A SURGICAL tSOLATOR

Field of the Invention

The present Invention Is directed to a surgical isolator and cradle, syllable for the use ^{! '} a surgical method for removal of premature eggs from birds in order to generate

Background to the I wan j ton

The first surgical isolators were developed for use in gnotobiotrcs where germ-free laboratory animals were obtained by delivering such animals from their parents by Caesareao section directly into an aseptic environment

European patent application no, 01650109 is directed to a method of rearing a bird of specified contamination free status. The method of this application comprises housing a bird as a pamni bird, surgically removing an egg in its shelf from the parent bird prior to transfer of the egg to the cloaca in the parent bird, incubating the egg and batching the egg to product a laying bird. The application also relates to the production of avian eggs of specified contamination free status.

The present invention Is concerned with a surgical isolator and cradle for use In sterile- procedures for obtaining eggs and birds of specified germ-free status.

in this specification, the term "contamination free/gemi-free" is used very broadly and relates to many pathogens and infections that can te carried by birds, particularly, poultry such as chickens and turkeys which are used wkfety to produce flocks of birds for breeding to produce fertile eggs for commercial production and to produce eggs and meat for human consumption, Further, such eggs and birds are used in the manufacture of a wide range of biological substances including vaccines, antibodies.

monodonaf antibodies, fibroblasts ami proteins, both for therapeutic and prophylactic use in people and animate. Thay are further used extensively for diagnostic tests and the prodυeϋon of transgenic eggs and birds _* Many of these uses require eggs and/or the birds produced from them to be free of either all or specified contaminants such as infections, including a variety of species of parasite, bacteria, mycoplasma, viruses, retroviruses, prions, DNA and RNA fragments. Sometimes, the viruses can be small viruses including pica ma and parvo viruses, Some of the bacteria from which eggs are often contaminated include Clostridia and Entero bacteria. There are many non-pathogenic organisms that should be controlled. Similarly, many of the micro-organisms which include parasites, aerobic and anaerobic bacteria, commensal species and species associated with the gut, are undesirable. Similarly, mycoplasma, viruses inducing retroviruses, prions, fungi, yeast and moulds are- also undesirable.

Therefore, the term "specified contamination free ^* or "germ-free status" could include some or all of these and is much broader than just free of specified pathogens. For example, conventional specific pathogen free (SPF) are not specified free from some viruses and indeed can be contaminated with bacteria. Thus, for certain uses, these may be sufficient. The use to which the eggs and the birds are to be put wall determine the contaminants that the egg or bird must be free of. Conventional germ- free and soma SPF eggs are derived by treating fresh naturally laid eggs with chemicals, including disinfectants and antibiotics, and placing them in isolators, Such naturally laid eggs are taken from selected parent stock birds. White these methods have been relatively successful ϊn the production of SPF eggs, they have not been truly successful h producing what are contaminant free eggs. However, the chemicals are not abte to eliminate contamination from, for example, bacteria entering the pores of the eggshell immediately after laying and before disinfection. Contamination of eggs, whether SPF, germ-free or gnotobiotic, results in loss of compliance with specifications and, in many instances, toss of commercial value and utility.

^

Statement of the Invention

According to a ffost aspect of the invention, them is provided a sterile surgical isolator and cnadta for a bind which provides a eon(aminanl«frø, atmosphere in which a S surgical procedure Is performed comprising

an isolator made of walls with at least two gloved ports, a solkl isolator floor which provides m operating surface and a surgical port within the solid isolator floor; 0 a cradle for receiving and positioning a bird during a sterile surgical procedure having an open and dosed position;

wherein the cradle enables the stable positioning of the operating surface ^S of the bird relative to the operating surface of the isolator such that the operating surface of the bind maybe operated on through the surgical pert and

wherein the cradle in the closed posiiion provides a complete and stable air seal to be maintained between a bird on the cradle and the surgical

According to a second aspect of the Invention, there is provided a method of using the surgical isolator and cradle according to any of the preceding claims wherein 5 the surgical port is sealed with a first layer of sterile transparent adhesive film;

the operating surface of the bird is cϊeaned and steri&ed and the bird is ^Q placed on the cradle in the open position;

a narrow strip of transparent adhesive film is placed on the sterile surface the binf;

a second layer of sterile transparent adhesive film is placed on top on the narrow strip of adhesive film; and

the cradle is moved into the closed position to ensure stable positioning of lhe operating surface of the bird, preferably the abdomen, relative to the operating surface of the surgical isolator;

wherein the first adhesive layer is in contact with the second adhesive layer am* a complete air seat is created between the bird and the surgical isolator _*

Datallad Description of the Invention

In general terms, the invention provides a surgical isolator and cradle and means for using it to provide a contain [nation free atmosphere In which a surgical procedure can bo performed. The surgical procedure involves the surg fear-removal of a premature egg Sn its shell from a bird.

The cloaca is the principle area of contamination within a bM. The cloaca is a chamber linked to both the digestive and reproductive systems of the bind, therefore an egg and faeces may be present in the cloaca at the same time. The egg, prior to entering the cloaca is free of contamination, However, as an eggshell Is porous external contamination when the egg passes from the reproductive system through the cloaca is a major problem, Specific methods are required to remove it from the parent bird whilst maintaining sterility and then, because of is prematurity, additional specific methods ana required to incubate and hatch it successfully and consistently.

Specicaliy. the present invention is directed to a surgical Isolator and cradle for use

in surgical procedures wherein a premature egg is removed in Is she! from a parent bird prior to the transfer of the egg to the cloaca, As the present invention is directed to obtaining a gemi-free egg, Jt is imperative that the surgical isolator and cradle provide a sterile environment for the procedure to be carried out in, £

Preferably, the surgical isolator should includes a sterile gaseous atmosphere.

According to one embodiment of the invention, the isolator suitable for surgical manipulation is made of walls, which may be flexible, with at toast two gloved ports 10 for surgical manipulation, a separate entry port and a solid isolator floor elevated above ground in Che isolator floor in front of the gloved ports, there is a special surgical port, Preferably, the surgical port may to approximately 200mm x 300 mm.

The birds are presented ami held for surgery using an adjustable stainless steel i 5 cradle which is pivotalty attached or hinged to the underside of the isolator floor below the surgical port.

The surgical isolator and associated equipment may to sterilised using a combination of methods including heat, moisture {steam}, radiailon and chemicals 0 such as peroxides and/or organic Iodine with alcohol. Sterilisation methods that do not impair the viability of the embryo should only be used, Furthennore, to maximise freedom from bacteria and spores, all surgical equipment should tie gamma-

S According to a preferred embodiment of the invention, surgical isolators should be thoroughly cleaned, dried, sprayed with alcohol (exposure time of at least 10 min}, dried, fumigated with 2-5% v/v peracetic acid (exposure time at least 20 minutes).

The atmosphere of the surgical isolator may be purged with sterile, filtered air to 0 avoid embryo-toxicity. Electrical and other equipment not suitable for steam, irradiation or peraeotio ackl sterilisation may be sterilized using commercial ethylene

⁵ O ~

o*kie sterilising systems applied mm, or preferably twice each for 24 hours.

According tø another embodiment of the invention adhesive, transparent, sterile, surgical plastic or adhesive films may fee used to achieve a stable air seal between S the sterilise ύ surface of the bird and the isolator.

According to a preferred embodiment of the invention, the surgical port is preferably seated with a transparent plasite or adhesive film to enable the formation of a complete and stable air seal between the sterilized surface of the bird and the i o opening to Jhe surgical isolator.

According to a most preferred embodiment the surgical port is seated with a first layer of sterile transparent adhesive film, a bird is placed on the cradie and the surface of the bird is sterilised, a narrow strip of transparent adhesive film is placed on the

1 s sterile surface the bird, a second layer, usually larger, of sterile transparent adhesive film is placed on top on the narrow strip of adhesive film, and the cradie is pivoted into the closed position to ensure stable positioning of the birds abdomen relative to the operating surface of the surgical Isolator; wherein the first adhesive layer is in contact with the second adhesive layer and a complete air seal is created between the birti 0 and the syrgical isolator.

The adhesive films should be applied to skin of the bird after complete removal of feathers, thorough preparation and sterilisation of the skin and removal of superficial keratocytes and sebum (using detergent, alcohol and organic iodine), 5

Maintenance of the integrity of the film and facilitation of surgical approach is enhanced by the use of a cradle, which allows stable positioning of the bird's abdomen relative to the operating surface of the surgical isolator.

S According to a still further embodiment of the invention confirmation of the seal Integrity may bo made using fluid test materials such as sterile helium gas released

asepiieafJy into the surgical isolator and teaks detected externally by use of a helium gas detector. Alternatively, sterile indicator liquids (such as iodine solution) may be used and detected by visual inspection

5 Preferably, surgical removal of the egg is best completed rapidly, at about less than 30 minutes from time of euthanasia, to avoid impairment of embryo viability. The prolonged use of anaesthetics or excessive delays between euthanasia of the parent bird and removal of the egg will adversely affect embryo viability.

0 According to a still further embodiment of the invention the surgical procedure for removal of the premature egg from the uterus of the bird includes incision through the adhesive film, incision of the skin of the bird, transaction though the subcutaneous, muscle, and peritoneal layers of the bird. The egg may be removed from *he bird either in the intact, sealed (e,g. clomped-off} uterus or directfy by incision of the s uterus. Ail direct contact between fluid forms of sterilising solutions should be avoided to counteract any risk of impairment of embryo viabitdy.

Alternatively, the surgical removal comprises:-

^Q performing a laparotomy incision and tying off the oviduct of the bird at both ends with sutures;

transecting the oviduct distal to each suture;

5 removing the egg enclosed in the oviduct;

sterilising the e-viduet;

removing the egg; and

sterilising the egg.

The surgical procedure used must ensure that the gut of the MtJ is not contaminated by waste material and that the egg is not damaged. Aseptic techniques must be used throughout, S

Preferably, the bird is sacrificed by euthanasia or kiting prior to removal of lhe egg in its shell. Alternatively, the bird may be euihanteed, Female parent birds may be either lϊvs or recently kilted. Live birds may, as consistent with ethical, legal and animal welfare considerations, be My conscious, sedated or anaesthetised. Eggs and ova 0 may lie either fertilised or u nfertifised conscious, sedated or anaesthetised.

According to a preferred embodiment of the invention, the cradle comprises two substantially parallel bars defining a space to receive a bird, in use, the cradfe Is oj ^j ened and a bird is placed in the cradle. Preferably, the bars are substantially s Identical and form an acute angle underneath the Isolator floor. The bird is placed in the cradfe head first, such that the operating surface of the bird {the uterus ami abdomen) is parallel to the isoϊator floor _* The tail end of the bird rests on the cradle bars such that the uterus and abdomen are positioned correctly. The release mechanism on the cradle Is then shut and the underside of the bird is then parallel to 0 the isolator lteor and surgical port, The surgical procedure for the removal of the premature egg can then take place. The gloved ports in the wails of the Isolator allow access to the underside and operating surface of the bird.

The surgical isolator generally comprises an enclosure surrounding a working area 5 surface having gloved ports, viewing areas and an access panel in the form of a surgical port. There is also a further access panel which enables the aseptic transfer of the surgically-derived egg from the surgical isolator to a transfer unl for subsequent transfer to a incubation isolator,

G Preferably, the removal of the egg is at a time prior and as close as possible to the transfer time when the egg would transfer naturally to the cloaca in the parent bird.

The surgically removed egg may then be placed in a sterile container and seated. The container should allow the egg to cool and bo of suitable design and sfee to for egg storage, A sterile container is one with an approximate volume 10 times that of the egg, with the egg supported and protected by a plastic frame.

According to a further embodiment of the invention, sterility of the entire surgical procedure may be confirmed. This includes collection and evaluation of samples such as microbiological swabs (taken as moist swabs and immediately placed in transport media) from the egg and reproductive tract for isolation of micro-organisms using suitable culture media for bacteria, mycoplasma, viruses and fungi, including liquid broth enrichment media, in aerobic, anaerobic and micrø-aerophilic environments.

Once the sterile surgically-derived premature egg is obtained, the egg may then be incubated in a sterile environment and hatched and reared to produce a laying bird.

According to the invention, for maximum freedom from micro-organisms eggs should preferably be derived asepticaliy from parent females prior to entry of the egg into the cloaca (unless they are also gemi-free or gnotobiotic) and the life-cycle should be completed in sterile isolators. The ife-cycle may be completed outside isolators when SPF eggs and birds are produced.

Infectious organisms that may be controlled by the invention include organisms that can be pathogenic or non-pathogenic to the relevant species. These include avian secies (typically chickens, fowls and turkeys), humans and other mammals {typically dogs, cats, ho ^j rs.es, cattle, pigs, sheep, goats, rats and mice). For the purposes of the invention, micro-organisms include parasites, bacteria (including anaerobic and aerøbie species, commensal species and species associated with the gut), mycoplasma, viruses (including retroviruses), prions, fungi, yeasts, moulds and ONA and RNA fragments,

The parent bfrd is the bird from which the surgically-derived egg is obtained. The surgicaly-derived egg Ss then incubated and hatched to form a toying bird. The surgically-derived egg and subsequent laying bird are genn-free.

S in one. embodiment of the iπventbn the parent -bird is chosen from a flock of similar birds all reared under the same conditions.

in another embodiment of the invention, the parent bird is hatched naturally in ø sterile environment from a flock of birds of similar existing contamination free status, 0

In a further embodiment of the invention the parent bird is one of a flock of birds which are of another contaminant free status having been produced by suitable selection and natural rearing methods under controlled conditions and the method is used to provide binds of a different contaminant free status, s

Preferably a laying bird forms part of a flock and after the laying birds arø hatched, a sample of the laying binds is remove?! and tested for specific contaminants to provide a measure of the contaminant free status of the flock _* Ideally when the specified contaminant free status is mi achieved in the faying bird, the laying bird is useci as a o parent bind in the method.

Preferably the parent bird is chosen as a day old bird.

In one embodiment Ih e laying bird is removed from the sterile environment to lay s eggs which are, in turn, hatched to produce further laying birds,

In another embodiment the laying bird is removed from the sterile environment and fed with food containing normal gutflora. The birds produced by this method, having normal gυtftera, preferably without avian and zoonotic pathogens, are suitable for 0 consumption or use in the food industry.

Typically the bird is a chicken.

Preferably when a bird is hatched from a laying bird having the specified contaminant free status ami is not a laying bird, the bird so hatched is reared in a sterile S environment for subsequent fertilisation of laying birds of the same or tower contaminant free status,

According to a further aspect of the invention, the invention further provides a method of providing an egg of a specified contaminant fee status comprising Sn a sterile Q environment:

housing a laying bird having the same or better contamination free status as provided in accordance with the method of the invention;

S using the laying bird to fay lhe egg; and

removing the egg to another sterile environment

0 The laying tørct may then be used to lay an egg, which may bo the end product itself, or which may hatch into a bird which could either fonn a flock of birch of gomvfree status or if it is not a laying bird, be used to fertilise a laying bird to reach tower contamination status.

S If fertile eggs are used to produce offspring or derived birds, then the eggs may be hatched, reared, maintained and bred m either conventional husbandry systems, SPF systems or in isolators to contra t the entry of micro-organisms.

It will be appreciated that in certain circumstances, when taking selected birds as Q parent birds, lhe laying binds produced may not in fact be sufficiently free ol contaminants to produce laying birds of the right quality. It may then be necessary to

carry out the same steps again using the eggs produced from such laying birds and artificially removing the eggs from these laying birds to provide further laving birds which hopefully will bo contaminant free,

S Indeed, the sterility can ba further improved by feeding the laying birds, in ihe sterile environment, with food containing normal gulftøra or sterile food. Preferably, the birds are given, in a sterile environment, sterile food and water. Alternatively, normal flora may be administered to the chick p re- hatching or orally to the bird at any stage after hatching* The normal flora may be one without pathogens, Il wfli be appreciated that i o when birds are hatched which are not laving birds, they will then be retained for subsequent fertilisation of the laying birds. In this way, the whole flock can be sterile.

It will be possible, in the present invention, to produce simply the eggs tor subsequent use. When eggs are required of a germ-free status, the first thing to do is to incubate 1 S ihe eggs by using the desired parent birds. Then, when the parent birds have been tested tor specified contaminants to provide a measure of the germ-free status, house that laying bird in another sterile environment and use that laying bird to lay eggs which will have a germ-free status,

0 A still further embodiment of the invention provides a method for incubating and haithing the surgically derived eggs _s and then rearing and breeding from Ihe subsequent birds.

Surgicaliy-derived eggs are premature and their development, for example 5 gastrulation, may te delayed, Also, the eggs may tøck certain features of a toll-term naturally laid egg, for example there may be reduced cuticle on the shelf and pore formation in the shell may be impaired. The shell, its pores and cuticle modulate respiratory gas exchange and hydration of the developing embryo, Therefore standard hatchery practices for normal laid eggs may not be appropriate for optimum 0 viability and can require modification to achieve consistently high hatch of healthy birds from surgically-derived eggs, The specifics conditions required vary with, for

example, the species and stage of development when removed from tho parent bird. Rearing and breeding ^e derived bird in a healthy and productive state whites maintained ϊn a specified contamination free or sterile environment requires adjustments to nutrient contents of diets, especially organic micrαnutrienls such a S vitamins, to compensate for losses occurring during sierifeation of the diets and from an absence of supply from commensal microorganisms,

Preferably, the eggs shoufd be allowed to cool after removal from the parent bird. They can then be stored, undisturbed for, in the case of chickens at least 24 hours o and not mom than 72 hours. Storage conditions may have HEPA filtered afr and, for chicken eggs a temperature between 15 and 23 ⁰ C, relative humidity of 50-75% anti be free from vibration or sudden jarring.

According to a further embodiment of the invention, for the first 24 hours standard 5 incubation conditions for the species of egg may be used. Thereafter each egg should be carefully monitored for weight loss, incubation temperature, relative humidity and, if appropriate respiratory gaseous exchange especially carbon dioxide and oκygen concentrations in air. The incubation and hatching conditions can be adjusted according to the invention. Jdealiy, for 5Sg surgically-derived premature egg 0 a target weight foss of from approximately G,4g/day is desirable. Jncubation temperatures of approximately 37,2*C are preferable initially on Day 0 until Day 18 of incubation and then temperatures of approximately 3(3.5 ⁸ C are desirable until hatching. Relative humidity may initially he set at approximately 40% but should be adjusted according daily according to ventilation rate and daily egg weight loss until S Day iδ when relative humidity shoufd be increased to approximately 85%.

λ sυiiahJe environment for hatched chicks, rearing birds, laying and reprøductively aciive birds is a rigid waited isolator, with HEftMIttered air. The air is maintained at positive pressure and exchanged at frequent intervals (e.g. 10 times / hour for adult 0 birds, taking into account cubic capacity of the isolator and stocking density), floor area of 0 ^, 2 - 0.4 nv*/ bird, gloves on entry ports protected from damage by birds _* and

air temperature and lighting controlled to provide conditions similar to those lor conventional birds of the same species and stage of life-cycle.

Rearing and breeding a bird in n heathy and productive state whilst maintained in a 5 specified contamination free or sterile environment requires specialised diets to compensate for lhe lack of certain nutrients normally produced by, for example, (lie contaminants found in the gut or on the skin of a bird in a conventional environment,

Jt will be understood that Ih fs invention applies to all avian and reptilian species, IO including but not limited to chickens, turkeys, quail, ducks, geese, guinea fowl, pheasant, partridge, parrots and grouse _*

The invention will be more clearly understood from the following description of the Figures and Examples. I S

Figure 1 shows a surgical isolator and cradle according to the invention.

Figure 2 Is a diagrammatic representation of the method of the invention using the surgical isolator and cradle of Figure 1 _* 20

Figure 1 shows a sterile surgical isolator and cradle for a bind according to the invention. This isolator provides a contaminant-froβ atmosphere in which a surgical procedure takes place, The isolator (1 ) comprises an isolator made of flexible walls with at least two gloved ports {not shown )„ a solid isolator floor (2) which provides an 5 operating surface and a surgical port (3) within the solid isolator floor. The surgical oort provides an aperture through which the person carrying out the surgery can access the operating surface of the bird. Attached to the isolator floor is a cradle (4} for receiving and positioning a bird during a sterile surgical procedure _* The cradle is pivotally attached or hinged to the underside of the isolator floor below the surgical 0 port, as shown by the hinge {5), The surgical port is adapted to receive the bird in the cradte and to provide a complete and stable air sea! to be maintained between a bird

IS

on the cradle arid the surgical port during surgery. The surgical port ajjerture is preferably covered with a sterile adhesive film {shown in hitch). This sterile adhesive film provides the stable air seal, The cradle also provides the advantage that it enables the stable positioning of the birds abdomen relative to the operating surface S of the surgical isolator.

Figure 2 shows a diagrammatical representation of the method of using the cradle according Io the invention.

I ^Q fig 2a shows the surgical Isolator (1) not in use end in the closed position which comprises an Isolator floor (2), surgical port and adhesive film (3), cradle (4) and hinge {5)> The cradle Is in the closed position. The surgical port (3) is sealed with a first layer of sterile transparent adhesive film.

15 Figure 2b shows the surgical Isolator fn an open position such that a chicken may be placed Jn the cradle. The chicken is placed head first into the cradle and the operating surface of the bird is defealhered and sterilised. The air seal between the bird isolator may be tested before and after surgery using sterile fluid test materials which are released into the surgical isofator to enable external teaks to be detected. 0

Figure 2c shows a second sterile adhesive frfm placed on the sterilised underside of the chicken ^, Preferably, a narrow strip of transparent adhesive film (not shown) is pfaced on a sterile surface the bird. λ second iayer of sterile transparent adhesive film is placed on top on the narrow strip of adhesive fϊfm. At this stage the cradle is S pivoted into the closed positron to ensure stable positioning of the birds abdomen relative to the operating surface of the surgical isolator. The bird's abdomen should be parallel to the floor of tho surgical isofator to facilitate surgical manipulation, legs should be held in the fully flexed position and ctoaca sealed,

0 Figure 2d shows the cradle in the dosed position such that sterile adhesive Him of the surgical port and adhesive film on the bjiti come Into contact to form a stable aif seal

within the surgical Isolator and provide a sϊβrile surface to bø operated on. When the first adhesive layer on the port is in contact wflh the second adhesive layer on the bind, a complete air seal is created between IhG bfrd and the surgtea) isolator.

The air seal between the bird and ihe surtjical fsoJator may be tested before and after surgery using sterile fluid materials which are released into the surgical isolator to enable teaks to be detected.

^ ^. 7 * _^ .

Example 1

Method

Fifty aduft female and five adult male chickens of known SPF status were maintained on selected diets ant! allowed to breed naturally. Timing of egg laying (evasion) was recorded individual^ for each female over a few-week period, The mean time of day (lime. L) when an egg was laid was calculated for each female. The time of day for L-3h was calculated and the period from L-3 to L was nominated as the derivation interval. This interval was ihe time in which aseptic surgical laparotomy was performed for removal of the most developed eggs in each bird.

for ih ^β procedure, birds were euthanased by cervical dislocation and shortly afterwards prepared. Birds were submerged in a disinfectant solution for 5 minutes, Feathers were removed from the ventraf thorax and abdomen and the exposed skin sterilised using a S0% solution of iodine in alcohol heated to 37*C, Each bird was then placed under a specially adapted surgical isotøtor sterilised with a 5% solution of peracetic acid and containing sterile instruments and a SOO ml fiask containing iodine in alcohol The bird was covered with a sterile drape and a sterile entry port of ihϋ isolator was then placed over the drape _* A laparotomy incision was made and the oviduct (typically ihe uterus) was tied off at both sides of the egg using suture material The oviduct was then transected distal to each of the sutures from the egg and the oviduct containing the egg was removed from the females' abdomen. The uterus-enclosed egg was then placed in the iodine/alcohol solution for five minutes after which the oviduct-enclosed egg was transferred via an entry port from the surgical isolator to a receiving isolator, in the receiving isolator; ihe oviduct was incised, the egg removed, swabbed with a disinfectant solution and transferred to an isolator adapted as a hatchery incubator.

Within one day of hatching, live chickens were removed from the hatchery isolator ami transferred to two large-scale rearing isolators suitable for rearing groups of

young chickens. Chickens were reared on commercial diets sterilised by radiation, At 18 days of age. five chickens were removed from each of the rearing isolators, eulhanased and sampled for bacteriology by aerobic and anaerobic culture. Samples included fiver, spleen, heart blood, vagina/cloaca, caeca! aritl smail Intestinal digesla 5 and faeces.

Results

Viable chickens were hatched successfully from the artificially derived eggs 1.0 (hatthabity >S0% more often >90%). No anaerobic or aerobic bacteria were isolated from the chickens sampled.

S A sate and highly effective method for artificial production of germ -free fertile eggs In chickens was established. Eggs were viable and produced viable germ-free chickens which were successfully maintained in isolators.

0 A series of further studies were conducted in accordance with Example 1 , Variables anticipated which affect sterility and viability of surgically derived fertile eggs were evaluated in this example. Evaluations made in the studies and the results obtained are provided below,

S The results Identified the variables that are critical control points required to consistently produce gemvfree eggs of high viability High viability is defined as about >S0% hatchabity and about »80% reaching reproductive maturity.

Example 2Aι o Effects of different techniques for sterilising equipment including surgical isolator and instruments. Evaluation included types, quantity and time of radiation, and chemical

l θ

sterilants (peroxide typos, halogen liquids, alcohol) in liquid and vapour forms for different temperatures and times, with and without egg-washing detergent solution on embryo vf ability and sterility.

Exposure of surgically- removed eggs to alcohol, alcohol ana ^* organic iodine a! 20 _« 32 or 37 ^! 'C ₍ and to peracetic acid solution (2-5% v/vj reduces halchataility from 60*100%

Exposure to peracetie aeW vapour for >5 mϊo reduces hatchabiiy by 0-29%, Exposure to iodine solution reduces høtchability by S0%.

Example SBr

Effects of time (aero to 180 minutes) between euthanasia and egg removal on viability and ease of surgical manipulation, halchabiϋty and sterility

Sterility 100%, surgery increasingly difficult after 30 minutes, hatchabily <3G% after 60 minytes.

Example 2Cϊ

Effects of different types and methods of application of adhesive films, to create art air seal between parent bird and surgical isolator; methods of testing the seal; devices far positioning bird during surgery to optimise seal formation and surgicaϊ manipulation,

gesMJs,

De*fatting ami sterilising skin aro important for obtaining consistent seal between film and tasrd skin.

Us© of at least two layers of film are required, one adhered to bird, anti another in the surgical port of the surgical isolator with adhesive layer adjacent to bird.

Birds' aWomen should be parallel to floor of surgical isolator to facilitate surgical manipulation, legs should be held in fully flexed position, cloaca seated. Test integrity of seal before/after surgery using helium gas and /or iodine iodfcato* solution, maintain positive pressure and sterile air, S

Example 3

Example ZAt

Effects of difference in lime between objected ovi- position ami surgical removal of i o tint egg from the parent bird on egg viability and sterility and ease of surgical manipulation.

Results

Halchafailitv: control (naturally laid eggs) 85-100% hatch, JMM 00% sterile; IS eggs removed from uterus within 30 min of anaesthesia or euthanasia, 13-40% hatch, 80-100% sterile; eggs removed from uterus 60 mm after euthanasia, 14% batch, 80-S2% steήle,

.i&se_oLsurtieal . rn a n ipufa t inn: control not applicable; ϋ 30 minutes, good, tissues easily eievatetf;

60 minutes, difficult, tissues difficult to elevate /early rigor mortis)

Example 3S:

Effects of timed ovi-position versus palpation versus combination of these techniques s or* proportions of eggs with complete shells versus soft shells, viability (usually life chicks at time of hatching! and on ease of surgical manipulation, Res.yite,

Timing atorie, soft shells and no suitable egg for removal β-71%, viability 13-50%, sterility 75-100%, ease of surtjical manipulation, variable;

Palpation atone, soft shells S no suitable egg for removal 13-71%, viability 13-54%, 0 sterility 89*100% , ease of surgical manipulation, good;

Timing combined with palpation, soft shells and no suitable egg for removal 10-23%,

viability 14-57%, sterility 92-100%, ease of surgical manipulation, good).

Example 3C:

Effects of antibiotics (e,g, OJially administered f I uit^ui notaries) on elimination of 5 transovarian bacterial until mycoplasmal infections and an viability and sterility of embryos and subsequent chicks,

Resujlg,

Without antibiotics, viability 22-60%. and sterility 86-92%;

With antibiotics, viability 13-57%, and sterility 89-100%. id

Example Wn

Effects of preincubation storage time φ to 5 days) and conditions, (temperature, humidity, egg orientation, ventilation, vibration), incubations conditions (temperature humidity, ventilation, orientation, weight loss), handling and hatching conditions on 15 embryo viability

Besyits.

Storage times of 0, 1-3, 4-5 days, hatcbability was 90, 90 and 60%, respectively.

Temperature, ventiat ion/ vibration for storage of 3 days: 25C and with vibration/ventilation, approximately ^* 2OC and no vibration /vsnttelωn; hatchabily was

orientation and weight loss; eggs flat on side, weight loss 7,8%, eggs domed- end upper-most, weight loss 10.2 -13.1 ^* %; hatehability 30% and &0-90%,

5 Conclusion

As expected given the wide rangts of variable investigated, a wide variety of røsulls for viability and sterility were obtained. The results identified the variables that are critical control points required to consistently produce germ-free eggs of high viability High viability is defined as about >50% hatchabiity and about >βθ% reaching reproductive maturity.