WO2009063512A1

WO2009063512A1 - Multistep process for the establishment of hybrids of livestock animals

Info

Publication number: WO2009063512A1
Application number: PCT/IT2007/000807
Authority: WO
Inventors: Giuseppe Bartolucci
Original assignee: Giuseppe Bartolucci
Priority date: 2007-11-15
Filing date: 2007-11-15
Publication date: 2009-05-22

Abstract

The present invention relates to a process for the establishment of hybrids of livestock breeds belonging to the class of mammals, wherein the genetic traits fall within a pre-established genetic plan with the aim of increasing the production and/or quality of the meat and/or milk to which they refer. The process envisages various selection stages, aided by genetic markers, wherein the desired traits are introduced by means of laboratory techniques and embryo transfer and artificial insemination techniques in order to guarantee uniformity of the traits, and is particularly applicable to bovines and swine.

Description

MULTISTEP PROCESS FOR THE ESTABLISHMENT OF HYBRIDS OF LIVESTOCK ANIMALS

DESCRIPTION

BACKGROUND OF THE INVENTION

The present invention refers to the biological livestock breeding sector, more

5 particularly to multipath cross-breeding processes for the establishment of hybrids derived from animal breeds belonging to the same species of livestock breeding animals such as swine, bovines, buffalos, yak, goats, sheep and 0 rabbits.

STATE OF THE ART

For some time now, many researchers, including Lush, Lerner, Bettini, Falconer and others, have indicated that a satisfactory 5 solution to the problems associated with meat production, just as with other animal produce, is that of the heterotic hybrid.

Heterosis, or hybrid vigour, is the superiority of offspring with respect to the mean 0 value of the populations to which the parents belong. Superiority is essentially manifested in the low hereditability traits (fertility) , but with the hybrid, there may also be a favourable combination of the traits possessed individually 5 by the various genetic types, more rapidly with respect to pure selection.

These are essentially the reasons that have directed various countries towards hybrids, especially in Europe, where considerable industrial plants have arisen, which, in percentage terms, participate significantly to national meat production. Thus, in the animal production operational plan, the term hybrid is used to describe the end products of a range of crosses of varying complexity, obtained in three or more steps, that may be somewhat different from one another and requiring very different technical times, capital investments and work involvement, due to the varying levels of complexity of the scheme on which they are based, the original genetic types (breed, man-made lines) , and the results obtained (the value of the final, or end product, i.e. livestock animal for meat production) .

For the majority of them (e.g. for the British and Dutch) various limitations in the comparison of the end results are in any case implicit in the production model itself.

In order to clarify this point, it will be appropriate to firstly describe a real hybrid model, with reference, by way of example, to the pig-breeding sector. According to the first European cross- breeders (the UK) improvements in pig-breeding have been the result of two initiatives: a) the switch in 1966 from progeny testing to performance testing, and the number of heads tested from 300 to 500 boars/year; b) in 1969 farm genetic checks were introduced and genealogy records abandoned as a tool for genetic improvement (surviving essentially as a result of the record-keeping significance) . Nowadays, approx. 70,000 breeders/year are monitored in the farms .

1972 saw the start of hybrid pig testing. In 2000, hybrid pig producers had captured approx. 70% of national production in Europe. The registration of productivity data was abandoned because it was shown to be incapable of influencing improvement, and because it was rather costly.

In order to understand the great revolution that gave rise to hybrids, it is essential to remember that with pure breeds, the market products are typically the offspring of the same pure-bred subjects, and that the intensity of selection and the turnover (alternation) applied to the parents can only be rather modest and these are preserved for as long as possible, while they are much faster in the hybrid.

Under certain aspects, with 3 multiplication stage hybrid, the situation changes profoundly, even though the end result is not substantially different : a) the intensity of selection and turnover applied to the first stage (the nucleus) may be incomparably greater, because the cost of selection is diluted through the multiplication centres; b) given the greater productive scale and the technology used, it is possible to market a relatively uniform product at any desired size.

These are the strengths of the three stage hybrid, starting from two breeds, even though this is essentially lacking the characteristics of the boar hybrid, namely complementarity and heterosis.

In summary, in the pure breed, meat production comprises two stages, breeding, in pure breed form, where genetic improvement is implemented, and the commercial stage in the

"fattening centre" where pigs are produced for the market, where genetic improvement is entirely due to the former.

The three stage model comprises the insertion of "multiplication centres" between the nucleus and the fattening centres.

Like the breeding females, the males are first of all used in the nuclei and then subsequently moved to the multiplication centres so that the animals themselves become GPs (grand parents) and Ps (parents) : this guarantees a genetic gradient between the two and a maximum rate of improvement.

But, starting from two breeds, the end product can only be a ³^ blood, which in fact excludes both complementarity and heterosis.

The cross-breeding process forming the subject of the present invention introduces an important innovation to the sector, in that it allows the production of a complex hybrid, in that it has complementarity of the characters possessed individually by the various genetic types and minimal heterosis in' the parental female and complementarity and heterosis in the final offspring, thus adapting optimally to the great Asian markets (approx. 65% of the global market, estimated forecast for 2007) .

Essentially, a product is provided that differs from the current commercial products because it is a true 5 stage hybrid wherein there is complete complementarity of heterosis, also in the final offspring.

Another significant innovation is represented by the fact that this result is achieved through the aid of molecular genetic markers allowing evaluation of the percentage of parental genome present in the hybrids. This guarantees the possibility of optimally exploiting the existing biodiversity in the parental lines used for the production of the hybrids, inserting the desired phenotypic characteristics into the latter thanks to the aid of the molecular markers . Unlike bovines, for swines, assisted reproduction technologies are not fully used commercially for various physiological and anatomical reasons pertaining to the species. However, in recent years, it has been possible to appreciate the enormous number of biotechnology facilities that can be of help in production centres. Deep uterine insemination with sexed semen, rather than classic mating techniques, is the technique of choice for propagating the genetic characteristics of the best males. Superovulation a^~rn3 embryo transfer are the techniques for best exploiting the characteristics of the female line. Furthermore, the possibility of using female recipients with phenotypes typically adapted to the commercial production areas may improve the selection of females with stable genetic characteristics (lack of epigenetic phenomena) .

The difficulties frequently encountered in the routine application of assisted reproduction techniques in pig breeding can only be solvable through the careful selection and use of dedicated instruments.

To date, the sexing of semen is the subject of debate, especially due to the rate with which a quantity of semen, suitable for use for insemination, is obtained. This problem has been solved by the flow cytometer with built-in separator. This technique makes it possible to obtain sexed semen with 97% accuracy. Sexed semen has drawbacks associated with preservation and, especially, motility. In order to deal with this drawback, the pig-breeding industry has developed flexible catheters that can be used for deep intrauterine insemination. In addition, the ability to identify ovulation times through the use of ultrasound equipment has especially improved the results that can be obtained.

Superovulation by means of special protocols, in vivo fertilisation using sexed semen and subsequent embryo transfer, using females phenotypically adaptable to the areas of destination as recipients, form the basis for obtaining superior genetic lineages.

The cost of the recipients is sustained by the lower number of donors (superovulates) and the possibility of selecting the females that are most stable, from the epigenetic viewpoint.

Furthermore, the possibility for early PCR- based diagnosis of communicable diseases, both in the semen and in the embryonic culture fluid, during embryo transfer, make such techniques extremely safe from the healthcare viewpoint.

An additional characteristic of the invention is that of resorting to the use of vasectomised males during the deep uterine insemination and embryo transfer steps. Indeed, it is known that the purely physiological aspect of mating has advantages, such as the use of the male besides for the mere oocyte fertilisation but also for the preparation of the uterus for embryo implantation by means of the proinflammatory action of large quantities of semen, typical in pigs.

Hence the present invention makes use of vasectomised males for superovulated females and for recipients with the purpose of making the uterine environment suitable for fertilisation using sexed semen and for embryo implantation respectively.

The process described envisages the following stages: A) experimental synthetic lines, B) Grand Parents, C) Parents, D) Great grand parents, E) final hybrid. Each of these stages is aided by the analysis of molecular markers .

From the comparative tests conducted, the final hybrid has been superior to the pure-bred animal for the value and uniformity of the characters from meat, the rate of growth, the conversion index, meat quality, for the absence of exudative diseases (which especially affect the breeds and types selected for leanness, being the most susceptible to stress) , and finally for resistance to certain respiratory diseases, while the procedure used in its formation allows the replication of the end product, as parents at any biologically achievable scale (thousands, tens of thousands, hundreds of thousands of individuals) .

The process of the invention will be clearer from the following detailed description, making reference to a preferred embodiment pertaining to the breeding of pigs.

In developing the process according to the invention, a series of interbreed crosses are envisaged starting from ten or eleven different breeds within the same species, each of which is selected for particular hereditary traits.

For each of the starting breeds, previously characterised genetically by means of molecular markers allowing the genetic introgression to be followed in the hybrids, is selected the bloodline positive for the most easily inherited traits, the so-called "dominant traits", with respect to a genetic plan envisaging five multiplication stages, as described below:

1) production of the synthetic foundation line, indicated by the initials SFL, constituting the mother strain free from genetic defects, born in a sterile environment, checked for growth and resistance to external stress. It consists of 500 animals. Six female breeds are used in the preparation of the synthetic female lines: the core consists of the Large White breed, to which has been added 12ths of the blood of another three or four breeds from the remaining five (Landrace, Hampshire, Duroc, Cinto Senese, Chester White) both in order to have greater docility and rusticity, and as a function of the country of destination of the animals. Thus, the expectation is: a pure founding breed (Large White) , two breeds as a blood stock and three breeds depending on the types of geographical areas where production is localised. In this regard, the areas are subdivided on the basis of the possible climatic temperature ranges that may be encountered, i.e.: a) constant temperature areas; b) areas where the temperature changes from winter to summer; c) areas with sudden significant temperature changes. On the basis of this, the following are provided for subsequent multiplications:

2) the foundation lines FLs which are obtained by crossing the founding synthetic female lines SFLs with a male of breed 1 (see fig. 1) obtained by crossing at least seven different blood lines of Italian, Dutch French and Canadian strains, which must confer the male animal with iminunogenetic resistance and antistress traits;

3) The great grand parent GGP line, which is obtained by multiplying the female foundation lines FLs with a male from breed 2 (male of different breed from the male of breed 1) obtained by crossing at least six blood lines, all different from one another, sharing the characteristic of having a dorsal fat thin thickness) ;

4) the GP (grand parent) line obtained by multiplying the GGP female line with a male of breed 3 (obtained by crossing at least eight blood lines, all different from on another, sharing prolificacy as a characteristic) ;

5) the parental females obtained by crossing the GP line with a male of breed 4 (from at least fifteen blood lines, all different from one another) sharing the major characteristic of rate of growth and milk production) .

The parental females are handed over to the breeders for the production of:

6) the final hybrid, denoted TWB, obtained by crossing parental females with semen from a male of breed 5 (selected from the best six blood lines for the characteristics of low food consumption and growth) . According to one particular characteristic of the invention, in order to secure uniformity, at least in the first three multiplication stages, namely for the synthetic foundation line, the foundation line and the Great Grand Parent (GGP) line, artificial insemination and embryo transfer techniques are used.

Said technique envisages the following stages : the application of superovulation protocols to the sample sow (i.e. the one with all the required requisites) ; synchronisation of oestrus with females that are phenotypically adaptable to the hybrid destination areas; - the use of vasectomised males and ultrasound platforms for optimisation of ovulation times; non-surgical and/or surgical deep intrauterine insemination with sexed semen from a single male, using a flow cytometer with built-in separator and/or immunogenetic techniques (FISH) , checked for the presence of pathogens;

- non-surgical and/or surgical transfer of embryos, harvested and checked from a healthcare viewpoint, into females that are phenotypically adaptable to the hybrid destination areas. According to one particular characteristic of the process, a different feeding system is used for each blood line sequence type.

The multiplication cycle will now be described by way of example.

The process begins with:

100 males, split into 60 + 30/40 reserves and 900 females for the female lines of 6 different breeds (150 females per breed) ; and 50 males and 500 females for the male lines of 5 different breeds.

30,000 head are obtained in the first year.

From these, on the basis of the parameters itemised below, 10,000 are selected for the female line females and 4,000 for the male line males .

The remaining 16,000 are not carried forward to reproduction and are eliminated.

The selection parameters for the two groups of 10,000 and 4,000 are as follows:

1) rate of growth: must not be less than 800 g per day within the 20 to 100 kg range;

2) must not present any type of disease or disease symptoms;

3) the limbs must be perfect;

4) sows must have a minimum of 14 teats;

5) they must have a feed conversion index no greater than 2.4 (the quantity of feed required to produce one kg of meat) . ANALYSIS OF THE TWB OFFICIAL COMPARATIVE TESTS

With the purpose of ascertaining the fa^'tstock value, comparative tests have been conducted by the Universities of Bologna and Naples, the ϋmbria Development Institute and the Ministry of Agriculture Experimental Animal Production Institute in Modena, over the course of which, all the TWB hybrids have been compared with other European types: Cotswold and Camborough of British origin, Hypor of Dutch origin, and the most famous pure breeds: Large White, Landrace, Pietrain and relevant crosses.

TWB gave exceptional results, both for its capacity to adapt, and for its meat characteristics.

The TWB hybrid has been shown to be endowed with: a high growth and accretion capacity (90 kg in 150 days) ; an optimal conversion index (2.60 F. U. per kg of living weight growth in the 15 to 90 kg range) ; high prolificacy: 20-30 piglets weaned/year per sow (corresponding to approx. 25-30 quintals of meat (living weight, 100 kg) ) produced on average from each breeder/year; significant rusticity; a high lean meat yield (approx. 58%) . TESTS CONDUCTED BY THE UMBRIA DEVELOPMENT INSTITUTE (PERUGIA - ITALY)

The Umbria Development Institute has conducted a series of comparative tests on 2,093 animals belonging to the following types: the Landrace (German, Dutch and Swedish line) and Large White (British and Dutch lines) breeds, 5 Landrace and Large White half-breeds, the British Cotswold hybrid, the TWB hybrid.

They studied growth rate, feed conversion index, breeding stress resistance and adaptation to various environmental conditions. The animals 0 have been subjected, to a forced diet, under typical local breeding conditions.

From the results summarised in table 1 it may be deduced that, in the TWB hybrid, mean daily growth is approx. 20% higher, allowing a 5 comparable reduction in feed costs. The absence of mortality shows the greater rusticity and adaptability of TWB.

TABLE 1

TESTS CONDUCTED BY THE NATIONAL FEDERATION OF BUTCHERS (ROME - ITALY)

The National Federation of Butchers has conducted a series of slaughter tests on 240 specimens taken from a group of 2,903 animals belonging to the same breeds and types indicated above .

Table 2 reports the results with the technical-economic evaluation of the carcasses and the mean score obtained by summing the scores attributed to the various cuts. It has emerged that the leg, shoulder, breast, marrowbone and the meat scraps from the TWB hybrid have given higher meat yields, with lower percentages of bone and fat. That corresponds to a commercial value for the TWB hybrid that is between 5 and 20% higher, with respect to other breeds and types .

TABLE 2

Technical-economic evaluation of the carcasses : Mean score attained by each cut obtained from lean pigs subj ected to the experiment .

TESTS CONDUCTED BY THE INSTITUTE OF ANIMAL PRODUCTION, UNIVERSITY OF BOLOGNA- ITALY)

The Institute of Animal Production of the University of Bologna has conducted a series of tests on the meat - fat ratio or on the yields of various cuts, comparing the carcasses of the following breeds and types (slaughtered at a mean live weight of 92 kg) :

Table 3 shows daily growth and the conversion index for the TWB hybrids, for the production of heavy pigs for Parma-type ham. As may be observed, mean daily growth is clearly higher with respect to all the Large Whites considered.

TABLE 3

(1) The age of the pigs in all groups at the start of the test was 80 days.

(2) The mean daily increase in the T.W.B. hybrids has been significantly more favourable (PO.05) with respect to all the Large Whites both hi the between sex and average comparison

Table 4 pertains to the yields of the carcasses in various cuts, from where it may be deduced that the TWB hybrid has the best yield in ham, loin and shoulder.

TESTS CARRIED OUT AT THE INSTITUTE OF EXPERIMENTAL ANIMAL PRODUCTION OF THE MINISTRY OF AGRICULTURE AND FORESTRY, MODENA

These were carried out in order to compare breeding data (growth rate and conversion index)

10 for the following breeds and types:

The data pertaining to the TWB hybrid have confirmed the higher mean growth index and the improved conversion index (2.86 F. U.) .

These data have been deduced by comparing animals within the 38 kg to 92 kg weight range. The Modena Institute for Experimental Animal Production has published the results of comparative tests between heavy TWB pigs for industrial use and pig breeds traditionally used for the cold cut industry belonging to the English and Dutch Large White breeds, as well as the respective crosses. Again, in this test, the heavy TWB strain has given truly surprisingly positive results, reaching a weight of 150 kg in just 90 days, compared to 210 days for the English Large Whites. In addition, there ware also feed savings of at least 76 kg of feed for every 100 kg of meat produced in comparison to the Dutch pigs, and at least 97 kg when compared to the English breed.

Similarly, positive results have been observed with less loss of preserved meats (hams, coppa (rump seasoned pork meat), etc.) and greater desirability. ANALYSIS OF THE CONCLUSIONS OF THE COMPARATIVE TESTS

In conclusion to the above-mentioned points, it may be stated that these comparative tests confirmed that TWB hybrid pig has the following positive characteristics, with respect to the more established breeds and hybrids: greater productivity during rearing; high degree of rusticity and resistance to rearing stress; higher yield at slaughter, and in superior cuts; improved and consistent meat organoleptic qualities and tenderness, allowing the mark of quality, documented in the texturometric, colourimetric and chemical data: it cannot be ascertained whether texturometric and colourimetric data have been made available for other pig breeds.

Claims

1) A process for the establishment of hybrids of livestock animal breeds belonging to the same mammal species, by means of mixed techniques, characterised in envisaging the following stages: a) the production of a synthetic foundation line (SFL) , consisting of the mother strain, free from genetic defects, born in a sterile environment, checked for growth and resistance to external stress, using six different female breeds, previously genetically characterised with molecular markers that allow following genetic introgression in the hybrids; the various female lines being crossed with males belonging to the same breed but different blood lines; b) the foundation lines (FLs) which are obtained by crossing the synthetic foundation lines (SFLs) with a breed male (1) obtained by crossing at least seven different blood lines, previously genetically characterised using molecular markers, capable of conferring the male animal with immunogenetic resistance and antistress traits; c) the great grand parent (GGP) line, which is obtained by multiplying the foundation lines (FLs) with a breed male (2) (male of different breed from the male of breed 1) obtained by crossing at least six different blood lines, all sharing the characteristic of having a dorsal fat thin thickness, as characterised previously by means of molecular markers; d) the line (GP) obtained by multiplying the line (GGP) with a breed male (3) (obtained by crossing at least eight different blood lines sharing the characteristic of prolificacy) previously genetically characterised_ by means of molecular markers; e) the parental females (PFs) obtained by crossing the line (GP) with a male (breed 4) (from at least 15 different blood lines) sharing the major characteristic of rate of growth and milk production, previously characterised genetically by means of molecular markers) ; f) the final hybrid (FH) obtained by crossing parental females (PFs) with males (breed

5) (selected from the best six blood lines for the characteristics of low food consumption and growth) previously genetically characterised by means of molecular markers; where the phenotypic characteristics are inserted in the hybrid in a controllable manner, thanks to the presence of the aforementioned molecular genetic markers, allowing the evaluation of the percentage of parental genome present in said hybrids; and where, in order to secure uniformity for the female lines in the first three multiplication steps, i.e. for the synthetic foundation line, the foundation line and the grand parent line, embryo transfer techniques are used in the crosses . 2) The process according to clam 1 characterised in that the synthetic foundation lines (SFLs) are obtained starting from a pure founding breed, two breeds as blood stock and three breeds according to the type of geographical area where production is localised.

3) The process according to claim 2 characterised in that the geographical areas on which selection of the three starting breeds is based, besides that of the founding breed, are subdivided on the basis of the possible climatic conditions that may be encountered, i.e. constant temperature areas, areas where the temperature changes from winter to summer and areas with sudden significant temperature changes.

4) The process according to in claim 1 characterised in that the embryo transfer technique envisages the following stages: the application of superovulation protocols to the sample sow (i.e. the one with all the required requisites) ; synchronisation of oestrus with females that are phenotypically adaptable to the hybrid destination areas; - the use of vasectomised males and ultrasound platforms for optimising ovulation times, both for superovulated females and for recipients; non-surgical and/or surgical deep intrauterine insemination of the sample sow with sexed semen from a single male, using a flow cytometer with built-in separator and/or immunogenetic techniques (FISH) , checked for the presence of pathogens;

- non-surgical and/or surgical transfer of embryos, harvested and checked from a healthcare viewpoint, into recipient females that are phenotypically adaptable to the hybrid destination areas.

5) The process according to the preceding claims characterised in that, in the case of pigs, Large Whites are used as the foundation breed in the preparation of the female synthetic foundation line.

6) The process according to the previous claims characterised in that embryos are transferred, following careful healthcare checks, into females not belonging to the synthetic lines that are phenotypically adaptable to the hybrid destination areas. 7) The process according to claim 3 characterised in that in the male lines, embryo transfer techniques are used for the scope of production of the final parental boar. 8) The use of molecular probes and/or molecular markers for the genotypic selection of gamete cells in a process for the establishment of hybrids of livestock breeds belonging to the same mammal species, by means of mixed artificial insemination and embryo transfer techniques.