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Summary of full report
By Dr Irek Malecki and Prof Graeme Martin
December 2005
RIRDC Publication No 05/200 RIRDC Project No UWA-71A/88A
Executive Summary
What the report is about
This report describes the
outcomes of a series of studies on ratite fertility: i) an analysis of
data on sperm-egg interactions in vivo and in vitro; ii) the development
of techniques for the assessment of male and female fertility; iii) the
assessment of sperm quality; iv) the development of techniques for semen
collection and artificial insemination; and v) the morphology of the ostrich
blastoderm before and during incubation. This work has delivered new technologies
for measuring fertility, diagnosing infertility, and assessment of the
efficiency of use of sperm. It has also led to technologies for the in
vitro assessment of sperm function after short- and long-term storage,
and provides a better understanding of the use of the behaviour of emus
and ostriches for the purpose of semen collection and artificial insemination.
Finally, the studies of the ostrich blastoderm have provided a basis for
development of a new tool for diagnosis of wastage caused by problems with
early embryo development.
Who is the report targeted
at?
The report is primarily
for managers and researchers working in the emu and ostrich industries,
although it is also relevant to those involved in poultry and game bird
industries.
Background
Our ratite industries need
technologies for quantitative examination of the fertility of males and
females, beginning with deposition of spermatozoa in the female reproductive
tract and finishing with the fertilised egg. These technologies allow measurement
of fertility and diagnosis of infertility in flocks and individuals and
will benefit our emu and ostrich industries by reducing reproductive wastage
– in the short-term, problem birds can be eliminated; in the long term,
breeders can use fertility assessment as a selection criterion, thus accelerating
genetic progress with both natural mating and artificial breeding. The
focus of this project was the development of ‘perivitelline techniques’
so we could quantify fertility in emu and ostrich flocks, improve semen
storage and cryopreservation protocols, advance development of techniques
for semen collection and artificial insemination, and study the requirements
of the ostrich blastoderm.
Aims/Objectives
i) To develop a sperm-egg
interaction assay for measurement of sperm quality in emu and ostrich;
ii) To develop an objective
method for diagnosing reproductive wastage in emu and ostrich flocks;
iii) To develop methods
for semen collection from male ostriches;
iv) To optimise in vitro
sperm storage and sperm cryopreservation protocols for emu and ostrich;
v) To determine the requirements
of the ostrich blastoderm for successful embryo development.
Methods used
We used abrupt separation
of the sexes to study the duration of sperm storage and fertility in female
emus and ostriches, and we measured the distribution of spermatozoa trapped
in the perivitelline membrane above the germinal disc to quantify the efficiency
of sperm utilization as a new measure of fertility. Sperm-egg interaction
was investigated in vitro to develop a bioassay of sperm function that
could be used for assessing the quality of fresh and stored sperm. Sperm
storage and cryopreservation was tested under different conditions of storage
temperature and time, freezing and thawing rates.
Behavioural development of male emus, and male and female ostriches, was studied in birds from hatch until maturity to understand the conditions required for successful development of semen collection and artificial insemination. We investigated the requirements of the ostrich blastoderm for storage temperature and time by determining blastoderm size and the stage and development of embryos during incubation.
Results/Key findings
Female emus and ostriches
lay fertilized eggs for up to 4 weeks after separation from males. Male
ostriches supply more sperm than male emus and female ostriches appear
to use sperm more efficiently than female emus. An in vitro sperm-egg interaction
assay has been developed and has proven to be a useful tool for assessing
sperm function in fresh, stored and frozen-thawed sperm.
Protocols for storage and cryopreservation have been developed further and now yield quality sperm after storage and a satisfactory number of functional sperm after cryopreservation. Female and male ostriches raised under frequent human contact developed behaviour patterns that facilitated mounting of a dummy teaser by male ostriches and crouching behaviour by female ostriches. Ostrich blastoderm size and stage of development are improved as egg storage temperature increases – the optimum egg storage temperature lies between 20 and 25°C.
Implications for relevant
stakeholders
Emu and ostrich farming
can now take advantage of the perivitelline techniques and objectively
assess the fertility of individual males and females, or of their entire
flocks. These techniques can either be used to diagnose problems or to
estimate the breeding values of males and females. Fertility, the most
important measure of reproductive efficiency, is now better understood
and can be controlled.
Two assays for testing sperm function, the hypo-osmotic swelling test (HOS) and fertilising ability (SE) following storage and cryopreservation, will help to optimise diluents and conditions for storage and cryopreservation of emu spermatozoa. Advances made for emu sperm should be transferable to ostrich sperm, which should help accelerate the development of optimum storage and cryopreservation protocols for ostrich sperm.
The ostrich industry can now begin to take advantage of artificial insemination technology. It is clear that semen collection and insemination methods can be developed for ostriches because we can develop desirable behaviour patterns in males and females. However, more research is required on the recognition of suitable behaviours and the conditions required for their successful development.
This project has developed tools for measuring fertility in flocks and individuals, and for diagnosing infertility. It has improved storage and cryopreservation of emu spermatozoa and developed an in vitro sperm-egg interaction assay to test sperm function. The project has shown that understanding and developing desirable behaviour of male and female ostriches are essential for development of semen collection and artificial insemination. The ostrich blastoderm appears to favour temperatures between 20 and 25°C but further studies are needed on embryo and post-hatch chick development.
Recommendations
The perivitelline techniques
should be used to evaluate the breeding efficiency of emus and ostriches
and to diagnose and eliminate fertility problems. They can be applied in
vivo and in vitro and males and females can be ranked using fertility measures
as selection criteria, so this important trait is improved in breeding
programs.
Artificial insemination can be used in emu breeding now, even though the technology is not perfect.
We need to continue to develop systems for sperm storage and cryopreservation, and to better define the semen diluent. Emu semen can be stored undiluted for up to a day but mechanisms protecting sperm from damage need to be better understood. Emu spermatozoa need protection from cooling and, so far, only limited protection is offered by our “Emu 3” diluent. Freezing efficiency is still unsatisfactory – more cooling and freezing rates need to be explored and the thawing process needs to be better controlled. Emu spermatozoa are cold-sensitive, unlike poultry species, so they require a unique approach, possibly one of those used for mammalian spermatozoa. Since the emu is a close relative of the ostrich, it is probable that same principles will apply to ostrich spermatozoa.
Artificial insemination as a breeding method will be feasible for the ostrich industry after more research on the behaviour of both males and females. We do not quite have sufficient understanding of
Results/Key findings
Female emus and ostriches
lay fertilized eggs for up to 4 weeks after separation from males. Male
ostriches supply more sperm than male emus and female ostriches appear
to use sperm more efficiently than female emus. An in vitro sperm-egg interaction
assay has been developed and has proven to be a useful tool for assessing
sperm function in fresh, stored and frozen-thawed sperm.
Protocols for storage and cryopreservation have been developed further and now yield quality sperm after storage and a satisfactory number of functional sperm after cryopreservation. Female and male ostriches raised under frequent human contact developed behaviour patterns that facilitated mounting of a dummy teaser by male ostriches and crouching behaviour by female ostriches. Ostrich blastoderm size and stage of development are improved as egg storage temperature increases – the optimum egg storage temperature lies between 20 and 25°C.
Implications for relevant
stakeholders
Emu and ostrich farming
can now take advantage of the perivitelline techniques and objectively
assess the fertility of individual males and females, or of their entire
flocks. These techniques can either be used to diagnose problems or to
estimate the breeding values of males and females. Fertility, the most
important measure of reproductive efficiency, is now better understood
and can be controlled.
Two assays for testing sperm function, the hypo-osmotic swelling test (HOS) and fertilising ability (SE) following storage and cryopreservation, will help to optimise diluents and conditions for storage and cryopreservation of emu spermatozoa. Advances made for emu sperm should be transferable to ostrich sperm, which should help accelerate the development of optimum storage and cryopreservation protocols for ostrich sperm.
The ostrich industry can now begin to take advantage of artificial insemination technology. It is clear that semen collection and insemination methods can be developed for ostriches because we can develop desirable behaviour patterns in males and females. However, more research is required on the recognition of suitable behaviours and the conditions required for their successful development.
This project has developed tools for measuring fertility in flocks and individuals, and for diagnosing infertility. It has improved storage and cryopreservation of emu spermatozoa and developed an in vitro sperm-egg interaction assay to test sperm function. The project has shown that understanding and developing desirable behaviour of male and female ostriches are essential for development of semen collection and artificial insemination. The ostrich blastoderm appears to favour temperatures between 20 and 25°C but further studies are needed on embryo and post-hatch chick development.
Recommendations
The perivitelline techniques
should be used to evaluate the breeding efficiency of emus and ostriches
and to diagnose and eliminate fertility problems. They can be applied in
vivo and in vitro and males and females can be ranked using fertility measures
as selection criteria, so this important trait is improved in breeding
programs.
Artificial insemination can be used in emu breeding now, even though the technology is not perfect.
We need to continue to develop systems for sperm storage and cryopreservation, and to better define the semen diluent. Emu semen can be stored undiluted for up to a day but mechanisms protecting sperm from damage need to be better understood. Emu spermatozoa need protection from cooling and, so far, only limited protection is offered by our “Emu 3” diluent. Freezing efficiency is still unsatisfactory – more cooling and freezing rates need to be explored and the thawing process needs to be better controlled. Emu spermatozoa are cold-sensitive, unlike poultry species, so they require a unique approach, possibly one of those used for mammalian spermatozoa. Since the emu is a close relative of the ostrich, it is probable that same principles will apply to ostrich spermatozoa.
Artificial insemination as a breeding method will be feasible for the ostrich industry after more research on the behaviour of both males and females. We do not quite have sufficient understanding of those behaviours that could be used in development of reliable semen collection techniques. Although female behaviour is more amenable than male behaviour, the decline in rate of ovulation when males are removed requires attention and methods to prevent it need to be developed.
Our data suggest that, amongst female ostriches, there are birds that produce blastoderms of poor viability. Confirming this would allow us to identify and eliminate another source of wastage. We need more research into blastoderm viability, the “physiological zero” temperature, and the relationships with the stage of development of the ostrich (blastoderm to embryo), hatchability and chick development. There is still no hatchability technology that could be adopted as satisfactory standard practice.
The industry can directly adopt some of our diagnostic tools but others require specialized equipment.
A professional laboratory would be needed to provide the more complex services to the farms. If put in place, these technologies could substantially benefit the ostrich and emu industries, and both industries need these technologies because current genetic gains are slow. Genetic progress could be accelerated significantly because breeders could make use of the reservoir of elite genes that already exist in captive populations. This would lead to improvement of the flocks, by genetic selection or by introduction of new genes, and both industries would be more competitive on international markets.
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