Improving Reproduction and Genetics in Game Birds and Ratites

RIRDC Publication No. 11/061

RIRDCInnovation for rural Australia

By Irek A. Malecki and Graeme B. Martin

October 2011

RIRDC Publication No. 11/061 RIRDC Project No. PRJ-000450

ISBN 978-1-74254-245-4 ISSN 1440-6845

Improving Reproduction and Genetics in Game Birds and Ratites Publication No. 11/061 Project No. PRJ-000450

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.

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Researchers Contact Details Associate Professor Irek A. Malecki Winthrop Professor Graeme B. Martin School of Animal Biology MO92, School of Animal Biology MO92, Faculty of Natural and Agricultural Sciences, Faculty of Natural and Agricultural Sciences, University of Western Australia University of Western Australia Crawley 6009 Crawley 6009 Western Australia Western Australia

Phone: +61 8 64887025 Phone: +61 8 64883424 Fax: +61 8 64881029 Fax: +61 8 64881029 Email: ireneusz.malecki@animals.uwa.edu.au Email: [email protected]

In submitting this report, the researchers have 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: 02 6271 4100 Fax: 02 6271 4199 Email: [email protected]. Web: http://www.rirdc.gov.au

Electronically published by RIRDC in October 2011 Print-on-demand by Union Offset Printing, Canberra at www.rirdc.gov.au or phone 1300 634 313

Foreword

Game bird production is not efficient because of low rates of fertility and hatchability, variable egg production and growth rates, inadequate nutrition, and lack of quality breeding programs. The game bird industries would benefit from the transfer of fertility and hatchability technologies that already exist in the emu industry so that problems in reproductive inefficiency can be objectively identified, quantified, prioritised and eliminated. Breeding flocks and individuals within the flocks could then be evaluated for their genetic value and this would improve breeding programs and lead to greater genetic gains.

It is important for the Australian game bird industries to recognise that improvements are feasible because there is genetic potential in our breeding populations of ducks, pheasants and pigeons, but each needs to either revise current breeding objectives to take into account existing variation in breeding populations or introduce better genetics from overseas.

The ratite industries are plagued by similar problems, many of which could be overcome by artificial insemination (AI) technology, so there is a strong need for reliable methods for semen collection, sperm storage and artificial insemination, and an understanding of factors responsible for ejaculate quality and sperm supply rates.

AI technology would aid the ratite industries in achieving rapid genetic improvements. This has been largely achieved by the project, so we now have reliable semen collection methods using teaser and dummy females, and artificial insemination methods that are effective and ‘animal-friendly’.

The project has demonstrated that there are production constraints in the management, reproduction, nutrition and genetics of game birds, so the next step is to develop breeding and husbandry strategies to improve the efficiency of production. For that to occur, assistance is needed to maintain progress until the breeding programs and standard management practices are in place. Every game and ratite industry in Australia clearly has the potential to grow and become more economically viable.

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 2000 research publications, forms part of our New Animal Products R&D program, which aims to provide knowledge for diversification of the Australia’s rural industries.

Most of RIRDC’s publications are available for viewing, free downloading or purchasing online at www.rirdc.gov.au. Purchases can also be made by phoning 1300 634 313.

Craig Burns Managing Director Rural Industries Research and Development Corporation

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Acknowledgments

The authors wish to acknowledge numerous contributions from game bird, emu and ostrich producers, the technical and research staff in Australia and overseas who, in various ways, contributed to this project. This report would not have been possible without a valuable input provided by:

Glenn Fraser and John Millington (Luv-a-duck, Victoria), Kevin and Ros O’Bryan (Olson’s Game Birds, Victoria), Leona McLaren, Pat Benett, Kelvin Jenzen (Pigeon Growers, Victoria), Mike Cowie and Rob Stout (Wangara Poultry, Victoria) and Ian Milburne (Glenloth Game, Victoria); Paul Kent, Danny Singh and Tanya Nagle (Queensland Poultry Research and Development Centre, Alexandra Hills, Queensland) and Michael Evans (Applied Nutrition, Queensland); Anne and Peter Cook, Ronald Boald, Sue Fallen, Jo Reid, Trevor Want and Richard Tan (Pigeon Growers, Queensland); Rudy Kopecny (Pigeon Grower, NSW); Kip Venn (Free Range Emu Farm, Western Australia); Dya Maharani, Janine Wojcieszek, Kristy Glover, Daniel Malecki (University of Western Australia). Zanell Brand and Schalk Cloete (Western Cape Institute of Agriculture: Elsenburg and Oudtshoorn Research Farm (South Africa).

Ewa Lukaszewicz and Artur Kowalczyk from the Wroclaw University of Life and Environmental Science (Poland), Jaroslaw Horbanczuk (Institute of Genetics and Animal Breeding, Polish Academy of Science, Jastrzebiec, Poland) and Henryk Naranowicz, the owner of ‘Stypulow Ostriches’ whose dedication and support of farm-based research was exemplary and allowed for the major international breakthrough in ostrich semen collection.

Acknowledgements also to Paulina Rybnik-Trzaskowska (PhD student, Institute of Genetics and Animal Breeding, Polish Academy of Science, Jastrzebiec, Poland) who studied semen collection in ostriches and Sushil Sood (PhD student, School of Animal Biology, University of Western Australia) who studied factors affecting storage of emu sperm in vitro. These people were funded from outside the project and some of their work forms part of this report.

We sincerely thank RIRDC for providing the funds and Dr Peter McInnes (Research Manager, RIRDC) for his encouragement and advice during the course of this project.

Abbreviations

AC – artificial cloaca AI – artificial insemination AME – apparent Metabolisable energy ANOVA – analysis of variance BW – body weight CP – crude protein CV – coefficient of variation DM – dry matter EM – early mortality GD – germinal disc GE – gross energy HBW – high body weight

HolesIPVL – sperm holes made in the IPVL IPVL – inner perivitelline layer LBW – low body weight LM – late mortality Log – logarithm ME – Metabolisable energy MJ – mega joules MM – middle term mortality N – nitrogen NSW – New South Wales OPVL – outer perivitelline layer PBS – phosphate buffered saline QLD – Queensland QDPI&F – Queensland Department of Primary Industries and Fisheries QPRDC – Queensland Poultry Research and Development Centre

SpermOPVL – sperm trapped in the OPVL SQUAB – young pigeon UWA – University of Western Australia VIC – Victoria WA – Western Australia YO – year old

Contents

Foreword ...... iii Acknowledgments ...... iv Abbreviations ...... v Contents ...... vi Executive Summary ...... x 1. Introduction ...... 1 2 Objectives ...... 2 3. Methodology ...... 3 3.1. Development of fertility and hatchability technology for the duck, pheasant and pigeon ...... 3 3.2. Quality of game bird breeding stocks ...... 3 3.3. Development of semen collection and artificial insemination for the ostrich ...... 8 3.4. Identification of factors affecting quality and quantity of the emu and ostrich ejaculates ...... 9 3.5. Determination of the duration of in vivo sperm storage and rate of lay in the absence of copulation in the ostrich ...... 10 4. Development of fertility and hatchability technology for the duck, pheasant and pigeon ...... 12

4.1 Visualisation of SpermOPVL, HolesIPVL and germinal disc morphology ...... 12 4.2. Pigeon early embryo development...... 14 5. The quality of game bird breeding stocks ...... 17 5.1 Pigeon ...... 17 5.2 Pheasant ...... 23 5.3 Duck ...... 25 6. Semen collection and artificial insemination for the ostrich ...... 26 6.1 Semen collection methods ...... 26 6.2 Artificial insemination (AI) of female ostriches ...... 27 7. Factors affecting the quantity and quality of emu and ostrich ejaculates ...... 29 7.1 Emu – effect of collection temperature on ejaculate quality ...... 29 7.2 Ostrich – effect of male age on sperm quality ...... 29 8. Duration of sperm storage and rate of lay in the female ostrich in the absence of copulations ...... 31 8.1 Duration of sperm storage ...... 31 8.2 The rate of lay ...... 31 8.3 Additional findings ...... 32 9. Discussion of Results ...... 33 10. Implications for relevant stakeholders ...... 39 11. Recommendations ...... 40 12. References ...... 42 Appendix 1. Communications resulting from this project ...... 44

Figures

Figure 1. The relationship between duration of incubation (days) and embryo wet weight...... 16 Figure 2. The relationship between duration of incubation (days) and embryo wet weight...... 16 Figure 3. Squab numbers (mean ± sem) per processing day of the month in a 12-month production cycle in Queensland (left) and in Victoria (right)...... 17 Figure 4. Mean (± SEM) squabs per grade after daily processing in plants in Queensland and in Victoria...... 17 Figure 5. Mean numbers of squabs in each carcass grade processed daily from July to June (12- month production) in Queensland (left) and in Victoria (right) ...... 19 Figure 6. The oviposition rate of the pheasant breeding flock as related to female age and month (open bar – 1 yo females; closed bar – 2 yo females)...... 24 Figure 7. Effect of flock age on the mean (±SEM) numbers of sperm holes in duck eggs...... 25 Figure 8. Effect of pairing combination on sperm penetration rates...... 25 Figure 9. Effect of male type on sperm penetration rates...... 25 Figure 10. Effect of temperature of the collection vessel on numbers of normal, bent and live emu spermatozoa after 24 h storage...... 29 Figure 11. The relationship between age of male ostriches and numbers of sperm found in the perivitelline layer of fertilised eggs laid by their companion females ...... 30

Figure 12. Decline in numbers of OPVLsperm over successive days of the fertile period in the female ostrich ...... 31 Figure 13. Changes in oviposition interval in relation to time of fitting the ‘apron’ (0 days)...... 32 Figure 14. Decline in numbers of female ostriches laying eggs after fitting the ‘apron’ onto their male companion...... 32

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Tables

Table 1. Pigeon reproductive traits recorded on collaborating farms and at UWA...... 5 Table 2: Allocation of diets to pigeons (5 male pigeons/pen)...... 6 Table 3. Squab carcass grades...... 17 Table 4. Egg and squab production by colonies from NSW, QLD and VIC between February and June 2006 (120 days)...... 20 Table 5. Egg production from NSW, QLD and VIC colonies between July and September 2006 in the absence of incubation (50 days)...... 20 Table 6. Mean, Min and Max values for reproductive traits of pigeon pairs held at the Shenton Park Field Station (UWA)...... 21 Table 7. Mean, minimum and maximum values for reproductive traits of pigeons in colonies (VIC 1, QLD 1 and 2) and individual pairs (VIC 2 and QLD 3), their pair efficiency and squab yield...... 21 Table 8. Mean, Min, Max and CV(%) values for production traits of farmed pigeons...... 22 Table 9. Effect of crude protein (CP) levels [%] in feed on squab performance...... 22 Table 10. Effect of lysine levels (%) on squab body weight...... 23 Table 11. Feed and gross energy (GE) consumption of adult pigeons on different diets...... 23 Table 12. Nitrogen corrected energy values of the feed consumed by adult pigeons...... 23 Table 13. Fertility (Mean ± SEM) of pheasant eggs estimated in incubated and in fresh eggs...... 24 Table 14. General characteristics (Min-Max values) for ostrich ejaculates...... 27 Table 15. Effect of male age on ejaculate characteristics in ostriches (Mean ± SEM)...... 30

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Photos

Photo 1. Commercial-type pigeon loft with 20 pairs per colony pen...... 4 Photo 2. Pigeon pen with plastic drums as nest boxes (two per pair) and a self-feeder in the centre...... 4 Photo 3. Colony pen with 4-tier double nest boxes...... 4 Photo 4. A self-feeder with maze, peas, wheat and sorghum (left to right)...... 4 Photo 5. Pigeon shed at QDPI&F...... 5 Photo 6. Pigeon wire nests at QDPI&F...... 5 Photo 7. Galvanised cages used in energy requirement studies at QPRDC...... 6 Photo 8. Male pheasant with two hens at the commercial facility...... 7 Photo 9. Duck breeding pen at UWA facility with a nest box (right), feeder (left of the box). and a bath tub (left)...... 7 Photo 10. Selection of young male ostriches for the dummy method: 3 males showing interest in a human by kantling, pushing the fence or staying close and flapping wings...... 9 Photo 11. Selection of pairs for the teaser method. A pair of ostriches is being accustomed to a handler being near them during copulation...... 9 Photo 12. Standing quietly female is examined for ease of the artificial insemination...... 9 Photo 13. Crouching female ostriches being selected for the artificial insemination...... 9 Photo 14. Male ostrich wearing apron returning to his female...... 10

Photo 15. Ostrich SpermOPVL counted in the germinal disc area for estimation of the sperm loss or sperm supply rates...... 10 Photo 16. Sperm holes in the yolk membrane following staining with Schiff’s reagent in bright field view...... 12 Photo 17. Trapped sperm in the yolk membrane visualised with DAPI...... 12 Photo 18. Male ostrich approaching a crouching teaser female...... 26 Photo 19. The phallus is redirected into the artificial cloaca...... 26 Photo 20. The phallus is held in the artificial cloaca during ejaculation while the collector squats behind the birds...... 26 Photo 21. Following ejaculation, the male dismounts and the collector leaves the pen...... 26 Photo 22. Hemp sack dummy for collecting semen. The AC is shown in the right hand corner insert...... 27 Photo 23. Male ostrich mounting the dummy...... 27 Photo 24. Artificial insemination of female ostriches that stand still after blindfolding...... 28 Photo 25. Artificial insemination of a female ostrich that is crouching for the inseminator...... 28

Plates

Plate 1. Appearance of the fertilised and unfertilised germinal disc (GD) in the duck, pheasant and pigeon eggs...... 13 Plate 2. Early embryo morphology in the pigeon, before and after incubation for up to 3 days...... 14 Plate 3. Early embryo morphology in the pigeon after artificial incubation for 4 to 6 days...... 15

Executive Summary

What the report is about The report describes the development and application of fertility and hatchability technologies in commercial production of Peking ducks, pheasants and meat pigeons, and artificial insemination technology for ratites. It clearly identifies a number of major constraints to efficient production of ducks, pheasants and pigeons. Ostrich producers are provided with new methods for semen collection and artificial insemination. The ostrich fertile period has been determined and some factors affecting the quality of the emu and ostrich ejaculates have been identified. Who is the report targeted at? The report is primarily for managers and researchers working in production of poultry, game birds, emus and ostriches, although it is also relevant to those involved in breeding of other bird species, as well as conservationists. Background Game bird industries in Australia are affected by inadequately performing breeding flocks, as is evident from the low ratio of wholesale birds to laid eggs. This is a problem because the Australian industry is small compared to those in Asia or Europe so we already have difficulty competing on international markets. Therefore, our game bird industries must improve their efficiency if they are to remain viable on the domestic scene, or if they want to compete internationally (RIRDC Report: Opportunities for exporting game birds - Publication No 03/106, 2003). On the other hand, our industries have great potential for increased production and processing with little capital expenditure, so could become globally competitive. Objectives Objective 1: Develop fertility and hatchability technology for the duck, pheasant and pigeon industries. Objective 2: Determine the quality of game bird breeding stocks and identify strategies for improving their efficiency. Objective 3: Develop semen collection and artificial insemination methods for the ostrich industry. Objective 4: Identify factors affecting quality and quantity of emu and ostrich ejaculates. Objective 5 Determine the duration of in vivo sperm storage and rate of lay in the absence of copulation in the ostrich. Methods used New techniques were developed for the assessment of fertility, hatchability, embryo mortality, semen collection and artificial insemination. The efficiency and reproductive potential of meat pigeons were assessed from data provided by processing plants and breeding data collected from selected pairs on farms. Protein and energy requirements of Australian pigeons were studied under controlled conditions using formulated diets. The work was conducted at UWA and QDPI&F research facilities, and on collaborating pheasant, duck, pigeon and ostrich farms in Australia, Poland and South Africa. Results/key findings Progress has been made in fertility and hatchability assessment of eggs and breeding birds for the Peking duck, pheasant and meat pigeon. The reproductive potential of pheasants, ducks and pigeons is high but the constraints are species-specific so each industry needs a different approach to improve production. In the ostrich, methods for semen collection and artificial insemination have been developed and used to reveal the major influences of age, season and genotype on ejaculate quality. The duration of sperm storage in vivo in the ostrich is about 2 weeks, so females could potentially produce up to 6 fertilised eggs from a single insemination.

Implications for relevant stakeholders The production efficiency of pheasants, pigeons and ducks could be improved quite rapidly, but different approaches are needed for each species. Either production needs to strictly adhere to a designed breeding program or changes to the current program are needed. Emu production is a step closer to improvement through improved efficiency of semen handling and processing for storage and cryopreservation, while the ostrich industry is on the cusp of a major breakthrough with the availability of artificial insemination. These advances, if implemented, would benefit producers and the local, industry and research communities, but we need continued efforts from government and industry bodies to maintain, or even increase, research effort and infrastructure funding if we are to achieve economic viability of game bird and ratite production. Recommendations It is recommended that techniques for assessment of ‘true’ fertility and hatchability are adopted by game bird producers as they can provide them with objective means for identifying and quantifying reproductive problems. In the absence of sufficient knowledge or equipment on farm, such assessment could be provided by a specialist laboratory. Industries such as pheasant and pigeon require controlled breeding programs to take advantage of existing and introduced genetics in order to speed up improvements in production efficiency. Further research is needed to optimise nutrition of breeding pheasants and pigeons. The ostrich producers should take advantage of artificial insemination now that practical methods are available. The research now needs to concentrate on development of effective protocols for semen storage, freezing and artificial insemination.

1. Introduction

The success of the game bird industries in Australia is limited by inadequately performing breeding flocks, as is evident from the low ratio of saleable birds to laid eggs. The industries produce about 17 million birds per year with a retail value of A$290 million, but our industry is small compared to Asian or European countries and this makes it difficult to compete on international markets. The game bird industries must improve their efficiency if they are to remain viable, even on the domestic scene, or if they wish to compete internationally (RIRDC report Opportunities for exporting game birds - Publication No 03/106, 2003). Our industries have the potential to increase production and processing with little capital expenditure and could become globally competitive if flock performance was improved. Over the years, significant knowledge has been gained about different production systems for game birds, and the industries have received some scientific input, but more is needed. Game bird production relies on random mating and uses either breeders from pure genetic lines and crosses (quail, duck) or crossbreeds with desired phenotypic characters (pheasant, pigeon). Irrespective of the species and management system, the breeding stocks have a low reproductive efficiency, ranging from 50% (quail) to 70% (pigeon). While the production systems differ between these industries, the basic problem is the same: for the number of eggs laid, too few birds reach the market. Both fertility and hatchability appear to be low, but until they are objectively measured we will not be able to identify, quantify or prioritise the sources of wastage, let alone find solutions. ‘True’ fertility has never been measured in game birds in Australia so fertility rates are not actually known – it is feasible that fertility is high but poor outcomes are due to early embryo mortality. If that is the case, hatchability technology can then address the problem. These two important aspects of reproductive efficiency very much depend on genotype, diet and management. Reproductive efficiency in relation to genotype, age, season and management have been studied in duck, pheasant and squab production systems. In the absence of information on protein requirements for squab-producing pigeons in Australia, feeding trials were conducted using pelleted diets formulated with different protein and lysine levels. In addition, an energy level required by pigeons was estimated. The ratite industries, on the other hand, after surviving the ‘boom and bust’ times, have revised their breeder and grower management strategies and moved to a stage of selective breeding. Both the emu and the ostrich have tremendous potential that is yet to be realised. Markets have been drawn to the quality of oil (emu) and leather (ostrich) as well as the red meat. Consistent quality and quantity of these products is needed to assure market stability and this can be best achieved through genetic gain. However, selective breeding programs need AI technology because emus are monogamous and ostriches are at best only partly polygamous. The project has made advances in development of AI technology for ratites by working in Australia as well as with international collaborators. The emu industry can now take advantage of AI technology, although semen quality, storage and cryopreservation conditions need to be further optimised. The ostrich industry, by contrast, awaited reliable methods for semen collection and AI, techniques long perceived as very difficult because of the unfavourable behaviour of males and females in relation to humans. By developing criteria for selecting suitable individuals for training, reliable semen collection and artificial insemination methods have now solved this problem.

2 Objectives

Objective 1: Develop fertility and hatchability technology for the duck, pheasant and pigeon industries. Objective 2: Determine the quality of game bird breeding stocks and identify strategies for improving their efficiency. Objective 3: Develop semen collection and artificial insemination methods for the ostrich industry. Objective 4: Identify factors affecting quality and quantity of emu and ostrich ejaculates. Objective 5 Determine the duration of in vivo sperm storage and rate of lay in the absence of copulation in the ostrich.

Methodology

3.1. Development of fertility and hatchability technology for the duck, pheasant and pigeon

‘True’ fertility The appearance of the fertilised and unfertilised germinal disc on the egg yolk surface was used to determine true fertilisation status of the germinal disc in fresh or stored but non-incubated duck, pheasant and pigeon eggs. The appearance was studied by detailed observations under a stereoscopic microscope. A description of the unfertilised chicken and turkey germinal discs (Kosin 1944; Bakst et al. 1998) and the germinal discs from a sample of eggs laid by non-mated pheasants and ducks was used as a guide.

Sperm supply and fertilisation rates – the in vivo sperm-egg interaction assay (HolesIPVL) We determined sperm supply and penetration rates in breeding flocks by counting the holes made by sperm when they enter the egg cytoplasm (HolesIPVL). This technique was originally developed for poultry (turkey, chicken, quail) and we transferred them to emu. Now we develop them for the pheasant, duck and pigeon. Egg membrane was collected on a filter ring, cleaned of residual yolk with PBS, and placed on a glass slide. The membrane was then stained with Schiff’s reagent (Bramwell et al. 1995) and allowed to dry overnight on the lab bench at 18-20°C (room temperature) and 40-50% relative humidity. After the filter ring had dried out, it was removed leaving a stained membrane on the glass. The holes made by sperm were then viewed using standard light microscopy and counted in five successive fields of view under 20X objective starting in the centre of the GD. Pigeon embryo mortality – determining the age of embryo death

To determine the age of pigeon embryos at the time of death in the early to middle stages of development, the growth rate was studied for up to 8 days from the commencement of egg incubation. The eggs were collected on the same day they were laid and placed in a chicken incubator set at 37.5°C and 50-60% relative humidity. After incubation, the eggs were opened, the embryo was photographed in the shell using a DP70 digital camera (Olympus Pty. LTD, Australia) and its morphological characteristics were noted. The embryo was then removed and its wet weight estimated. The wet weight was determined in embryos from Day 3 of incubation. The data (n = 49) for embryo weight were plotted against incubation time. The growth rate fitted an exponential curve so the data were log-transformed and the equation was computed with the wet weight of the embryo as a dependent variable.

3.2. Quality of game bird breeding stocks

3.2.1 Pigeon

Abattoir production The abattoir data from two processing facilities, one in Queensland and one in Victoria, were collected to estimate the range of carcass grades for processed squabs (young pigeons), and the within and between-month variation. Data were obtained for the 12-month production cycle between July 2006 and June 2007. In Queensland, 215,936 squabs, supplied by up to 49 growers, were processed over 202 processing days. In Victoria, on the other hand, only 28,800 squabs supplied by up to 7 growers were processed over 52 processing days in the year.

Reproductive performance on farms The studies were conducted on farms using pairs maintained in colonies and as single pairs. Three farms participated, one in Victoria and two in Queensland. For the study, each farm allocated 1 loft containing a colony of 20 pairs (Photo 1) with an average production of about 11 squabs per pair. Each pair had access to two nests, either wooden or plastic drums (Photo 2). Individually penned birds (20 pairs) were on two farms (10 pairs each), one in Victoria and one in Queensland. The pairs were taken from several colonies and placed in a separate loft containing pens for single pairs with double nest boxes. The reproductive performance of pairs (Table 1) was recorded for 12 months between July 2006 and June 2007.

Photo 1. Commercial-type pigeon loft with Photo 2. Pigeon pen with plastic drums as 20 pairs per colony pen. nest boxes (two per pair) and a self-feeder in the centre.

Assessment of reproductive performance at UWA Shenton Park Field Station 30 pairs, each producing 10-14 squabs per pair was imported from growers in Victoria, New South Wales and Queensland. The pairs from each state were housed in separate 3 x 3 x 2.5 m pens. Each colony was provided with a set of nest boxes (Photo 3), a minimum of 2 nests per pair. Four grains (maize, peas, wheat and sorghum) were on offer and water was provided ad libitum (Photo 4). The diet was supplemented with shell grit and mineral-vitamin mix. The reproductive traits (Table 1) were initially recorded for 3 months (settling in phase) and then for 7 months.

Photo 3. Colony pen with 4-tier double nest Photo 4. A self-feeder with maze, peas, boxes. wheat and sorghum (left to right).

The rate of lay in the absence of incubation In the absence of any information on the ability of pigeons to lay in the absence of incubation and rearing, we studied the rate of lay by removing the eggs as soon as they were laid. The rate of lay was determined in pairs kept at the UWA Shenton Park Field Station. The nests were inspected daily and all new eggs were removed and recorded. The rate of lay was calculated by dividing the total number of eggs laid over the duration of the studied period.

Table 1. Pigeon reproductive traits recorded on collaborating farms and at UWA. Reproductive traits Explanation Egg weight Measured as soon as laid (24-48 hours) Hatch BW Chick Body Weight measured on hatch day 7D BW 7-Day Body Weight 14D BW 14-Day Body Weight 28 D BW 28-Day Body Weight Wean BW Body weight of squab weaned on day 28 Egg number/pair All eggs laid in studied period Squab number/pair All squabs weaned in studied period Egg number/clutch All eggs laid in a clutch Cycle duration (days) Interval between the egg of proceeding clutch and first egg of next clutch Clutch number/pair All clutches containing 1 egg or more

Pigeon nutrition The effect of crude protein, lysine and Metabolisable energy levels were studied at the Queensland Poultry Research & Development Centre (QPRDC) in consultation with Dr Michael Evans. Current pigeon diets were revised and a pellet diet was developed. Pigeon pairs were imported from growers in Victoria (n = 18 pairs) and Queensland (n = 19 pairs) and housed in a pigeon shed (Photo 5) subdivided into 12 pens, 2.55 x 1.50 m each. Each pen was stocked at 0.3 sq. m/bird, equipped with six mesh nest boxes (Photo 6) 60 cm wide x 40cm high x 40cm deep divided into two with a 12 cm perch, a bell drinker and poultry feeder.

Photo 5. Pigeon shed at QDPI&F. Photo 6. Pigeon wire nests at QDPI&F.

Crude protein (CP) and lysine levels The effect of crude protein levels (26 vs 20%) was studied over 152 days between June and October 2006. The effect of lysine levels (0.59, 0.72 and 0.85%) was studied over 150 days between February and June 2007. The number of eggs laid, egg size, number of clutches, number of squabs weaned, body weight at weaning and growth rate were recorded.

Metabolisable energy (ME) levels In the absence of current Australian research information on energy levels for squab producing pigeons, a feeding trial was conducted using pelleted diets formulated with three different lysine levels: 0.85% (Diet 1); 0.72% lysine (Diet 2) and 0.59% lysine (Diet 3). Celite (diatomaceous earth) was added to each diet as a marker at the overall rate of 2.0% of the prepared diets. The trial was conducted in a fully insulated, air-conditioned building specifically designed for work on avian Metabolisable energy. This building provided complete environmental control by the use of an integrated air-conditioning system and maintained at the temperature between 23°C and 25°C for the duration of the study. Pairs were separated about a month before the trial and respective males were grouped in large pens according to their final cage destination to help minimise territorial disputes. Five male pigeons were chosen from each of samples from Queensland and Victoria, with each state randomly represented over each of the three diets (Table 2). Males were chosen in preference to females to avoid potential contamination with broken eggs. Each cage was constructed of mesh and measured 95 cm (L) x 70 cm (W) x 40 cm (H).

Table 2: Allocation of diets to pigeons (5 male pigeons/pen). Origin Pen no Body weight Diet No at start (g) 1 3734.6 2 Queensland 2 3587.1 3 3 4055.4 1 4 3995.1 2 Victoria 5 4215.8 1 6 3907.2 3

Cages were fitted with a 50 mm x 25 mm wooden perch through the length of the cage and the galvanised mesh floor was covered with a small 15 mm x15 mm plastic mesh to compensate for the pigeons small feet and toes (Photo 7). Pigeons were stocked at 1330 cm2/ bird and were provided with ad libitum feed at three stations and a water supply, also at three places in each cage, in order to reduce the risk of territorial disputes.

Photo 7. Galvanised cages used in energy requirement studies at QPRDC.

3.2.2 Pheasant Egg production, fertility and hatchability were investigated on a commercial pheasant farm in Victoria. A 600-female breeding flock of the ring neck pheasant (Phasianus colchicus) was studied over two breeding seasons (2005-06 and 2006-07) to determine the effect of season and female age on egg production and fertility. The flock was maintained in 12 pens of 50 females in each with the sex ratio in the mating system of one male to five females (Photo 8). The flock consisted of 7 pens containing 350 one-year-old (1 yo) and 5 pens containing 250 two-year-old (2 yo) females.

Season 2005-06 Daily rate of lay per female and egg production performance were estimated from daily egg records. Egg fertility was estimated from the proportion of fresh eggs and from incubated eggs. Fresh eggs were collected daily from each pen (one egg per pen) over a two-week period three times during the season, in mid-November, December and January. Eggs were stored at 8°C and at the end of 2-week collection period they were sent to the UWA laboratory. Within a few days, the eggs were broken open to determine the morphology of the germinal disc. If the germinal disc did not contain a blastoderm, the egg was deemed unfertilised. Using incubated eggs, fertility was estimated from a proportion of all eggs set containing a detectable embryo, combining eggs that hatched, pipped but died in shell, and those that did not pip. The eggs were sampled from the incubator trays (n = 18) three times during the breeding season in early November, December and January. The proportion of infertile eggs was determined in eggs that did not pip; 4% (310 eggs in total) of such eggs from the incubator trays were frozen and then sent to UWA laboratory for analysis. Before incubation, eggs were stored for up to 8 days.

Season 2006-07 In the second year, fresh egg fertility was estimated from eggs collected over two days in order to account for all producing females. Eggs were collected from 8 pens (4 pens with 1 yo hens and 4 with 2 yo hens) four times, in the middle of November, December, January and February. After each collection period, eggs were sent to UWA laboratory and processed as above.

Photo 8. Male pheasant with two hens at the Photo 9. Duck breeding pen at UWA facility commercial facility. with a nest box (right), feeder (left of the box). and a bath tub (left).

The effect of female age and month on fertility was estimated.

3.2.3 Duck Effect of genotype on sperm supply and penetration rates This study was carried out at UWA using birds provided by the commercial producer “Luv-a-duck” (Victoria) from their Low and High body weight lines. In the High Body Weight (HBW) line, males weighed 5.1 ± 0.5 kg (Mean ± SD) while females weighed 5.0 ± 0.3 kg. In the Low Body Weight (LBW) line, males weighed 3.3 ± 0.6 kg and females 3.2 ± 0.2 kg. The experiment lasted 24 weeks during which every male was mated with every female in the following male x female combinations: LBW x LBW, LBW x HBW, HBW x LBW, HBW x HBW (3 pairings per combination). The pairs were kept in the individual pens (Photo 9) and the males were rotated every 7 days for 12 weeks and then every 14 days for another 12 weeks. Eggs were collected daily, stored for 3 days at 8°C then broken open and processed. Effect of flock age on sperm supply and penetration rates In commercial duck flocks, high rates of hatching are maintained until 36 weeks of age, and then, over the course of 16 weeks, they decline by about 10%. The proportion of clear eggs (infertile) rises between 36 and 44 weeks of age. The underlying cause of reduced hatchability in the second half of the egg cycle is not understood, but one possibility is reduced fertility as the flock ages (Brillard 2003). This study was undertaken to test whether such a trend is reflected in numbers of sperm supplied during mating to fertilise the eggs. The effect of age was studied in the commercial duck flocks of the same genotype. Eggs were sampled in flocks ranging from 20 to 65 weeks of age in August 2006. After collection the eggs were shipped to the UWA laboratory, stored on arrival at 4°C and processed within 10 days. For both duck studies, the eggs were broken open, fertilisation status was confirmed by examining the morphology of the germinal disc (GD) and the GD image was acquired using DP70 digital camera (Olympus Australia). The yolk membrane was then collected with the filter paper ring and the yolk residue washed off with PBS. The membrane was stained with Schiff’s reagent (Bramwell et al. 1995) and allowed to dry overnight on the lab bench at 18-20°C (room temperature) and 40-50% relative humidity. The dry filter ring was then removed leaving a stained membrane on the glass. Starting in the centre of the GD, sperm holes were counted under normal light microscopy in five successive fields of view using 20X objective.

3.3. Development of semen collection and artificial insemination for the ostrich The manual massage method for collecting semen from ostriches has always been difficult because it stressed both the birds and the handlers during restraint and during the manual extrusion of the phallus (von Rautenfeld, 1977; Irons et al., 1996; Hemberger et al., 2001). We therefore aimed to develop stress-free methods for semen collection that used an artificial cloaca (AC). The study was carried out at the Stypulow Ostrich Farm in Poland where more than 50 breeding adults and up to 200 juveniles were made available for the project. In the first stage, the behaviour of adult male and female ostriches was studied for expression of favourable behaviours that would facilitate training to two methods, the teaser female or the dummy female.

3.3.1 Semen collection Males were selected for the dummy method if they expressed the following behaviours towards their regular handlers: lack of fear, lack of aggression, an approach to the fence associated with wing flapping and pecking, lifting wings and pushing the enclosure fence, or squatting and kantling (Photo 10). Males were selected for the teaser method if they expressed the following behaviours: docile temperament with little aggression and willingness to mate with a crouching female in the presence of a regular handler (Photo 11).

Photo 10. Selection of young male ostriches Photo 11. Selection of pairs for the teaser for the dummy method: 3 males method. A pair of ostriches is showing interest in a human by being accustomed to a handler kantling, pushing the fence or being near them during copulation. staying close and flapping wings.

3.3.2 Artificial insemination We focused on the development of methods in which the female does not need to be physically restrained. The first method involved the female standing with or without a blindfold (Photo 12), and the second was with the female crouching in a mating position when approached by a human (Photo 13). We chose females that showed no fear of humans and would stand quietly for the procedure or those that would crouch for a human when approached.

Photo 12. Standing quietly female is Photo 13. Crouching female ostriches being examined for ease of the artificial selected for the artificial insemination. insemination.

3.4. Identification of factors affecting quality and quantity of the emu and ostrich ejaculates

In the emu, the effect of semen collection temperature on sperm quality was investigated. Emus breed in winter and semen collected into an artificial cloaca (AC) can be exposed to low ambient temperatures, perhaps leading to reduced quality and subsequent poor sperm survival in vitro. We used male emus maintained in the UWA Shenton Park Field Station. Ejaculates were collected by AC from 4 males trained to either the teaser or the non-teaser method (Malecki et al. 1997a). The AC was fitted with thermally insulated vials maintained at 5°C, 10°C or 20°C. Semen was diluted in UWA-E diluent (1:2) and stored for 0, 6, 24 and 48 h at 5°C, 10°C or 20°C. Sperm viability was determined by

estimating the numbers of live/dead and normal/abnormal sperm under a light microscope at 40X magnification with a digital camera mounted on a microscope. In the ostrich, we addressed the effect of male age on ejaculate quality in collaborative projects in Poland and South Africa. In Poland, we collected semen into an artificial cloaca (AC) from the dummy-trained males (Rybnik et al. 2007) that were 2-4 years old and determined ejaculate volume, sperm concentration, total number of spermatozoa and motility. Sperm concentration was estimated with a haemocytometer (Malecki et al. 1997b) and motility by subjective scoring of mass motility of spermatozoa (scale 0 – 5) under 10X objective (Allen and Champion 1955). In South Africa, the effect of male age on ejaculate quality was studied indirectly by estimating the sperm supply to eggs. This work was done in collaboration with the Elsenburg Institute of Agriculture in South Africa and carried out at the Oudtshoorn Agricultural Research Centre. Ostrich pairs aged 3-9 years were used. Eggs were collected and broken open so the fertilisation status of the germinal disc determined. A 2 cm diameter circle of the vitelline membrane directly overlying the GD was collected on a filter paper, cleaned of yolk with PBS, and stained with 1 µg/mL DAPI in PBS (Wishart 1987). Sperm nuclei were visualised with fluorescence microscopy (Olympus BX60-FL, Olympus Optical CO., LTD) using a “U” filter cube with 372 nm excitation and 456 nm emission wavelengths (Photo 15). Sperm were counted in five successive fields of view of 20X objective starting in the centre of the GD.

3.5. Determination of the duration of in vivo sperm storage and rate of lay in the absence of copulation in the ostrich

Data on the duration of sperm storage in the female is essential for the development of the artificial insemination technology, yet we still have limited knowledge of the duration or the between- and within-female variation. To improve our existing data (Malecki et al. 2004), this study was carried out in collaboration with the Elsenburg Institute of Agriculture in South Africa over two consecutive seasons (2005-06 and 2007-08) at the Oudtshoorn Agricultural Research Centre. We used 21 pairs (15 in 2005-06 and six in 2007-08; different pairs in the two years) that were maintained individually in 50 x 50 m paddocks. The males were captured and, after fitting the ‘apron’ (bottomless feed bag stitched to a harness – Photo 14) to prevent copulation, they were released back to their females. Eggs were collected and processed as above (1.4).

Photo 14. Male ostrich wearing apron Photo 15. Ostrich SpermOPVL counted in the returning to his female. germinal disc area for estimation of the sperm loss or sperm supply rates.

Estimating sperm storage duration, fertile period and rate of lay

The duration of sperm on the vitelline membrane was defined as the number of days from last copulation to the day of the last egg containing SpermOPVL. Consequently, the fertile period was defined as the number of days over which sequentially laid eggs were fertilised after the last copulation. Last copulation is regarded as the time of the last sperm deposition in the female and is used to define the start of the fertile period. In our study, the time of the last copulation was determined indirectly from the changes in numbers of sperm on a series of eggs (Malecki & Martin 2002). The last increase in sperm numbers was interpreted as evidence of the last copulation before the apron was fitted. A correction for the duration of the 2-day egg cycle was made since the earliest that a sperm increase could be detected in the perivitelline membrane was 2 days after copulation. The rate of lay was estimated for eggs laid for up to 10 days before fitting the apron and to the last egg of the laying sequence after fitting the apron. The laying sequence was deemed terminated when no egg was laid for 6 consecutive days (3 egg cycles). Rate of lay in female ostriches reared without males We tested whether female ostriches that had been reared in isolation from males would lay eggs when they reached maturity and were maintained without males. This preliminary study was carried out on the ostrich farm in Poland. We reared female ostriches in a female-only flock from 12 months of age and in the next year (year of sexual maturity, age 18-24 months), we chose 15 females that tended to remain in close proximity to humans. We moved them to a separate paddock and studied their response to human approach. The paddock was entered every 4 hours, 3 times a day, for 3 days and female response to human approach was recorded (1 – crouch, 0 – no crouch). The scoring was repeated a month later, in the peak of the breeding season for 6 days, 3 times per day (n = 18 observations). When the next breeding season started, egg laying was recorded and the rate of lay was estimated.

Statistical analyses Where indicated, data were either treated with analysis of variance (ANOVA) or regression. Where necessary, data were subjected to log or square root transformation before analysis. In general, descriptive statistics are given and the results are mainly presented as Mean ± SEM unless otherwise specified. Where comparison of means was needed, Fisher’s LSD was used unless otherwise stated and differences were considered significant if P < 0.05. For statistical software, we used SuperAnova (Abacus Concepts, Berkeley CA, USA), SPSS or SAS.

4. Development of fertility and hatchability technology for the duck, pheasant and pigeon

4.1 Visualisation of SpermOPVL, HolesIPVL and germinal disc morphology

Identification of sperm holes in the duck egg membrane (Photo 16) was successfully developed using the emu HolesIPVL protocol (RIRDC 2005). Sperm trapped in the yolk membrane (Photo 17) were visualised by the same technique used for poultry and ratites species (1.4 and 1.5 – this report).

Photo 16. Sperm holes in the yolk Photo 17. Trapped sperm in the yolk membrane following staining membrane visualised with DAPI. with Schiff’s reagent in bright field view.

The gross appearance of fertilised and unfertilised germinal discs of the duck, pheasant and pigeon resembled those of emu and ostrich, as we have reported (RIRDC 2005) and as described in the literature for poultry (Kosin 1944; Bakst et al. 1998). An unfertilised GD consisted of a group of white yolk droplets surrounded by a yellow ring, within which numerous vacuoles were present (Plate 1, Photo 1A-3A). A fertilised GD contained blastoderm that, depending on interval between oviposition and blastoderm examination, had either two distinct areas, a clear concentric one in the centre (area pellucida), surrounded by an opaque ring (area opaca), or had the opaque centre surrounded by a concentric and opaque band (Plate 1, Photos 1B-3B). We found very little visible difference between the fertilised discs of ducks, pheasants and pigeons, or between unfertilised discs of those species.

Plate 1. Appearance of the fertilised and unfertilised germinal disc (GD) in the duck, pheasant and pigeon eggs.

Unfertilised GD Fertilised GD

1A 1B

Duck

2A 2B

Pheasant

3A 3B

Pigeon

4.2. Pigeon early embryo development

4.2.1. Early morphological changes Fresh eggs opened on day of lay had either a concentric blastoderm or a blastoderm assuming a pear shape if the egg was found warm under a bird. Thus a proportion of eggs were collected warm and when opened in the laboratory they showed embryo progression (Plate 2, Photo 0 to 0+).

Plate 2. Early embryo morphology in the pigeon, before and after incubation for up to 3 days.

0 days 0+ days

2 days 3 days

On day 2 of incubation, the embryo would elongate to an avocado shape when entering the primitive streak formation stage (Plate 2-2). Some were more advanced with a clearly delineated head fold and spine but the heart was not yet well defined. On day 3, most embryos had a clearly visible heart and, on day 4, the eye started to be visible. Eye pigmentation became stronger on days 5 and 6, and beginning the limb development was noted from day 5 (Plate 3).

Plate 3. Early embryo morphology in the pigeon after artificial incubation for 4 to 6 days.

4 days 4 days

5 days 5 days

6 days 6 days

4.2.2 Growth changes The rate of embryo development for up to Day 8 of incubation was the same for all 3 colonies. Over 5 days, from Day 3 to Day 8, the embryo increased in size by 35-fold (0.015 ± 0.010 g to 0.525 ± 0.011g) in an exponential manner. The origin of the embryo (colony) had no effect on embryo growth and there was no interaction between colony and day of incubation.

4.2.3 Embryo age When embryo weight was considered as an independent variable and plotted against incubation time as the dependent variable, the relationship appeared logarithmic (Fig. 1). The equation describing this relationship was then used to determine embryo age for wet weights up to 0.6 g. Due to unavailability of embryo weight data for incubation between 9 to 18 days, it was not possible to develop an equation for estimating age of embryo death in the middle to late term of incubation. The likely relationship could be one described by the equation in Figure 2. This graph was plotted for

demonstration only using embryo weight data for up to 8 days of incubation and then extrapolating to the weight of 1-day-old chicks.

Figure 1. The relationship between duration Figure 2. The relationship between duration of incubation (days) and embryo of incubation (days) and embryo wet weight (y = 1.05 ln(x) + 8.11, r2 wet weight (y = 2.014 ln(x) + 10.56, = 0.86). r2 = 0.951).

5. The quality of game bird breeding stocks

5.1 Pigeon

5.1.1 Squab processing

Annual pattern Between July 2006 and June 2007, squab processing in Queensland and Victoria increased in spring (September – November) through to summer, peaking in December, and declined to reach the lowest levels in late autumn and the beginning of winter (P < 0.001). This seasonal pattern appeared to be more pronounced in Victoria than in Queensland (Fig. 3).

Figure 3. Squab numbers (mean ± sem) per processing day of the month in a 12-month production cycle in Queensland (left) and in Victoria (right). Carcass grade Squabs were graded according to their carcass weight (Table 3). The carcass numbers showed a similar distribution amongst grades and that pattern was similar in every month (data not shown), irrespective of the region (Fig. 4).

Table 3. Squab carcass grades.

Weight (gram) Grade

200-249 2.0 250-299 2.5 300-349 3.0 350-399 3.5

400-449 4.0 450-499 4.5 500-549 5.0 Figure 4. Mean (± SEM) squabs per grade after daily 550g and more 5.5 processing in plants in Queensland (open bars) and in Victoria (closed bars).

Figure 5. Mean numbers of squabs in each carcass grade processed daily from July to June (12-month production) in Queensland (left) and in Victoria (right). Standard errors are omitted for clarity.

Most squabs (68%) produced were in grades C3.5 to C4.5 (Fig. 4) so 32% were outside that range. The distribution tended to be affected by variation in squab size within and between producers. The most variable sizes were C5.0, 4.5, 3.0 and 2.5 (Fig. 5). In Queensland, numbers of C5.0 and 4.5 were markedly reduced between November and April. Numbers of C3.5 and 3.0 squabs changed in the opposite manner to those for C5.0 and 4.5. In Victoria, C3.5 and 3.0 numbers were distributed in a similar fashion to those in Queensland, whereas numbers of C5.0 and 4.5 squabs were fairly stable throughout the year. Numbers of C2.5 also varied between months, again without a clear pattern.

5.1.2 Pigeon reproductive performance

Pigeons at UWA Production performance during acclimatization During the 3-month adaptation to the Field Station environment, the NSW colony had more productive pairs, produced more squabs and was more efficient than the two other colonies. Egg production per breeding pair was similar (Table 4).

Table 4. Egg and squab production by colonies from NSW, QLD and VIC between February and June 2006 (120 days). Number of chicks Number Number of Number of eggs laid Group weaned Colony of pairs in pairs efficiency^ Per breeding Per breeding stock breeding Total Total (%) pair pair NSW 11 11 67 6.1 53 4.8 79 QLD 10 8 48 6.0 27 3.8 56 VIC 10 7 44 6.3 22 3.1 50 ^Efficiency = number of chicks weaned/number of eggs laid x 100.

Egg production in the absence of incubation In the absence of a normal production cycle that includes egg incubation and chick rearing, egg laying continued. Egg production was similar between NSW and QLD colonies but it was low in VIC colony (Table 5). Table 5. Egg production from NSW, QLD and VIC colonies between July and September 2006 in the absence of incubation (50 days). Colony Eggs/colony Eggs/pair Eggs/day Eggs/day/female Predicted eggs/female/year NSW 92 8.4 1.8 0.17 61 QLD 78 7.8 1.6 0.16 58 VIC 55 5.5 1.1 0.11 40

Production performance The performance of pigeons during a 7-month period varied within and between colonies. Body weight did not differ by Day 14 after hatch but, by Day 28 and by weaning, squabs produced by NSW pairs weighed more than those produced by pairs from Victoria and Queensland (Table 6). In every colony, differences between the lowest and the highest body weight were nearly double for every weighing interval. Queensland pairs averaged more eggs per pair than two other colonies. The number of squabs was the same for Queensland and NSW pairs while VIC pairs produced the least. The average cycle duration was similar between NSW and VIC colonies but QLD pairs had shorter cycle.

Table 6. Mean, Min and Max values for reproductive traits of pigeon pairs held at the Shenton Park Field Station (UWA). Colony Reproductive trait VIC NSW QLD Mean Min-Max Mean Min-Max Mean Min-Max Egg Weight* 23 17-29 24 21-31 23 16-29 Hatching BW** 22 13-35 24 15-35 25 16-46 7 D BW*** 150 122-212 161 91-233 182 115-292 14 D BW 422 346-520 446 310-609 474 326-622 28 D BW 644 543-745 734 382-860 679 552-820 Wean BW 665 600-756 745 586-905 666 534-797 Number of eggs/pair 6 1-8 6 1-10 9 1-15 Number of squabs/pair 3 0-6 5 1-8 5 0-8 Number of clutches 3 1-4 4 2-6 5 4-8 Number of eggs/clutch 2 1-2 2 1-2 1 0-2 Cycle duration (days) 48 21-76 49 33-127 32 23-56 *All weight traits are in grams; **Hatching Body Weight; ***7-Day Body Weight. Pigeons on farms On average, squabs grew at a similar rate on all farms, increasing their body weight about 10-fold in the first week then 2-fold in the second week. By Day 14, squabs would weigh about 70% of their 28- day body weight. Colony VIC 1 averaged fewer eggs per pair than any other colony but the proportion of squabs weaned from those eggs was greater. Cycle duration tended to be longer in VIC 1 than in other colonies. Very short cycles were probably caused by loss of eggs or chicks during the cycle. The most efficient colony was VIC 1 but, for squab yield, VIC 1 was similar to QLD 3 (Table 7).

Table 7. Mean, minimum and maximum values for reproductive traits of pigeons in colonies (VIC 1, QLD 1 and 2) and individual pairs (VIC 2 and QLD 3), their pair efficiency and squab yield.

Breeding flock Pairs in colonies Individual pairs VIC 1 QLD 1 QLD 2 VIC 2 QLD 3 Trait Mean Min-Max Mean Min-Max Mean Min-Max Mean Min-Max Mean Min-Max Egg weight* 24 18-39 24 8-40 25 17-31 24 20-33 25 18-35 Hatching BW** 20 15-25 23 14-32 23 15-31 20 15-23 26 16-39 7D BW*** 180 100-280 215 14-298 223 73-295 168 110-212 221 64-293 14D BW 452 220-615 491 334-670 489 215-700 437 304-560 517 237-696 28D BW 704 300-905 676 512-888 685 378-915 704 545-810 688 436-865 Wean BW 712 540-910 676 522-888 685 400-915 697 570-820 701 518-890 Eggs/pair 16 12-20 21 6-28 20 13-25 19 16-22 24 16-30 Weaned 13 9-20 11 3-17 11 2-20 12 11-14 14 9-18 squabs/pair Eggs/clutch 2 1-2 2 1-3 2 1-4 1 1-2 1 1-2 Cycle duration (d) 42 13-93 34 5-119 34 5-80 37 5-66 35 6-103 Clutches/pair 8 6-10 11 4-14 11 7-19 10 9-12 12 8-15

Pair efficiency 84 52 58 64 57 (%) Squab yield 9.3 7.2 7.7 8.6 9.6 (kg/pair) *All weight traits are in grams; **Hatching Body Weight; ***7-Day Body Weight.

Pigeon traits The number of clutches, cycle duration and the number of chicks per clutch were the most variable traits (Table 8). Only about 50% of eggs produced chicks. The growth rate of squabs appeared to be rapid: body weight increased 10-fold from hatch to Day 7 but then only doubled in the next 7 days reaching 70% of the weaning weight by Day 14. The number of hatched chicks was considerably less than the number of egg per clutch but most hatched chicks survived till weaning. While some pairs showed very poor performance, raising as few as 4 chicks, the other pairs produced up to 19 chicks (Table 8).

Table 8. Mean, Min, Max and CV(%) values for production traits of farmed pigeons (n = 76 pairs, colony and individual pairs combined). Reproductive trait Mean Min Max CV% Number of clutches 10 6 14 57 Cycle duration (days) 36 4 116 36 No of eggs/clutch 2 1 4 13 No of chicks/clutch 1 0 4 65 Egg weight (g) 25 8 40 11 Body Weight At hatch (g) 23 14 96 29 7 day (g) 212 14 464 27 14 day (g) 480 215 964 17 28 day (g) 704 300 915 11 At weaning (g) 701 400 915 11 Number of eggs/pair 19 12 28 20 Number of chicks hatched/pair 12 5 21 24 Number of 7-day squabs/pair 12 4 21 26 Number of 14-day squabs/pair 12 4 21 26 Number of 28-day squabs/pair 11 4 19 26 Number of weaned squabs/pair 11 4 19 27

5.1.3 Pigeon nutrition

Crude protein and lysine levels for breeding pigeons Breeding pairs receiving a pelleted diet containing 26% protein produced squabs that were 50 g heavier at 28 Day than pairs receiving the diet containing 20% protein (Table 9). There was no effect of CP levels on egg weight and 14-day squab BW. Levels in between might have had similar effects to the 26% diet but they have not been tested. Table 9. Effect of crude protein (CP) levels [%] in feed on squab performance. CP level (%) Egg weight (g) 14 day Squab BW (g) 28-day Squab BW (g) 20% 25 495 666 26% 25 501 720

The minimum lysine level required to yield the heaviest squab was 0.72% (Table 10). The protein content of the pigeon diet should be formulated to contain 0.72% lysine (7.2g/kg feed).

Table 10. Effect of lysine levels (%) on squab body weight. Lysine level (%) Egg weight (g) 14 day Squab BW (g) 28-day Squab BW (g) 0.59 25 432 587 0.72 25 476 664 0.85 24 480 621

Feed Energy Values for Breeding Pigeons Diets were fed for four days and then excreta samples were collected over the next four days. Excreta samples were dried and chemically analysed. The gross energy consumed was between 10.44 and 11.61 MJ/Kg (Table 11). When corrected for energy excreted as nitrogen, the energy (AME) of the diet that was retained was just below 10 MJ/Kg (Table 12). Table 11. Feed and gross energy (GE) consumption of adult pigeons on different diets. Diets State of Pen Feed Feed Feed Gross Energy origin No weighed returned consumed (MJ/Kg) Diet 1 Vic 85 13000 12395.5 604.5 11.61 Diet 1 Qld 86 13000 12755.8 244.2 11.22 Diet 2 Vic 83 13000 12506.5 493.5 10.79 Diet 2 Qld 81 13000 12271.1 728.9 11.57 Diet 3 Qld 84 13000 12647.7 352.3 10.55 Diet 3 Vic 88 13000 12638.5 361.5 10.44

Table 12. Nitrogen corrected energy values of the feed consumed by adult pigeons. Diet No DM basis Nitrogen Nitrogen Nitrogen Energy excreted AME N (g) % diet intake (g) excreted (g) as N (MJ) corrected Diet 1 347.91 6.66 40.29 23.17 0.59 9.36 Diet 1 250.47 7.14 17.42 17.88 -0.02 *5.61 Diet 2 337.78 5.81 42.35 19.62 0.78 9.99 Diet 2 317.02 6.80 33.59 21.56 0.41 9.18 Diet 3 214.53 5.69 20.03 12.21 0.27 9.25 Diet 3 261.79 5.87 21.25 15.37 0.20 8.31 * The low feed intake for this group could not be explained

5.2 Pheasant

5.2.1 Effect of age and month on egg production performance and fertility Egg production performance varied with age and month. Laying commenced in October (mid-spring) and reached a maximum daily rate in November (late spring), then declined steeply over summer and terminated in February (Fig. 6). The seasonal pattern of lay was similar for 1 yo and 2 yo female although the rate of lay depended on month and age. Overall, 1 yo females laid at a higher rate than 2 yo females (0.40 ± 0.02 vs 0.30 ± 0.02), particularly in November, December and in January. There was no significant difference between age groups in October or February.

Figure 6. The oviposition rate of the pheasant breeding flock as related to female age and month (open bar – 1 yo females; closed bar – 2 yo females).

Overall, the fertility of fresh pheasant eggs was 91.5 ± 1.1% (mean ± SEM) for the season. It was not affected by female age. Fertility estimated in fresh eggs was highest in November and declined by about 5% in December and a further 5% in January (Table 13). Fertility estimated from incubated was lower than fertility estimated from fresh eggs. Less than 50% of non-pipping eggs were infertile.

Table 13. Fertility (Mean ± SEM) of pheasant eggs estimated in incubated and in fresh eggs. Month of lay Fertility of non-pipping eggs (%) Fertility of set eggs1 (%) Fertility of fresh eggs2 (%) November 60.4 ± 4.9 83.2 95.8 ± 1.5a December 53.3 ± 6.7 83.0 91.1 ± 1.8b January 66.1 ± 4.7 86.3 87.5 ± 1.7b 1Note – Calculated from total number of fertile eggs/total number of set eggs x100. Total number of fertile eggs = number of hatched chicks + number of dead embryos in pipping eggs + number of dead embryos in non-pipping eggs. 2Means in a column with different superscript differ significantly (P < 0.05; t-test).

5.2.2 Effect of pen, time of season and collection day on egg fertility In the second year of study, we attempted to account for every female in a pen so eggs were collected over two consecutive days. Fertility of fresh eggs sampled in 8 selected pens was 90.7 ± 1.3%. Season and female age had no effect on fertility. Egg fertility varied between pens, ranging from 76.9% to 96.6% (both 1 yo). This confirmed the fertility values of the previous year. Egg fertility was similar on Days 1 and 2 of collection and there was no difference in the mean number of eggs collected between Day 1 (19.7 ± 2.0 eggs) and Day 2 (21.1 ± 2.0 eggs).

5.3 Duck

5.3.1 Effect of flock age on sperm penetration rates. Sperm penetration rates (numbers of sperm holes) were maximal when the flocks reached 36 weeks old. The numbers of sperm holes were then fell markedly by 44 weeks and remained low (Fig. 7).

Figure 7. Effect of flock age on the mean (±SEM) numbers of sperm holes in duck eggs.

5.3.2 Effect of body weight line on sperm penetration rates There were more sperm holes in eggs from LBW x LBW pairings than for any other combination. The numbers did not differ between LBW x HBW and HBW x LBW combinations (Fig. 8).

Figure 8. Effect of pairing combination on Figure 9. Effect of male type on sperm sperm penetration rates. penetration rates.

Males varied in their sperm penetration rates but there was no interaction between male and BW line. Two males from the LBW line ranked the highest and the lowest while the third male was in a median position(Fig. 9). Based on the number of holes, the ranking order of males, from the highest to the lowest sperm supplier was as follows: 1 - LBW; 2 – HBW; 3 – HBW; 4 – LBW; 5 – HBW; 6 – LBW. Mating duration had no effect on the numbers of sperm holes but there was an interaction between pairing combination and mating duration. The LBW x LBW pairing produced less sperm holes during the 7-day mating than during the 14-day mating, while LBW x HBW combination produced more sperm holes during 7-day than the 14-day mating. Combinations involving HBW males had similar numbers of sperm holes for the two mating durations.

6. Semen collection and artificial insemination for the ostrich

6.1 Semen collection methods

Two methods for semen collection have been developed: teaser female and dummy. In the teaser method, the male follows the female in a courtship display and, when the female submits (Photo 18), the male mounts the female. The phallus is redirected into an artificial cloaca (AC) into which the male ejaculates (Photo 19). The phallus is held in the AC until the male is about to dismount, at which time the collector can leave the enclosure (Photos 20, 21).

Photo 18. Male ostrich approaching a Photo 19. The phallus is redirected into the crouching teaser female. artificial cloaca.

Photo 20. The phallus is held in the artificial Photo 21. Following ejaculation, the male cloaca during ejaculation while the dismounts and the collector leaves collector squats behind the birds. the pen.

In the dummy method, males are trained by taking advantage of male courtship behaviour directed towards humans. Attempts by the male to mount a human are redirected onto a dummy placed between him and the collector (Photos 22, 23). The male then performs the kantling display and mounts the dummy (Photo 23). After locating the AC located inside the dummy, the male ejaculates while expressing the same behaviour observed either during mating or teaser method of collection.

Photo 22. Hemp sack dummy for collecting Photo 23. Male ostrich mounting the semen. The AC is shown in the dummy. right hand corner insert.

Of the 6 males selected for training, 4 were trained to mount a dummy and 3 were trained to mount a teaser female. All of the successfully trained males accepted the artificial cloaca and, after the training was completed, 4 consecutive ejaculates from all 7 males were used to estimate the volume, sperm concentration and motility (scale 0 to 5) in an ejaculate (Table 14). The volume, concentration and numbers of live sperm did not differ between methods of collection.

Table 14. General characteristics (Min-Max values) for ostrich ejaculates. Collection Volume Concentratio Sperm per Sperm Live sperm method (mL) n (x109/mL) ejaculate (x109) motility (%) Teaser 0.5-2.9 1.6-4.8 82-98 Dummy 0.2-2.0 1.5-6.7 78-96 Mean ± SEM 1.09 ± 0.13 4.21 ± 0.27 4.67 ± 0.62 4.3 ± 0.1

6.2 Artificial insemination (AI) of female ostriches We developed two insemination methods that do not require physical restraint. The first method is an approach where the female is gently held against the body of an assisting person, the shed wall or the yard fence. After blindfolding the bird, she stands for the procedure secured by one person while the second person performs the artificial insemination procedure (Photo 24). Females varied considerably in their willingness to accept this approach but, in general, they allowed AI and remained still, although they needed time to become accustomed to the procedure. The second method uses females that crouch on their own accord. It was not necessary to restrain the birds physically and the stimulus of applying pressure on the female’s back was used as part of the strategy to gain access to the cloaca. In this method, the female is followed until she crouches to a mating position. Then the inseminator crouches behind the female and places hands on her back to provide additional stimulation. The female ostrich usually responds by either bending her neck or keeping it upright, or by putting her neck on the ground and clapping her beak, raising her tail and exposing the vent (Photo 25).

Photo 24. Artificial insemination of female Photo 25. Artificial insemination of a female ostriches that stand still after ostrich that is crouching for the blindfolding. inseminator.

Insemination procedure A hand is introduced into the proctodeum, passing above the female phallus (phallus phemininus), and then the fingers are directed to the left through the urodeum and left to the oviduct opening. The hand and fingers then guide the insemination straw into the vagina and semen is deposited at the desired depth. In both the standing and crouching approaches, a second person may be required to apply pressure on the female’s back because this keeps seems to keep her in a steady position. Both approaches appear to be stress-free for the female and her calm behaviour allows for a gentle procedure.

7. Factors affecting the quantity and quality of emu and ostrich ejaculates

7.1 Emu – effect of collection temperature on ejaculate quality

Ambient temperatures are low during the emu breeding season in Australia so we thought it feasible that cooling during collection would affect ejaculate quality. We found no difference in numbers of normal sperm among collection vessel temperatures (Fig. 10). The numbers of live sperm were marginally lower after collection at 10°C than at 5° or 20 °C. The numbers of bent sperm (abnormal type of sperm) were higher after collection at 5 and 20°C than at 10°C. For all measures, there was no interaction between collection temperature and the subsequent storage temperature of emu semen for up to 24 h. There was also no interaction between collection temperature and storage time. Thus, emu ejaculate quality should not be compromised when semen collection is carried out at low winter temperatures and this should not adversely affect their sperm quality when semen needs to be stored in vitro for up to 24 h. This is something that need not concern the industry and also allows the use of simple procedures.

Figure 10. Effect of temperature of the collection vessel on numbers of normal, bent and live emu spermatozoa after 24 h storage.

7.2 Ostrich – effect of male age on sperm quality

7.2.1 Effect of male age on ejaculate parameters In the absence of reliable methods for semen collection, the factors that affect ostrich semen quality were not known. Our success with techniques for collection of ejaculates allowed us to test the effects of the age of the male donor. Ostriches are thought to fully mature by 4 years of age even though they begin reproductive activity when 2 years old. In Poland, we studied ejaculates collected from the same four males (all trained to the dummy method) over 3 consecutive years, from when they were 2 years old until they were 4 years old. Semen volume, the concentration and total number of spermatozoa, and motility varied with age (Table 15). Ejaculate volume and total number of spermatozoa were lower in 2- than in 3- and 4-year-old males. The concentration of spermatozoa was lower in 3- than in 2- and in 4-year-old males. Motility was higher in 3- and 4-year-old than in 2-year-old males.

Table 15. Effect of male age on ejaculate characteristics in ostriches (Mean ± SEM).

Male age Semen Concentration of Number of Motility (years) volume (ml) spermatozoa (x109/ml) spermatozoa (x109) 2 0.71 ± 0.12c* 4.10 ± 0.34a 3.19 ± 0.59c 3.91 ± 0.17b 3 1.27 ± 0.07b 3.45 ± 0.11b 4.59 ± 0.35b 4.70 ± 0.05a 4 1.74 ± 0.13a 4.10 ± 0.15a 6.99 ± 0.53a 4.80 ± 0.06a a,b,c * – means in columns having different superscript differ significantly (P<0.05, ANOVA). Semen quality and quantity is the lowest in 2 yo males and is likely to improve by the time the males is 3 or 4 years of age. This also indicates that serving capacity of 2-year-old males in an AI program may be lower than that of older males.

7.2.2 Effect of male age on sperm supply to eggs In the absence of any information on ejaculate characteristics of males older than 4 years, we studied the effect of age indirectly by estimating the numbers of sperm trapped in the perivitelline membrane. The sperm numbers supplied to females during mating was similar between 3 and 7 yo males but the negative slope of this relationship indicates sperm numbers would decline once males become older than 7 years.

Figure 11. The relationship between age of male ostriches and numbers of sperm found in the perivitelline layer of fertilised eggs laid by their companion females (y= -1.31x + 16.75, r2 = 0.21, n = 11 pairs).

8. Duration of sperm storage and rate of lay in the female ostrich in the absence of copulations

8.1 Duration of sperm storage Not all pairs adapted to the ‘aprons’: after fitting, 50% of the females either produced infertile eggs or stopped laying within 2-4 days. Eleven pairs continued laying so their eggs were used for estimating the duration of sperm storage. The mean duration was 14.4 days (range 6-22). There was considerable between-pair variation in the numbers of sperm found in eggs, as evidenced by the variance in Figure 12. Sperm numbers declined at about 23% per day. The numbers of sperm on the perivitelline membrane could be predicted from the equation LogSpermOPVL = – 0.112 xday + 2.191. On this basis, sperm numbers would be less than 1 sperm per mm2 by Day 20 after last mating, reducing the probability of the egg being fertilised by that time to zero.

Figure 12. Decline in numbers of OPVLsperm over successive days of the fertile period in the female ostrich. Values are Mean ± SEM.

8.2 The rate of lay

From the time the ‘apron’ was fitted, the mean rate of lay remained unchanged for up to 6 days but then declined between Days 8 and 10 (Fig. 13). On Days 12 and 14, the between-oviposition interval was higher than on Days 8 and 10, extending it beyond 2 days. Almost all females were laying for up to 8 days. The number of laying females declined below 50% by Day 10 (Fig. 14) and by Day 18 less than 25% continued laying. Two females continued laying for up to 32 days and one for up to 36 days (data not shown). On average, laying was terminated in 12.1 days (range 2-36 days) after apron fitting.

Figure 13. Changes in oviposition interval in Figure 14. Decline in numbers of female relation to time of fitting the ostriches laying eggs after fitting ‘apron’ (0 days). the ‘apron’ onto their male companion.

8.3 Additional findings

Rate of lay in female ostriches reared without males At 20 months of age, out of 15 randomly chosen females that were scored for voluntary crouch (1 – crouch, 0 – no crouch), nine females that did not crouch were excluded. The remaining 6 females scored 6.3 ± 3.7 from 9 observations. When scoring for those 6 females was repeated a month later, during the peak of the laying season (3 times per day over 6 days, a total of 18 observations), the mean score was 17.3 ± 1.1. In the next breeding season, those females laid eggs at the same rate as females that were maintained with males (0.20±0.2 vs. 0.19±0.01 eggs/female/day respectively).

9. Discussion of Results

Fertility and hatchability technologies for the duck, pheasant and pigeon

The general methodology for visualising SpermOPVL in duck eggs was much the same as that used for other birds (Wishart 1987), but the wet membrane preparation technique that works so well for quail and chicken (Bramwell et al. 1995; Wishart et al. 2004), was not satisfactory. The holes made by duck sperm only became visible when we used dried vitelline membrane, the technique we developed for the emu (Malecki & Martin 2005). It appears that the duck vitelline membrane is too thick to see the sperm holes in wet preparations. This is the first time anyone in the world has produced a technique for visualising sperm holes in the duck vitelline membrane. We have also shown that our approach works for the goose.

Clearly, egg size determines which technique should be used and it can be generalised that, for eggs that are similar in size to the chicken egg or smaller, wet preparations of the vitelline membrane will probably work. However, for larger eggs (eg, duck, goose, emu), the membrane needs to be dried after staining with Schiff’s reagent.

The fertilised and unfertilised germinal discs of duck, pheasant and pigeon eggs resemble those of the chicken, turkey, emu and ostrich (Kosin 1944; Bakst et al. 1998, Malecki & Martin 2002, 2003; Malecki et al. 2005). Unfertilised and fertilised germinal discs can be satisfactorily differentiated based on the presence or absence of the blastoderm, although there are minor variations in blastoderm morphology between individuals and with storage conditions. Future studies need to determine changes in blastoderm morphology in relation to storage temperature and delay from oviposition to egg examination. Between-individual variation in these measures could be partly responsible for variable embryo development.

In this project, the pigeon embryo has been staged for the first 6 days of development. These gross morphological changes, combined with changes in embryo weight, should enable determination of the time of embryo death with reasonable accuracy. Additional work is needed to complete the table of development because pigeon eggs are incubated naturally and the time of commencement of incubation, as well as the incubation persistency of the parents, is often difficult to determine without constant observations. It can be expected that embryo growth rate will vary with pigeon type, cross or strain but, until these variables are clearly defined, the rate of growth needs to be generalised over the meat-type pigeon population.

Quality of game bird breeding stocks

Duck By investigating the levels of sperm supply and penetration rates in flocks from 20 to 60 weeks of age, we found the numbers of sperm holes reached maximum values by 36 weeks, declined to 44 weeks, and then remained low for the rest of the egg production period. This is in parallel with the rates of hatching – they are maintained at high levels until about 36 weeks of age and then, over the course of the next 16 weeks, they decline by about 10%. According to commercial data, the proportions of clear eggs (infertile) increase between 36 and 44 weeks of age. There is clearly a reduction in numbers of sperm penetrating the ovum after age 36 weeks. The increase in numbers of clear eggs around that time suggests the females receive insufficient numbers of sperm for fertilisation.

Male ducks commence sperm production at about 20 weeks of age, but 20 weeks later, females do not appear to be sufficiently supplied with sperm even though the reproductive life of the males can continue for up to 60 weeks. There are at least two likely causes of this decline – one related to the male and one to the female. The males could be mating less frequently, depositing variable ejaculates, or be more selective with the females; the females could be more selective with the males or be

retaining fewer sperm. In broiler breeder males, reduced reproductive performance is associated with reduced mating frequency and attainment of large body weight (Brillard 2004). However, no relevant studies have been carried out in ducks although it is known that body weight of breeder ducks needs to be controlled or their egg production and hatching performance are reduced (Scott & Dean 1991).

Reduced hatching performance could be linked to reduction in rates of sperm supply but whether this is caused by diminished rate of sperm production or reduced libido remains to be determined. Female ducks, on the other hand, may lose their ability to retain sperm in the sperm storage tubules as they age. In broilers, the rate of sperm efflux from the tubules is greater in older than in younger females (Brillard, 1993; Gumulka & Kapkowska 2005).

The effect of body weight on sperm penetration rates is clear. Males and females from the High Body Weight line contributed to reductions in sperm hole numbers when mated to males and females from the Low Body Weight line. Low numbers of sperm holes in the High Body Weight x Low Body Weight combinations suggest low sperm supply by males from the High Body Weight line, but a reduction in sperm hole numbers in the Low Body Weight x High Body Weight combination suggests the oviduct of High Body Weight females may have a low efficiency of sperm transfer. In both lines, however, high and low fertility males could be identified, suggesting that there is considerable within- line variation that should be studied further as it may provide a solution to reduced fertility in the High Body Weight lines.

Pheasant Commercial pheasant production in Australia has a short breeding season and is confounded by a highly seasonal pattern that affects rate of lay and fertility and that impacts adversely on the economics of production. Even though fertility is not affected by female age, breeding 2-year-old females appears to be disadvantageous due to a reduced rate of lay.

Several approaches could be attempted to improve the economics of production. Since the pheasant is a photoperiodic long-day breeder (Deeming & Wadland 2002), the season could be extended by manipulation of daylength. This requires light-controlled housing but more eggs might be produced. Given that egg fertility rates are satisfactory, a reduction in male numbers should be attempted to find the optimum mate to female ratio – probably around 1 male to 8 females (Deeming & Wadland 2002) as opposed to the current practice of 1 male to 5-6 females. Hatchability rates, on the other hand, seem to be low and further research under controlled conditions is needed. There is also considerable between-female variation in egg production performance, but the flock-mating system does not allow for accurate measurement of female performance, and therefore selection for higher egg production. A breeding program needs to be introduced to begin improvement – such a program would benefit from artificial insemination technology and there might be a need to introduce new genetics to the Australian population, but a study into the existing genetic structure of our commercial pheasant population is needed.

Pigeon There was wide variation in squab size and squab numbers processed by the abattoirs. The dominant squab size was 350-399 gram and about 30% were outside the market-desirable range. There was also considerable seasonal variation in carcass size in both Victoria and Queensland. The least variable in Victoria was 450-499 gram size but their numbers were low when compared to 400-449 gram size. In Queensland, on the other hand, 400-449 gram seemed the least variable with 450-550 gram as the most variable and also in low numbers between November and April. It appears that a carcass bigger than 450 grams is difficult to grow, whereas 400-450 gram is feasible under current conditions. However, numbers in this range are less than 30%.

Fewer squabs were processed in autumn and winter than in spring and summer, with Victoria being more affected than Queensland. Perhaps the cooler temperatures during the winter months in Victoria were responsible for reduced production. On the other hand, production in both states picked up at end of spring and summer, before declining thereafter, suggesting that late summer conditions affect production, although moulting may contribute to reduced performance at that time.

While the cold weather may cause a high-energy demand for parents and squabs, hot and humid summer conditions can lead to heat stress and high water demand. Pigeons are able to breed in high temperatures because of their ability to dissipate heat through cutaneous water evaporation (Arieli et al. 2002), although pigeons that are not heat-acclimated are less tolerant to high ambient temperatures than heat-acclimated pigeons. Poor tolerance to heat, accompanied by the demands of producing crop milk, could result in high water requirements for thermoregulation and compromised crop milk output. When high ambient temperatures combine with high humidity, heat dissipation through water evaporation would be reduced, exacerbating the problem. Production efficiency in hot regions of Australia might be improved by selection for greater heat tolerance.

The abattoir results were not reflected in the data from the farms. On average, the pairs produced heavier squabs than those processed for the market, so it seems that there is an overestimation of average squab weight on farms. Traits such as 7-Day and 14-Day body weight, number of eggs laid, squabs weaned and clutches produced per pair, were all highly variable on all farms. Given the body weight changes recorded, squabs are able to reach 70% of the 4-week BW by the first 14 days of age – by extrapolation, 90% of their weaning body weight is reached in the first 3 weeks of life (Aggrey & Cheng 1992). Clearly, Australian squabs have the potential to grow fast and it seems feasible to wean squabs sooner than 4 weeks, possibly allowing more breeding cycles, more eggs and squabs, for as long as skin and feathers reach processing maturity in that time. Squab BW is highly heritable and the correlation between genetic and parental factors is strongest for 7-, 14- and 21-Day BW (Aggrey & Cheng, 1993; 1995). This suggests that rapid gains could be made through selection for this trait. The major problem seems to be with the number of squabs hatching and weaned. While a desired weaning body weight of about 700 gram is achievable, only 60% of eggs is weaned. This poor efficiency can be attributed to poor fertility and hatchability, and high variation in parental performance. There are only limited observations and no specific studies yet undertaken to clearly establish cause-effect relationships.

Egg contamination is one important problem that was often reported during the project. Egg infection develops rapidly and may prevent embryo development at a very early stage. Spoiled eggs, therefore, may often be diagnosed as infertile. There were also incompatible or incompetent pairs – incompatible pairs would not produce fertile eggs whereas incompetent pairs would not incubate their eggs to term or would not look after their chicks.

A limited study of the nutritional needs of pigeons confirmed the protein and energy requirement reported in the literature, but further work is warranted with an emphasis on nutrient digestibility so the diet meets the needs of the parents and their squabs. Scientific evidence is still lacking on the requirements of breeding pigeons for amino acids, fatty acids, vitamins and minerals. Imbalances might be limiting crop milk production or affecting the quality of eggs. The parents are under considerable challenge due to egg production, incubation and feeding the young from the crop, whilst meeting their own maintenance requirement at the same time. It is generally agreed that 12% ME/kg and 12-18% CP content is sufficient to sustain high production (Sales & Jennsens 2003). In one study, a pelleted diet with 16% CP given with corn and supplemented with fats appeared as efficient for squab production as a 22% CP diet (Waldie et al. 1991). In another study, a pelleted diet containing 18-20% CP maintained higher egg production, tended to reduce mortality, and produced heavier squabs at weaning than a low CP diet (Bottcher et al. 1985; Meleg et al. 1999). However, while a high- CP diet may be necessary for high egg production and high squab weaning weight, the levels of fertility, hatchability or squab mortality are not satisfactory (Bottcher et al. 1985; Waldie et al. 1991; Meleg et al. 1999). Thus CP levels appear to have been optimised, but more research is needed to define the CP content in relation to dietary energy.

Without a scientifically based program for genetic improvement and breeder management, squab production will remain at the current level, hardly generating supplementary income. An improvement in production performance should not only be sought through better genetics and improved nutrition, yielding greater numbers of bigger squab, but also in breeder management (nest type and size, diet type, water availability, disease prevention and prevention of cross-contamination). These factors vary greatly amongst farms and should be standardised.

Development of methods for semen collection and artificial insemination in the ostrich

Semen collection Teaser and dummy methods have been developed for collecting semen from ostriches, similar to those we have developed for the emu (Malecki et al. 1997a). In principle, the teaser method relies on the response of a male to a crouching female (teaser) while the dummy method relies on spontaneous expression of male sexual behaviour to a human. The teaser method requires a crouching female and male ostrich that recognises it as a stimulus for copulation. The dummy method can only be applied if the male ostrich expresses sexual behaviour, such as courtship and mounting attempts, to a human. Once these behaviours are recognised in individual males, training can commence and success or failure depends on the male and female attributes, the training environment and the trainer.

Unlike previously reported concerns (Rozenboim et al. 2006), the use of the artificial cloaca was not a problem in the teaser method we developed. Fear of a human standing nearby is a factor to overcome but, once a human was no longer inhibitory, the male proceeded with mounting and the artificial cloaca became quite readily accepted. Ultimately, the appearance of a human becomes a stimulus for copulation and induces the female crouch and successful copulation. Trained male ostriches were kept with two females and, in most cases, one would crouch for a human when approached. This ensured the stimulus was readily available and the male could mount. It is unclear though how this method would work when the males were taken away from the female after each ejaculate collection. Due to space limitations in the yards, we could not test whether the teaser chosen by us would crouch each time the male was brought in or whether the males would readily accept removal from the female pen after collection. Removal of the male was successfully tried during the last project (Malecki & Martin 2005) suggesting males would co-operate. Removal of males would give control over copulations and allow more efficient utilisation of males in an AI program.

The success of our dummy method supports our earlier contention that male ostriches that display courtship behaviour to humans could be trained using this approach (Malecki & Martin 2005). The dummy method does not require a female but males suitable for training need to be identified. Males that show interest in a human by squatting and kantling, or pushing against the fence with an intention to mount a human, can be selected for training to a dummy. The critical point appears to be redirection of mounting intentions from humans to the dummy and, once this is successful, training to mount the dummy and ejaculate into an artificial cloaca inserted in the dummy can commence. In most cases, males become trained, although some take more time than others to learn mounting. Unlike the teaser method, this method provides better control over semen output but the maximum rate of collection still needs to be determined (Malecki et al. 1997b).

Both methods appear to be stress-free for the males and are not laborious for the handlers. Normal ejaculates can be collected regularly and are of satisfactory quality.

Artificial insemination We have developed two new approaches for artificially inseminating female ostriches. Both appear to stress the females less than physical restraint and immobilization in the crash box, although a study to compare the methods directly has not yet been done.

In the first method, where the female stands still for the procedure, all that was required was that the female accepts the insemination procedure. We first accustomed those females to our presence in their enclosure, and then accustomed them to accept being held gently against a person, shed wall or enclosure fence; finally, we accustomed them to cloacal manipulation. It needs to be stressed, however, that naturally calm behaviour by the females was a necessary pre-requisite. Nervous females would keep their distance from us, walking or even running away. With such females, it would take a very long time to calm them and, for some, it might never be possible.

The second approach is possible only with the females that crouch for a human. The ontogeny of crouching behaviour is unknown but it is believed to result from imprinting to humans. The imprinting process is complex but essential to bird survival in the wild. Ostriches are hatched artificially and raised on farms, so it seems likely that imprinting would be facilitated. Some mature females would spontaneously crouch for us, making them a candidate for either an artificial insemination or for teasing a male for semen collection. The insemination procedure requires further study, but it seems that it can be performed without complication, whether the female is standing or sitting, for as long as she remains still and relaxed.

Quality of emu and ostrich ejaculates

Previously, we have shown for the emu that the quality and quantity of ejaculates varies among individual males and with collection frequency (Malecki et al. 1997), and we were concerned that the collection technique itself and environmental factors might also contribute to variability.

By its nature, semen collection is usually associated with an abrupt reduction in semen temperature and this is detrimental to sperm viability in some birds (Sexton 1984) and mammals (Tao et al. 1995). Emu ejaculates are collected during winter months, so it was essential to test whether collection temperature affected sperm survival. We found that the proportions of live and normal spermatozoa and their motility were not affected by collection temperature (5 to 20°C) when the sperm were subsequently stored in vitro for up to 24 hours. This is important for on-farm practice because semen collection can be carried out without major concern for ambient temperature. If emu ejaculates need to be stored for longer than 24 hours, longer-term effects of collection temperature should be tested.

In the ostrich, on the other hand, we studied the effect of male age on ejaculate quality. As anticipated, the quantity and quality of ostrich ejaculates increased with age, with the total number of sperm per ejaculate and motility highest in 4-year-old males. This is the first evidence based on normal ejaculates that supports suggestions that male ostriches do not become mature until 3-4 years of age. Similar findings can be expected in the emu as they are thought to mature at 2-3 years of age, although we have yet to collect the experimental evidence. Ejaculate parameters need now be measured in males older than 4 years. It has been reported that female ostriches reduce egg production after about 9 years of age (Cloete et al. 1998) but no corresponding information on sperm production is available. In the study we carried out in South Africa, sperm numbers in the perivitelline membrane were high in eggs fertilised by 3-7-year-old males and low in eggs fertilised by 9-year-old males. This indirect estimate suggests that ejaculate quality begins to decline in males older than 7 years, although we know from work in other birds (broiler chicken) the female environment can influence sperm numbers reaching the ovum (Brillard, 2003; Gumulka & Kapkowska 2005). While the evidence for the effect of age on ejaculate quality in ratites is growing, given the evidence in poultry (Kelso et al. 1997; Gumulka & Kapkowska 2005), the age when ejaculate quality is at maximum should to be determined.

The duration of in vivo sperm storage and rate of lay in ostriches in the absence of copulation

Sperm storage We have confirmed that female ostriches lay fertilised eggs for about 2 weeks after an insemination. Our study was, however, complicated by an unexpected early termination of laying so less data were collected than anticipated. We predicted that female ostriches would store sperm for longer than female emus (Malecki et al. 2004; Malecki et al. 2006). If stress was responsible for the cessation of ovulation in some females, we cannot discount the possibility that it also was compromising the results in those females that continued laying, perhaps reducing the duration of sperm storage. Whether emus and ostriches are similar in sperm storage duration remains to be confirmed, possibly with artificial insemination experiments.

Since ostriches lay eggs every 2nd day, as opposed to every 3rd day for emus, ostriches can potentially produce more fertilised eggs than emus if they have the same duration of fertile period. Based on our current data suggesting a 2-week duration of sperm storage in the ostrich, we would expect production of up to 7 fertilised eggs from a single artificial insemination, although the optimal sperm still needs to be determined. Selection for laying consistency is required because only a regular 48-hour oviposition interval would guarantee maximum numbers of eggs. We also need a better understanding of the potentially inhibitory effect of stress on ovulation. The females in our study laid erratically perhaps as a result of stress due to the handling of the males during capture in the paddock for the fitting of ‘aprons’. In addition, the presence of the apron and the inability to copulate might be stressful for the birds.

Rate of lay in female ostriches reared without males

As suggested from observations in our previous RIRDC report (Malecki & Martin 2005), we again demonstrated that female ostriches lay eggs in the absence of males in their paddock. However, a couple of confounding factors need to be considered: i) the males were in close proximity, so visual or auditory contact may have been sufficient to stimulate ovulation; ii) imprinting to humans, reinforced by our frequent visits to their paddock, could provide a stimulus. It is clear that male and female ostriches develop a strong bond and that females, when separated from their partner, can stop laying even though they can still see the male through the wire fence (Malecki et al. 2004). Young females, when reared in isolation from males, appear to ovulate spontaneously although, again, ovulation may be caused by bonding to humans. Further studies are needed to clarify the effect of female-human interactions on ovulation in ostriches.

10. Implications for relevant stakeholders

• In the duck industry, the wide variation between males in sperm supply and rates of egg penetration within High and Low Body Weight lines may underpin fertility issues in commercial crosses, so a test to identify poor performers could prove useful. • In the pheasant industry, the unsatisfactory hatch rates suggest in-breeding depression (this hypothesis could be tested directly with DNA technology). Keeping a high proportion of 1 yo females could improve egg and chick production but selective breeding needs to be introduced to reduce variation and improve growth rate. • In the pigeon industry, squab production will benefit from improved management and selective breeding and these programs need to be introduced immediately. • The ostrich industry should move to selective breeding based on AI with our new and reliable methods for semen collection and artificial insemination.

11. Recommendations

Pigeon industry The general practice in Australia is for replacement birds to be selected from amongst those hatched in winter and spring months because they grow faster and mature sexually sooner than squabs hatched in summer or spring. However, as Marder & Gavrieli-Levin (1987) report, squabs that are exposed daily to high ambient temperatures fully develop cutaneous water evaporation as their major cooling mechanism. It is thus probable that squabs hatching in winter months would be less tolerant to high ambient temperatures than those hatching in summer months. Therefore, heat acclimation and selection for heat resistance should be addressed in future research.

Given that 14-day squab body weight could be used as a measure of parental performance, defined as feeding and caring for the young (Aggrey & Cheng, 1993), the trait of squab body weight appears to be simultaneously affected by selection for improved parental performance. With continuous selection for parental performance and squab body weight by 3 weeks of age, it is likely that squabs would achieve market body weight about week earlier. Thus, in the future, it seems feasible to shorten the squab cycle by about a week, allowing about 2 more cycles and the production of 4 more squabs per pair per year. Other traits, such as total egg production, fertility and hatchability, would also need to be considered. On the other hand, selection could be directed towards a higher number of squabs per pair, but this would probably be at the expense of squab body weight. The efficiency of traditional squab production depends on parental performance and is affected by genotype, diet and environment. The genetic potential for nutrient utilisation appears to have been reached, but further improvements in other genetic factors, plus nutrition and other technologies, are still likely to increase production efficiency and economic return. All those approaches could potentially increase squab output, but more research is needed before their feasibility can be demonstrated.

Ratite industries The development of artificial insemination (AI) technology for ratites has advanced greatly and is now in the stage of optimizing methods for most efficient use of males for semen collection and protocols for semen storage and preservation. Females can be inseminated artificially with minimal discomfort, especially if they crouch voluntarily for this procedure. Fresh semen can be used for inseminations and possibly stored for a few hours as well, although this is yet to be tested on farm.

The underlying mechanisms controlling ovulation in emus and ostriches need to be better understood if we are to achieve the most efficient male:female ratio. It is important to recognise that the two species provide us with different challenges, possibly because of inherent differences in their mating systems. While female emus rarely accept each other in close proximity, female ostriches seem happy to be in a group and lay eggs in the same nest, indications that they do not need a male to stimulate ovulation.

The industry is faced with considerable variation between males and between females in reproductive traits that impacts on the efficiency of production. On one hand, this variation will require intensive selection efforts if it is to be overcome but, on the other, it provides us with considerable opportunities for achieving substantial gains through genetics (Malecki et al. 2006). Commercial ostrich production that relies on natural mating can take advantage of those methods to develop the artificial insemination technology. If sustained and rapid genetic improvement is to be achieved, the ratite industries need to adopt artificial insemination technology and this can be accelerated by further development with support from the industry and government agencies.

Duck industry Further studies into factors affecting fertility are warranted. Reduced hatching performance appears to be linked to reduction in rates of sperm supply and the cause of it should be identified. Even though the effect of body weight on sperm penetration rates is clear a variation within the high body weight line should be explored. Pheasant industry It is well known that the pheasant is a photoperiod dependent species having a breeding season under long-days. Implementation of the lighting regime and the light controlled housing could see an extension of the season if greater production is needed. There appear to be too many males in breeding so either extra females are maintained for every male or male numbers are reduced. Given that hatchability rates are rather low, the Australian pheasant population may have reached the bottleneck. The population is based on a small number of founder birds, is relatively closely bred and small so there might be a need to introduce new genetics. A study into genetic structure of the existing commercial population is needed and new genetics should be imported. A controlled breeding program with pedigree and performance recording needs to be introduced based on both natural mating and artificial insemination in order to improve production and economic return.

12. References

Allen, CJ & Champion, LR 1955, ‘Competitive fertilisation in the fowl’ Poultry Science, vol. 34, pp. 1332-1342. Aggrey, SE & Cheng, KM 1992, ‘Estimation of genetic parameters for body weight traits in squab pigeons’ Genetics Selection Evolution, vol. 24, pp. 553-559. Aggrey, SE & Cheng, KM 1993, ‘Genetic and posthatch parental influences on growth in pigeon squabs’ Journal of Heredity, vol. 84, pp. 184-187. Aggrey, SE & Cheng, KM 1995, ‘Genetic correlation between genetic and parental effects on growth in pigeon squabs’ Journal of Heredity, vol. 86, pp. 70-72. Arieli, Y, Peltonen, L & Ophir, E 2002, ‘Cooling by cutaneous water evaporation in the heat- acclimated rock pigeon (Columba livia)’ Comparative Biochemistry and Physiology A, vol. 131, pp. 497-504. Bakst, MR, Gupta, SK, Potts, W & Akuffo, V 1998, ‘Gross appearance of the turkey blastoderm at oviposition’ Poultry Science, vol. 77, pp. 1228-1233. Bottcher, J, Wegner, R-M, Petersen, J & Gerken, M 1985, ‘Investigations on reproductive, growth and carcass performance of table pigeons (Pt. 2. Effect of dietary crude protein level and age at slaughter of squabs)’ Archive fur Geflugelkunde, vol. 49, pp. 63-72. Bramwell, RK, Marks, HL & Howarth, B Jr. 1995, ‘Quantatitive determination of spermatozoa penetration of the perivitelline layer of the hen’s ovum as assessed on oviposited eggs’ Poultry Science, vol. 74, pp. 1875-1883. Brillard, JP 1993, ‘Sperm storage and transport following natural mating and artificial insemination’ Poultry Science, vol. 72, pp. 923-928. Brillard, JP 2003, ‘Practical aspects of fertility in poultry’ World’s Poultry Science Journal, vol. 59, pp. 441–446. Brillard, JP 2004, ‘Natural mating in broiler breeders: present and future concerns’ World’s Poultry Science Journal, vol. 60, pp. 439-445. Cloete, SWP, Van Schalkwyk, SJ & Brand, Z 1998 ‘Ostrich breeding – Progress towards a scientifically based strategy’ In: Ratites in a Competitive World, p. 55-62, Editor F.W. Huchzermeyer, Proceedings of the 2nd International Scientific Ratite Conference, Oudsthoorn, South Africa, 21-25 September 1998. Deeming, DC & Wadland, D 2002, ‘Effect of mating sex ratio in commercial pheasant flocks on bird health and the production, fertility and hatchability of eggs’ British Poultry Science, vol. 43, pp. 16-23. Gumułka, M & Kapkowska, E 2005, ‘Age effect of broiler breeders on fertility and sperm penetration of the perivitelline layer of the ovum’ Animal Reproduction Science, vol. 90, pp. 135–148. Hemberger, MY, Hospes, R & Bostedt, H 2001, ‘Semen collection, examination and spermiogram in ostriches’ Reproduction in Domestic Animals, vol. 36, pp. 241-243. Irons, PC, Bertshinger, HJ, Soley, JT & Burger, WP 1996, ‘Semen collection and evaluation in the ostrich’ In Deeming, D. C. (ed.) Improving our Understanding of Ratites in a Farming Environment. Ratite Conference, Oxfordshire, pp. 157-159. Kelso, KA, Cerolini, S, Speake, BK, Cavalchini, LG & Noble, RC 1997, ‘Effects of dietary supplementation with h -linolenic acid on the phospholipid fatty acid composition and quality of spermatozoa in cockerel from 24 to 72 weeks of age’ Journal of Reproduction and Fertility, vol. 110, pp. 53-59. Kosin, IL 1944, ‘Macro- and microscopic methods of detecting fertility in unincubated hens’ eggs’ Poultry Science, vol. 23, pp. 266-269. Malecki, IA & Martin, GB 2002, ‘Fertility of the male and female emus (Dromaius novaehollandiae) as determined by spermatozoa trapped in eggs’ Reproduction Fertility and Development, vol. 14, pp. 495-502. Malecki, IA & Martin, GB 2003, ‘Sperm supply and egg fertilisation in the ostrich (Struthio camleus)’ Reproduction in Domestic Animals, vol 38, pp. 429-435.

Malecki, IA & Martin, GB 2005, Reproductive technologies for ratite farming, Rural Industries Research & Development Corportation, Barton, ACT, Publication No 05/200. Malecki, IA, Martin, GB & Lindsay, DR 1997a, ‘Semen production by the male Emu (Dromaius novaehollandiae) 1. Methods for semen collection’ Poultry Science, vol. 76, pp. 615-621. Malecki, IA, Martin, GB & Lindsay, DR 1997b, ‘Semen production by the male Emu (Dromaius novaehollandiae) 2. Effect of collection frequency on the production of semen and spermatozoa’ Poultry Science, vol. 76, pp. 622-626. Malecki, IA, Cloete, SWP & Martin, GB 2006, ‘Future directions in breeding of ratites’ proceedings of the Ratite Science Workshop of the XIII World Ostrich Congress, 12-15 October 2006, Sao Paolo, Brazil. Malecki, IA, Cloete, SWP, Gertenbach, WD & Martin, GB 2004, ‘Sperm storage and duration of fertility in female ostriches (Struthio camelus)’ South African Journal of Animal Science, vol. 34, pp. 158-165. Malecki, IA, Horbanczuk, JO, Reed, CE & Martin, GB, 2005, ‘The ostrich (Struthio camelus) blastoderm and embryo development following storage of eggs at various temperatures for up to three weeks’ British Poultry Science, vol. 46, pp. 652-660. Marder, J & Gavrieli-Levin, I 1987, ‘The heat-acclimated pigeon: an ideal physiological model for a desert bird’ Journal of Applied Physiology, vol. 62, pp. 952-958. Meleg, I, Dublecz, K, Vincze, L & Horn, P 1999, ‘Effect of dietary crude protein level on reproductive traits of commercial pigeons in different production terms’ Acta Agraria Kaposvariensis, vol. 3, pp. 247-253. Rosenboim, I, Navot, A, Snapir, N, Rosenshtrauch, A, El Halawani, ME, Gvaryahu, G & Degen, A 2003, ‘Method for collecting semen from the ostrich (Struthio camelus) and some of its quantitative and qualitative characteristics’ British Poultry Science, vol. 44, pp. 607-611. Rybnik, PK, Horbanczuk, JO, Naranowicz, H, Lukaszewicz, E & Malecki, IA 2007, ‘Semen collection in the ostrich (Struthio camelus) using a dummy or a teaser female’ British Poultry Science, vol. 48, pp. 635-643. Sales, J & Janssens, GPJ 2003, ‘Nutrition of the domestic pigeon (Columba livia domestica)’ World’s Poultry Science Journal, vol. 59, pp. 221-232. Scott, ML, & Dean, WF 1991, ‘Protein, energy and amino acid requirements of breeder ducks’ In: Nutrition and management of ducks. M.L. Scott of Ithaca Publisher, Ithaca, New York. Sexton, TJ 1984, ‘Effect of temperature and method of semen collection on the viability of turkey spermatozoa’ Poultry Science, vol. 63, pp. 844-846. Tao, J, Du, J, Kleinhans, FW, Critser, ES, Mazur, PC & Critser, JK 1995, The effect of collection temperature, cooling rate and warming rate on chilling injury and cryopreservation of mouse spermatozoa’ Journal of Reproduction and Fertility, vol. 105, pp. 231-236. Von Rautenfeld, DB 1977, ‘Mitteilungen zur kunstlichen besamung, geschlechts und altersbestimmung beim strau∫ (Struthio camelus australis, Gurney)’ Der praktische Tierarzt, vol. 5, pp. 359-366. Waldie, GA, Olomu, JM, Cheng, KM & Sim, J 1991, ‘Effects of two feeding systems, two protein levels, and different dietary energy sources and levels on performance of squabbing pigeons’ Poultry Science, vol. 70, pp. 1206-1212. Wishart, GJ 1987, ‘Regulation of the length of the fertile period in the domestic fowl by numbers of oviducal spermatozoa, as reflected by those trapped in laid eggs’ Journal of Reproduction and Fertility, vol. 80, pp. 493-498. Wishart, GJ & Staines, HJ 1995, ‘Assessing the breeding efficiency of broiler breeder flocks by measuring sperm transfer into laid eggs’ British Poultry Science, vol. 36, pp. 317-323. Wishart, GJ, Young, M, & Staines, HJ 2004, ‘Weekly monitoring of broiler breeder flock mating efficiency by sperm transfer into eggs’ British Poultry Science, vol. 45, pp. 400-403. Ya-jie, JI, Yan-bo, Y & Wu-zi, D 2001, ‘Studies on ostrich semen character and semen storage at low temperature’ Journal of Economic Animal, vol. 5, pp. 49-54.

Appendix 1. Communications resulting from this project

The research team at the University of WA, led by Irek Malecki and Graeme Martin, were the organisers and major editors of the 4th International Ratite Science Symposium, held in conjunction with the XXIII World’s Poultry Congress, 29 June-4 July 2008, Brisbane, Australia. Papers for this symposium were published in a Special Issue titled “Ratite Science for industry and conservation”, published by the Australian Journal of Experimental Agriculture, 2008, Volume 48 (10), 1247-1350. Eds. I.A. Malecki, P.C. Glatz, C.A. Anderson and L.E. Webb. CSIRO Publishing, Victoria, Australia.

As a satellite meeting to this major event, we also organised and ran a workshop on Clean, Green and Ethical ratite industries in Perth (June 2008) with participants from Australia, South Africa and Latin America.

Refereed Journal Articles Rybnik, P.K., Horbanczuk, J.O., Naranowicz, H., Lukaszewicz, E., and I.A. Malecki (2007). Semen collection in the ostrich (Struthio camelus) using a dummy or a teaser female. British Poultry Science, 48 (5), 635-643. Malecki, I.A., Rybnik, P.K. and G.B. Martin (2008). Reproductive technologies (AI) for ratites: a review. Australian Journal of Experimental Agriculture 48, 1284-1292. Special issue for the 4th International Ratite Science Symposium, 29 June-4 July 2008, Brisbane, Australia. Brand, Z., Cloete, S.W.P., Malecki, I.A. and C.R. Brown (2008). The genetic relationships between water loss and shell-deaths in ostrich eggs, assessed as traits of the dam. Australian Journal of Experimental Agriculture 48, 1326-1331. Special issue for the 4th International Ratite Science Symposium, 29 June-4 July 2008, Brisbane, Australia. Cloete, S.W.P., Brand, Z., Bunter, K.L. and I.A. Malecki (2008). Direct responses in breeding values to selection of ostriches for live weight and reproduction. Australian Journal of Experimental Agriculture 48, 1314-1319, Special issue for the 4th International Ratite Science Symposium, 29 June-4 July 2008, Brisbane, Australia.

Refereed Articles in Conference Proceedings Malecki, I.A. Cloete, S.W.P & G.B. Martin (2006). Future direction in breeding ratites. In Proceedings of the 1st Latin America Ratite Science Workshop. 26-28 October Sao Paulo, Brazil (invited paper). Malecki, I.A. & G.B. Martin (2006). The effect of season and hen age on egg production and fertility of commercial pheasants in Australia. In Proceedings of the Australian Poultry Science Symposium, 14-16 February 2007, Sydney, Australia. Malecki, I.A., Rybnik, P.K., Horbanczuk, J.O., Lukaszewicz, E. & Naranowicz, H. (2007). Semen collection in ostriches. In Proceedings of the XIV World Ostrich Congress, 19-20 October, Riga, Latvia, Society “Ostrich of Latvia” (Ed), pp 57-60 (invited paper). Rybnik, P.K., Horbanczuk, J.O., Naranowicz, H., Lukaszewicz, E., & I.A. Malecki (2007). Semen collection from male ostriches using a dummy female – preliminary results. XIX International Poultry Symposium Polish Branch WPSA, 14-15 September 2007, Olsztyn, Poland, p. 142. Malecki, I.A., Cheng, K.M. & P.J. Marini (2008). Squab production – a review. An invited review for the XXIII World’s Poultry Congress, 29 June-4 July 2008, Brisbane, Australia. Malecki, I.A. P.K. Rybnik, E. Lukaszewicz & J.O. Horbanczuk (2008). Crouching behaviour and oviposition rate in female ostriches reared without males. Australian Journal of Experimental Agriculture 48, xxi (1 page paper presented at the 4th International Ratite Science Symposium, 29 June-4 July 2008, Brisbane, Australia.

Malecki, I.A. & P.K. Rybnik (2008). Artificial insemination of female ostriches using voluntary crouch. Australian Journal of Experimental Agriculture 48, xx (1 page paper presented at the 4th International Ratite Science Symposium, 29 June-4 July 2008, Brisbane, Australia. Rybnik, P.K., Malecki, I.A., Horbanczuk, J.O. & E. Lukaszewicz (2008). Semen characteristics of 2- and 3-year-old male ostriches. Australian Journal of Experimental Agriculture 48, xxv (1 page paper presented at the 4th International Ratite Science Symposium 29 June-4 July 2008, Brisbane, Australia. Rybnik, P.K., Malecki, I.A., Horbanczuk, J.O. & E. Lukaszewicz (2008). Characteristics of ostrich ejaculate in the second half of the breeding season. Australian Journal of Experimental Agriculture 48, xxvi (1 page paper presented at the 4th International Ratite Science Symposium, 29 June-4 July 2008, Brisbane, Australia. Brand, Z., Cloete, S.W.P, Malecki, I.A. & C.R. Brown (2008). Preliminary results on the effect of genotype on embryonic position in dead-in-shell ostrich eggs. Australian Journal of Experimental Agriculture 48, xix (1 page paper presented at the 4th International Ratite Science Symposium, 29 June-4 July 2008, Brisbane, Australia.

Conference Attendance

26-28 October 2006 The World Ostrich Congress (WOC) and the First Latin America Ratite Science Workshop (LARSW) Sao Paulo, Brazil. Irek Malecki gave talks on "The Australian Ostrich Industry" for the WOC and on "Future directions in breeding of ratites" for the LARSW.

19-20 October 2007 The World Ostrich Congress (WOC) Riga, Latvia.

29 June-4 July 2008 XXIII World’s Poultry Congress and 4th International Ratite Science Symposium, Brisbane, Australia.

Other activities

June-July 2005: Participation in the ostrich project in Poland August-September 2005: Participation in the ostrich project in South Africa October 2005: Research visits to Poland and France and attendance of the 3rd International Ratite Science Symposium. October 2005: Game Birds visits to Queensland, Victoria and NSW. May-June 2006: Participation in the ostrich project in Poland October-November 2006: Participation in the ostrich project in South Africa March, September and November 2006: Game Bird Farm visits in Queensland, Victoria and NSW. 24 June- 29 July 2007: Participation in the ostrich project in Poland 11-28 November 2007: Participation in the ostrich project in South Africa 18 August and 9 September 2007: Game Bird visits in Queensland and Victoria.

Improving Reproduction and Genetics in Game Birds and Ratites

by Irek A. Malecki and Graeme B. Martin

Publication No. 11/061

This report describes the development and application of RIRDC is a partnership between government and industry fertility and hatchability technologies in commercial production to invest in R&D for more productive and sustainable rural of Peking ducks, pheasants and meat pigeons and artificial industries. We invest in new and emerging rural industries, a insemination technology for ratites. It clearly identifies a suite of established rural industries and national rural issues. number of major constraints to efficient production of ducks, pheasants and pigeons. Most of the information we produce can be downloaded for free or purchased from our website . The report is primarily for managers and researchers working in production of poultry, game birds, emus and ostriches, although RIRDC books can also be purchased by phoning it is also relevant to those involved in breeding of other bird 1300 634 313 for a local call fee. species, as well as conservationists.

Contact RIRDC: Level 2 15 National Circuit Ph: 02 6271 4100 Most RIRDC publications can be viewed and purchased at Barton ACT 2600 Fax: 02 6271 4199 our website: Email: [email protected] PO Box 4776 web: www.rirdc.gov.au www.rirdc.gov.au Kingston ACT 2604 Bookshop: 1300 634 313

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