areas.
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2.1.3 Statistical analyses The survival time data were analysed using a Cox’s proportional hazards model in Survival Kit V3.12 (Ducrocq and Sölkner 1994; 1998) to identify risk factors. In addition, the data were analysed using a competing risk approach whereby different hazards of mortality could be assigned for the six different causes of mortality (Southey et al. 2004) described in Section 2.1.2. All categories were assumed to be independent. Furthermore, different models were used to analyse the two different datasets (Porosus resource and Pedigree resource) and a 5% significance level was chosen to evaluate explanatory variables by backward elimination. 2.1.3.1 Porosus model The model used for the Porosus data was specified as ln[hijk(t)] = ln[h0(t)] + (βHDHDaysjk + βNoNoHatchjk + Yeark + Areacodej) where hijk(t) is the hazard function for the ith individual from the jth areacode in the kth year at time t, h0(t) is the unspecified baseline hazard function, HDaysjk is the number of days between hatching date and the 1st of January in that particular year for an individual from the jth areacode in the kth year; βHD is the regression coefficient for HDays; NoHatchjk is the number of live hatchlings in a particular clutch from the jth areacode in the kth year; βNo is the regression coefficient for NoHatch; Yeark is the fixed effect of the kth year (k = 2005, 2006, 2007); and Areacodej is the fixed effect of the ith area of collection (i = 1,….,13). 2.1.3.2 Pedigree model Two models were used to analyse the Pedigree resource to obtain estimates of variance components for heritability and breeding value estimation as follows. 1. Pair model ln[hijk(t)] = ln[h0(t)] + (βHDHDaysjk + βNoNoHatchjk + Yeark + Pairj + Clutchjk) 2. Animal model ln[hijk(t)] = ln[h0(t)] + (βHDHDaysjk + βNoNoHatchjk + Yeark + Animali) where hijk(t) is the hazard function for the ith individual from the jth pair in the kth year at time t, h0(t) is the unspecified baseline hazard function, HDaysjk is the number of days between hatching date and the 1st of January in that particular year for an individual from the jth pair in the kth year; βHD is the regression coefficient for HDays; NoHatchjk is the number of live hatchlings in a particular clutch from the jth pair in the kth year; βNo is the regression coefficient for NoHatch; Yeark is the fixed effect of the kth year (k = 2005, 2006, 2007); Pairj is the random effect of pair (assumed N(0,σ2Pair)); Clutchjk is the common environment (random) effect of a clutch produced by the jth pair in the kth year (assumed N(0,σ2Clutch)); and Animali is the random effect of the ith individual (assumed N(0,σ2Animal)).
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The Pair model log-survival heritability estimates were calculated as
h
2 logt
2 σ Pair = 2 2 σ Pair + σ Clutch + π6
2
2 2 using the estimates of the variance component, σ Pair and σ Clutch (Isberg et al. 2004), whilst the animal model estimates were calculated as
2 = h logt
2 σ Animal
2 σ Animal + π6
2
2 using the variance component estimate, σ Animal .
2.2 Histopathology Forty crocodiles were sacrificed from Darwin Crocodile Farm for the histopathology component of this study. The study was split into two phases. Phase 1 was conducted in November 2005 and Phase 2 in July 2007. Phase 1 of the study was a pilot phase which involved a wide range of standard veterinary pathological procedures. Phase 2 was designed to target data collection to specific parameters that were identified in Phase 1 as possible differences in runts compared to “normal” crocodiles. In both Phases, ten runt crocodiles and ten normal crocodiles were randomly sampled over the period of one week (Table 2). Crocodiles were fasted for 72 hours prior to sampling to remove any effect of recent feeding on blood or tissue parameters. Crocodiles were randomly selected from several pens. The inclusion of ten normal crocodiles from the same cohort in each phase of the project was to have a control group reared under the exact same conditions to allow direct comparison with runts and facilitate interpretation of results. This was necessary since baseline or “normal” clinical pathological parameters and the histological appearance of tissues are not well documented in crocodiles of this species and age group. Table 2. Descriptive statistics of the runts and “normals” in each phase of the study.
Year 2005 2007
Status Runt Normal Runt Normal
Av. Age (SD) 197 (36.44) 225 (15.15) 124.7 (11.61) 118.4 (2.46)
Av. TL (SD) 35.41 (1.41) 56.02 (4.73) 34.35 (1.47) 48.35 (3.77)
Av. Bwt (SD) 74.4 (24.06) 454.9 (127.44) 64.3 (6.48) 286.3 (76.86)
During Phase 1, crocodiles were transported from the farm to the laboratory where blood sampling, euthanasia and post-mortems were conducted over the ensuing four hours. For Phase 2, blood sampling and euthanasia occurred at the farm, immediately after removing the crocodile from the pen, with subsequent post-mortem sampling occurring over the next four hours after transport to the laboratory. Total length and body weight were recorded for each crocodile (Table 2). All crocodiles were blood sampled from the cervical sinus using the technique described by Lloyd and Morris (1999). The initial 0.5 ml of blood was reserved for haematological study and the remainder of the blood sample used for biochemical analyses. Crocodiles were humanely euthanised immediately following blood sampling with a lethal intravenous dose of pentobarbitone sodium into the ventral tail vein.
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In both phases, a full gross necropsy was performed on each crocodile, taking note of any grossly evident abnormalities or differences between organs and tissues of runts and normals that may signify a problem with a particular tissue or organ system. In Phase 1, samples were aseptically obtained for bacterial culture from each crocodile and faeces was collected from the colon for parasitological study. In both phases, the carcass was fixed in 10% neutral buffered formalin for histological processing.
2.2.1 Haematology Routine diagnostic veterinary haematology was used to evaluate the red and white blood cell components of the circulating blood. These components can provide information on general health status, bone marrow function and whether the animal may be suffering from infection. Haematological investigations were performed on all crocodiles in both Phases of the study using blood anticoagulated with ethylene diamine tetra-acetic acid (EDTA). Packed-cell volume (PCV), which measures the ratio of red blood cells to total blood volume, was determined by centrifugation of blood in microhaematocrit tubes. The differential white blood cell count was made from a direct smear of the blood using a fast Wright’s-Giemsa type stain (Diff Quik, Lab Aids Pty. Ltd., Narrabeen, NSW, Australia). The total white blood cell count was determined by diluting 25 µL of blood in a 1:32 ratio with phloxine B stain solution, counting the number of eosinophils and heterophils in nine large squares of a standard haemocytometer and using the differential count to calculate the total number of white blood cells. The percentages of lymphocytes and monocytes from the differential count and the total white blood cell count were then used to calculate the absolute numbers of lymphocytes and monocytes in the blood.
2.2.2 Serum biochemistry A wide range of serum biochemical analyses were performed on all crocodiles in both Phases. Analyses included evaluation of electrolyte status (sodium, chloride and potassium), which provides information primarily on nutrition, hydration status and organ (primarily kidney) function. Function and evidence for necrosis of the liver was investigated by measuring tissue enzymes (alanine aminotransferase (ALT), aspartate amino-transferase (AST), alkaline phosphatase (ALP) and gamma glutamyl-transferase (GGT)). Creatine kinase (CK) was measured as an indicator of muscle damage and to facilitate interpretation of liver enzymes, some of which may also be produced in muscle. Serum minerals (calcium and phosphorus) were measured since they are required at certain concentrations for proper bone formation, in addition to providing pertinent information on kidney function in reptiles. Serum proteins were evaluated to assess the ability of the liver to produce protein (albumin), nutritional status (albumin) and immune status (globulins). Total serum iron was measured to investigate possible reasons for anaemia. Uric acid level, and to a lesser degree creatinine and urea, may reflect kidney function in reptiles, and glucose provides an indication of nutritional status, liver and pancreatic function. All analyses with the exception of globulin were performed on an automated biochemistry analyser (Konelab 20i, Thermos Electron Corporation, Vantaa, Finland). Serum was harvested from blood that was allowed to clot in plain blood tubes left at room temperature for two to four hours. Globulin concentrations were calculated by subtracting albumin concentration from total protein concentration.
2.2.3 Corticosterone assay In Phase 2, serum corticosterone, the major stress hormone in reptiles, was measured using a corticosterone kit test according to the manufacturer’s directions (Corticosterone HS EIA, IDS Ltd., Boldon, U.K.). In addition to the 20 crocodiles in the main Phase 2 sample, an additional seven normal crocodiles and three runts were sampled from the same pens and during the same period, in order to increase sample size for this assay. Where the serum corticosterone exceeded the upper limit of the assay range, the result used was the highest detectable value of the kit (20 ng/ml). In order to reflect the ongoing background stress level exhibited by the crocodiles and minimise elevation of corticosterone due to prolonged handling and transport following removal from the pen, the assay was
7
performed using serum harvested from blood collected at the farm immediately following removal of the crocodile from its usual pen.
2.2.4 Bacteriology General bacterial culture of two filtering organs (liver and spleen) was conducted in Phase 1 to investigate the possibility that runt crocodiles were more likely to have bacterial infection compared to normal crocodiles. The liver and spleen were aseptically removed at the beginning of the postmortem for culture in all crocodiles. Additionally, sterile swabs for culture were used to sample the contents of enlarged yolk sacs noted in two crocodiles (one runt and one normal). Techniques for the culture of tissues involved aseptically homogenising the tissue, then applying the material to a sterile swab. Swabs were used to inoculate tryptic soy agar with sheep blood and MacConkey agar with crystal violet (Oxoid Australia Pty Ltd., Thebarton, South Australia). Agar plates were examined for colonies after 24 and 48 hours incubation at 35ºC. Gram negative bacteria were speciated using Microbact biochemical strips (Oxoid Ltd., Hants, UK).
2.2.5 Parasitology To investigate the possibility that intestinal parasites are associated with runting, faecal flotations were performed on all crocodiles in Phase 1. The technique involved collection of all faeces in the colon of the crocodile, emulsification in a zinc sulphate solution, centrifugation and microscopic examination of both the surface layer and sediment. This technique will reveal significant numbers of nematode, trematode, cestode or pentastomid eggs, as well as coccidial oocysts.
2.2.6 Histology Histological examination allowed a detailed examination of the microscopic architecture and cellular morphology of tissues, and is much more sensitive than gross examination (i.e. with the unaided eye) in the detection of abnormalities. In Phase 1, a complete range of tissues, encompassing all organ systems, was examined histologically (see below). In Phase 2, selected tissues were examined that were noted in Phase 1 being distinct between runt and normal crocodiles. For preparation of histological slides, tissues that had been fixed in 10% neutral buffered formalin were trimmed and placed in cassettes for routine histological processing. Tissues were sectioned at 5 µm and stained with haematoxylin and eosin, a routine stain for histological examination of tissues. Organs/tissues examined included heart with large vessels at the heart base, lung, trachea, kidney, liver, oesophagus, stomach, duodenum, jejunum, large intestine, yolk sac remnant, pancreas, fat body, spleen, tonsil, thymus, thyroid, adrenal and pituitary glands, brain, spinal cord, eye, skin, skeletal muscle, bone, joint and bone marrow. The specific tissue orientation, region of an organ and size of section were made as uniform as possible to maximise the ability to compare the tissue among individuals and between runt and normal crocodiles. For example, sagittal sections of the heart, incorporating the apex of the ventricle, atrium and great vessels at the base of the heart, and a complete sagittal section of the brain. The pancreas was sectioned in the mid-region where lobes are intermingled with duodenal loops, and intestinal segments were taken at approximately the same level along the intestine and villus height was compared to width at the base of villi as a means of assessing possible villus atrophy in runts. For assessment of the growth plate, bone marrow and a synovial joint, a sagittal section of the distal femur to the proximal tibia was used. For the thymus, a routine transverse section was made incorporating the tissues running down the ventral neck (trachea, oesophagus, blood vessels, thymus and surrounding fibrous connective tissue) at the level of the bifurcation of the trachea, which is the usual location of the bulk of the thymic tissue. Where thymic lobes appeared reduced or absent in the initial section, additional sections in the same vicinity were made to ensure an accurate histological picture of the thymus was being obtained. In most cases, all organs listed above were examined in all crocodiles in Phase 1, although in some instances, small
8
organs, such as pituitary glands, were missed in individuals. Crocodiles were sexed by histological examination of the gonads. Since histological examination of tissues is a subjective procedure, an attempt to control bias was made by not labelling slides as to whether the crocodile was a runt or normal.
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3. Survival Analysis Results 3.1 Porosus resource A total of 36,346 crocodiles hatched between 2005 and 2007 and were used in the overall survival analysis. These animals were allocated into 13 areas (Area 1 is all captive nests and 2-13 are wild nest areas) depending on their clutch of origin. There was a total of 5,043 crocodile deaths until the last day of the study (31st December, 2007) leaving 31,303 (63.13%) animals still in the production system. Dummy records were created for these animals and right censored with an average censoring time of 649 days. Less than one per cent of total records needed to be omitted for data integrity purposes. A summary of these data is shown in Table 3, whilst a summary of the number of deaths within each category are given in Table 4. Table 3. Summary statistics of the Porosus resource data used in the survival analyses from Darwin Crocodile Farm. Right censored records were animals that were assumed to still be in the production system at the termination of the study period (31st December, 2007). Some observations were omitted due to data integrity concerns. Year 2005 2006 2007 TOTAL
Total hatchlings 11,621 12,385 12,340 36,346
Total no. deaths 1,847 1,556 1,640 5,043
No. right censored records 9,774 10,829 10,700 31,303
Av. censor age (± st. dev.) 1020 (24) 641 (28) 287 (23)
No. omitted observations 134 170 53 357
Table 4. Total number of deaths in the Porosus resource including a breakdown into the six defined categories.
Year 2005 2006 2007 TOTAL
Total no. deaths 1,847 1,556 1,640 5,043
Reason for death Congenital defects Runt 25 855 31 408 106 1,191 162 2,454
Diseaserelated 158 392 34 584
Stressrelated 184 80 91 355
No visible ailments 572 386 194 1,152
Managemen t 53 259 24 336
3.1.1 Survival analysis results A summary of the results for each cause of mortality from the Porosus resource is given below. A summary of the significant explanatory variables is provided in Table 5. With the exception of disease-related deaths, the regression coefficients for HDays were all positive indicating that for each day later a clutch hatches, the risk of mortality also increases. The regression coefficients for number of hatchlings from each clutch (NoHatch) for each cause of mortality was negative indicating that the greater number of hatchlings to hatch from a clutch, the lower the risk of mortality. Year (Tables 5 and 6) and Areacode (Table 5) were significant for each cause of mortality. Figure 3 shows the different Areacode hazards of mortality for overall survival, congenital defects and runtism, whilst Figure 4 shows the hazards for the disease-related, stress-related, no visible ailments and management death categories. For comparative purposes, Area 1 (captive nests) was used as the baseline area.
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Table 5. Significance summary for explanatory variates used in the Porosus resource analyses for the different causes of mortality. Regression coefficients (SE) on the log-hazard scale are given for the significant (p=0.000) HDays and NoHatch terms. A 9 indicates if the term was significant for Year or Areacode. 8 indicates the term was non-significant.
Overall Congenital Runt Disease-related Stress-related NVA Management
HDays 4.40×10-3 (5.57×10-4) 4.48×10-4 (3.14×10-3) 1.09×10-2 (7.85×10-4) -1.17×10-2 (1.77×10-3) 8 8 8
NoHatch -2.23×10-2 (1.20×10-3) -5.22×10-2 (6.27×10-3) -2.32×10-2 (1.72×10-3) -2.06×10-2 (3.54×10-3) -1.46×10-2 (4.60×10-3) -2.65×10-2 (2.50×10-3) 8
Year 9 9 9 9 9 9 9
Areacode 9 9 9 9 9 9 9
3.1.1.1 Overall Porosus survival analysis This analysis was conducted using the data without distinguishing the cause of crocodile mortality. Therefore, there were 5,043 crocodile deaths and 31,303 censored records available for analysis. All explanatory variables were significant (HDays, NoHatch, Year and Areacode; Table 5). The antilog of the estimate for HDays (Table 5) is 1.004405 indicating that for each day later a clutch hatches, the hazard of mortality increases by 0.44%. This is further exemplified when calculated on a weekly basis whereby the hazard of mortality is increased by 3.12% for every week later a clutch hatches. Furthermore, the number of hatchlings that result from a clutch (NoHatch) is also significant in predicting the hazard of mortality. For each additional hatchling, the risk of mortality decreases by 2.25% (antilog of -0.223). Of the three years of data analysed for overall survival, 2005 had the greatest number of deaths observed (n = 1947) and was used as a base to compare the other years. Table 6 shows the regression coefficients (±SE), hazard ratios (exponentiated coefficients) and the antilog of the 95% confidence interval for the year effects expressed as a deviation from the base year (in this case 2005). Compared to 2005, the hatchlings in 2006 had a 12% lower hazard of mortality, whilst 2007 had a 29% greater hazard. Figure 3 shows the relative risk ratio of each collection area (Areacode) as a deviation of Area 1 (captive nests at Darwin Crocodile Farm). Areas 2 (24%), 10 (13%) and 12 (26%) had significantly higher risks of mortality, whilst Areas 3 (30%) and 7 (18%) had significantly lower risks of overall mortality. Areas, 4-6, 8-9, 11 and 13 were not significantly different from Area 1 (p>0.05) mainly due to their low number of uncensored records. 3.1.1.2 Congenital defects There were 162 deaths in the congenital defects category with an average failure time of 27.77 days (min-max: 1 - 515days). All explanatory variates were significant (Table 5). For every day later a clutch hatches, the hazard of mortality increases by 0.04% (0.31% per week), whilst for every additional hatchling produced from a clutch, the risk of mortality decreases by 5.08%. For this analysis, 2007 was used as the base year for comparison as it had the greatest number of uncensored deaths (Table 6). The reason for the increased number of congenital deaths in 2007 was most likely a management decision to give more animals the opportunity to survive rather than any actual year effect. In comparison, 2005 and 2006 had significantly lower hazards of 72% and 65%, respectively.
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Only Areas 9 and 13 were significantly different to Area 1, with increased hazards of 46% and 92%, respectively. All other areas were non-significantly different (p>0.05) due to the low number of observed failures in this category. Area 6 had no deaths in this category (Figure 3). Table 6. Estimates (±SE) of year effects for each mortality cause, their hazard ratios and the antilog of the 95% confidence interval (CI) using the Cox proportional hazards model. The hazard ratio is the antilog of the estimate and represents the risk of mortality. All are expressed as ratios relative to the year with the greatest number of observed deaths.
95% CI Year Estimate ± SE Overall survival 2005 2006 -0.13 ± 0.04 2007 0.26 ± 0.04 Congenital defects 2005 -1.26 ± 0.24 2006 -1.04 ± 0.22 2007 Runtism 2005 -0.67 ± 0.05 2006 -1.49 ± 0.06 2007 Disease-related 2005 -0.92 ± 0.10 2006 2007 -2.45 ± 0.18 Stress-related 2005 2006 -0.64 ± 0.14 2007 -0.00 ± 0.15 No visible ailments 2005 2006 -0.21 ± 0.07 2007 -0.72 ± 0.09 Management 2005 -1.73 ± 0.17 2006 2007 -1.00 ± 0.37
Hazard Ratio
Lower
Upper
1.00 0.88 1.29
0.81 1.20
0.95 1.40
0.28 0.35 1.00
0.18 0.23 -
0.45 0.54 -
0.51 0.23 1.00
0.46 0.20 -
0.57 0.25 -
0.40 1.00 0.09
0.33 0.06
0.48 0.12
1.00 0.53 1.00
0.40 0.75
0.70 1.34
1.00 0.81 0.48
0.70 0.40
0.94 0.58
0.18 1.00 0.37
0.13 0.58
0.25 0.23
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b
4.00 Overall Congenital
3.50
Runtism
b 3.00
Risk ratio
2.50 2.00
c
c 1.50 1.00
c
a
a
a
a bc
a
a c 0.50 0.00 1
2
3
4
5
6
7 Areacode
8
9
10
11
12
13
Figure 3. Probability of mortality and standard errors for each collection area (Areacode) for overall survival, congenital defects and runtism. Data points labelled with the same letters are significantly different from Area 1 (captive nests) for their respective cause of mortality.
3.1.1.3 Runtism analysis There were 2,454 deaths from runtism over the study period with an average age of death of 192 days (min-max: 5 - 1035 days). All explanatory variables were significant (Table 5). The hazard of mortality increases significantly by 7.91% for every week later a clutch hatches (1.09% per day). However, this hazard is offset by 2.29% for every additional hatchling produced from a clutch. Using 2007 as a base year, crocodiles that hatched in both 2005 and 2006 had significantly lower hazards of mortality (49% and 77%, respectively; Table 6). The reason for the large discrepancy between 2007 and the other years was largely due to management. This lead to a disease outbreak and early antibiotic administration which resulted in the majority of these animals never thriving and eventually they became runts. Although the initial animals were categorised in the stress-related cause of mortality, once the course of antibiotics was finished and no further bacteria were being detected (as reported by pathological studies at BVL), the animals that continued not to thrive were considered as runts. Areas 2 (59%), 10 (37%) and 12 (67%) had significantly larger hazards of mortality compared to Area 1, whilst Area 3 was significantly lower (36%; Figure 3). All other areas were non-significantly different to Area 1.
3.1.1.4 Disease-related analysis 584 disease-related deaths were recorded with an average age of death of 129.7 days (min-max: 2 – 596 days). Interestingly, the hazard of mortality for an animal dying due to a disease-related cause decreased by 7.84% for each week later the clutch hatched (1.16% per day), and decreased further (2.03%) for each additional hatchling that was produced from the clutch (Table 5).
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For this cause of mortality, 2006 was used as the base year for comparison and both 2005 (60%) and 2007 (91%) had lower hazards of mortality due to disease-related causes (Table 6). All areas were non-significantly different to Area 1 (p>0.05) with the exception of Area 3 which had a 25% lower hazard of disease-related mortality. Area 6 had no observed disease-related mortalities (Figure 4). 3.1.1.5 Stress-related analysis 355 stress-related mortalities were recorded during the study period (average age 292.77 days; minmax: 2 – 1,023 days). The time of hatch was non-significant, whilst for each additional hatchling produced in a clutch, the hazard of mortality decreased by 1.45% (Table 5).
2.50 Disease-related Stress-related
2.25
NVA 2.00
Management
Risk ratio
1.75 1.50 1.25 1.00
a b cd a
0.75
c
c b
d
d
c
d
d
0.50 0.25 0.00 1
2
3
4
5
6
7 Areacode
8
9
10
11
12
13
Figure 4. Probability of mortality and standard errors for each collection area (Areacode) for the disease-related, stress-related, no visible ailments (NVA) and management categories of mortality. Data points labelled with the same letters are significantly different from Area 1 (captive nests) for their respective cause of mortality.
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Although 2005 was used as a base year, 2007 was non-significantly different (p>0.05). However, 2006 had a 47% lower hazard of mortality compared to the other two years (Table 6). The greater hazard in 2007 was related to a management difficulty mentioned above in 3.1.1.3 where the initial cause of deaths were categorised as stress-related. Only Area 3 had a significantly different hazard compared to Area 1 (42% lower), whilst Areas 6 and 8 had no mortalities recorded in this category (Figure 4). 3.1.1.6 No visible ailments There were 1,150 deaths in this category with an average failure time of 235.53 days (min-max: 1 1035days). HDays was non-significant (Table 5), whilst an increase in NoHatch decreased the hazard of mortality by 2.62%. For this analysis, 2005 was again used as the base year for comparison (Table 6) as it had the greatest number of observed deaths. In comparison, 2005 and 2007 had significantly lower hazards of 19% and 52%, respectively. Areas 3 (30%), 7 (23%) and 9 (35%) had significantly lower hazards compared to Area 1, whilst all other areas were non-significantly different to Area 1 (Figure 4). 3.1.1.7 Management Management deaths occur occasionally on the farm due to unforeseen events. There were 336 deaths recorded in the Management category during the study period (average age 357.92 days; min-max: 1 – 957 days). As expected, neither HDays nor NoHatch were significant (Table 5) as these events occur randomly. Crocodiles that hatched in 2005 (82%) and 2007 (63%) had significantly lower hazards of mortality than animals that hatched in 2006 (Table 6). This was mainly a function of the newly constructed yearling pens where piling-up of animals became a problem due to delayed refilling of water into these pens. When the husbandry structure was optimised, these mortalities decreased. Accordingly, Management was the appropriate category for these animal deaths to be categorised under. Interestingly, there were significant area effects (Figure 4). Areas 7, 10, 11 and 12 had significantly lower hazards of mortality compared to Area 1 (59%, 34%, 33% and 52%, respectively).
3.1.2 Kaplan-Meier survival functions For each mortality category, a Kaplan-Meier estimate of the baseline survival function from hatch to 365 (Figure 5) and 1,035 (Figure 6) days of age was produced. The plot shows the probability of a crocodile surviving to any given day, and shows that the first year is definitely the period of highest mortality. The probabilities of crocodiles surviving to day 365 and day 1,077 for each cause of mortality are given in Table 7. A brief description of each survival function is given below. Congenital – 75% of congenital defect deaths occur by day 67 with the majority being animals with unabsorbed yolk sacs. The curve declined steeply to day 67 (99.72%) and then gradually becomes horizontal. There was only one death after day 365 (day 515) which was an animal with a spinal defect. Runtism – Deaths from runtism constitute the majority of deaths that occur on the farm. Few deaths classified as runtism occur before day 71. However, between days 71 and 250, the probability of survival decreases rapidly (90.69% at day 250). After day 250, the decline continues albeit at a slower rate.
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Disease-related - The probability of a hatchling crocodile surviving a disease outbreak to three months is 99%. However, between three and five months, around the same time the residual yolk sac is fully absorbed, the probability of a hatchling surviving a disease-related outbreak is reduced by 1.5% (97.5% survival probability at day 160). From this time, the probability of survival plateaus to the end of the first year (96.8%) and the remainder of its production life (96.7%). Stress-related – The trend for this cause of mortality was similar to that described above for diseaserelated mortalities, although it occurs later after hatching. The first 100 days is relatively free of stress-related deaths as no movements, etc occur during this time (probability of survival 99.78%). However, after day 100, management regimes such as grading and moving begin and the probability of survival decreases by 1.23% to day 424 (98.55%). The mortalities then plateau until day 616 (98.46%) and then drops again by 0.45% until day 829 (98.01%). No visible ailments – Deaths in this mortality category are consistent during the first 267 days with the probability of survival decreasing by 4%. This is proceeded by a continual decline at 94.29% to day 1077. Management – The probability of mortality due to management is negligible over the first 289 days (0.66%). However, by day 428, the probability had increased by 2.11% before plateauing until day 502 and then further increasing to day 822. The first drop in survival was mainly influenced by the slow re-filling of water into some pens, as previously mentioned in Section 3.1.1.7. Table 7. Probability of a crocodile surviving to day 365 and day 1077 for each cause of mortality.
Cause of mortality Overall survival Congenital defects Runtism Disease-related Stress-related NVA Management
Probability of survival to day: 365 1077 88.15% 83.39% 99.40% 99.39% 89.74% 86.05% 96.77% 96.71% 98.64% 97.85% 96.13% 94.29% 98.50% 96.24%
16
1 0.99 0.98
17
Estimated survivor function
0.97 0.96 0.95
Overall Congenital defects
0.94
Runtism Disease-related
0.93
Stress-related NVA
0.92
Management
0.91 0.9 0.89 0.88 0
50
100
150 200 Survival time (days)
250
300
350
Figure 5. Kaplan-Meier estimated survival functions for crocodiles between hatch and one year of age (365 days) for each cause of mortality. The y-axis has been scaled from 0.88 to 1.00 to provide a clearer view of the trend for each curve.
1 0.98
18
Estimated survivor function
0.96 0.94 Overall
0.92
Congenital defects Runtism Disease-related
0.9
Stress-related NVA
0.88
Management
0.86 0.84 0.82 0
100
200
300
400
500 600 700 Survival time (days) 365 days
800
900
1000
1100
Figure 6. Kaplan-Meier estimated survival functions for crocodiles between hatch and day 1077 (2.95 years) for each cause of mortality. The vertical line indicates one year of age (365 days). The y-axis has been scaled from 0.82 to 1.00 to provide a clearer view of the trend for each curve.
3.2 Pedigree resource Progeny records were collected from 67 “pair” families (53 sire families; 67 dam families). Parents were all known-breeding pairs that were originally wild caught and assumed to be unrelated. There was a total of 2,721 hatchlings of which 407 mortalities were recorded during the study period. Summary statistics are shown in Table 8. Those animals that were still in the production system at the end of the trial period were right censored (n = 2,314; 85.04%) with an average censoring time of 629 days. A summary of the number of deaths within each category are shown in Table 9. Computational problems in fitting the survival model occurred when analysing deaths due to the management category, consequently this cause of death was omitted from further analyses. Table 8. Summary statistics of Pedigree resource data used in the survival analyses from Darwin Crocodile Farm. Right censored records were animals that were assumed to still be in the production system at the termination of the study period (31st December, 2007). Year 2005 2006 2007 TOTAL
Total hatchlings 909 815 997 2721
Total no. deaths 143 104 160 407
No. right censored records 766 711 837 2314
Av. censor age (± st. dev.) 1007 (289) 628 (151) 252 (71)
Table 9. Total number of deaths in the Pedigree resource and a breakdown into the six defined mortality categories.
Year 2005 2006 2007 TOTAL
Total no. deaths 143 104 160 407
Reason for death Congenital defects Runt 4 63 1 30 2 108 7 201
Diseaserelated 12 16 9 37
Stressrelated 23 20 19 62
No visible ailments 39 29 22 93
Managemen t 2 8 0 10
3.2.1 Pair model survival analysis results A summary of the results from the Pedigree resource using the pair model are given below, whilst a summary of the significant explanatory variables are given in Table 10. Hdays was significant for all causes of death with the exception of congenital defects and no visible ailments (NVA), whilst NoHatch was non-significant for all causes except for disease- and stress-related deaths. Year was significant for the overall survival and runtism analyses. The random effect of Pair was significant in all of the analyses. Clutch was modelled as an interaction term between Pair and Year, and thus was only evaluated for the overall survival and runtism causes of death where year was also significant. However, Clutch was only significant for the runtism cause of death. Table 10 also shows the heritability estimate for each cause of mortality. The heritability estimates for all causes of death were 0.76 (SE 0.09) with the exception of runtism which is 0.71 (SE 0.08).
3.2.1.1 Overall Pedigree survival model The 407 deaths occurred at an average age of 192.23 days (min – max: 2 – 1,002 days). The antilog estimate for HDays (Table 10) indicated that for each day later a crocodile hatches the hazard of mortality increases by 0.59% per day or 4.23% per week. 2007 was used as the base year and in comparison, 2005 and 2006 had reduced hazards of mortalities of 72% and 62%, respectively.
19
Pair 1 had the lowest log hazard estimate of -1.59 (antilog estimate (e-1.59) = 0.204), whilst Pair 51 had the highest estimate of 1.75 (antilog estimate = 5.78; Figure 7A). This means that a juvenile from a clutch produced by Pair 1 has the highest chance of surviving to slaughter whilst juveniles produced by Pair 51 have the lowest chance, compared to all other breeding pairs. More specifically, if we denote S0(t) as the baseline survival function, that is the probability that an individual survives to age t, averaged across the population, then the survival function for offspring of Pair 1 will be [S0(t)]0.204 (increased survival) whereas those from Pair 51 will have a survival function of [S0(t)]5.78 (reduced survival). So in general, the survival function for offspring of a particular pair will be [S0(t)]R, where R is the hazard ratio for a particular pair, being the antilog of the BLUP estimate on the log hazard scale. The baseline survival function, S0(t), is routinely available in survival analysis output (Ducrocq and Sölkner 1994; 1998), and has been shown in Section 3.2.2 (Figures 12 and 13). Since the hazard of mortality changes with time, it was decided that the most appropriate time to approximate breeding values was at day 365 (or one year). Juvenile survival CBVs are expressed as a percentage difference in survival to 365 days, relative to the population average, and have been calculated as
⎧[S (365)]R i ⎫ − 1⎬ ×100 = [S0 (365)]R i −1 − 1 ×100 , CBVi = ⎨ 0 ⎩ S0 (365) ⎭
{
}
with approximate standard errors
{
}
SE(CBVi ) = − R i lnS0 (365) [S0 (365)]R i × SE(BLUP) × 100 where Ri and SE(BLUPi) are the hazard ratios (exponentiated hazard BLUP estimates) and standard error of the BLUP estimates, respectively (Isberg et al. 2004). Table 10. Significance summary for explanatory variates used in the Pair model analyses for the different causes of mortality. Regression coefficients (SE) on the log-scale are given for the significant (p
