ES2338960B1 - PROCEDURE FOR IMPROVING PRODUCTIVITY IN SHEEP. - Google Patents

Abstract

Productivity improvement procedure in sheep, of the type used in livestock farms, especially in the Aragonese Rasa breed, for the improvement of prolificity and the detection of infertility, characterized in that used as an element for the detection and selection of specimens more suitable for reproduction the existence of an allele of the gene Bone Morphogenetic protein 15 (BMP15).

The invention presented provides the main advantage of getting a safe selection of specimens with a higher prolificity index, avoiding those with infertility problems or with low prolificacy, and promoting genetic improvement that results in an increase in profitability economic exploitation of livestock.

Description

Productivity improvement procedure in sheep

The present specification refers to, as its title indicates, to a productivity improvement procedure in sheep, of the type used in farms livestock, especially in the case of the Aragonese Rasa breed, for the improvement of prolificacy and the detection of infertility, characterized in that it uses for the detection and selection of more suitable specimens an allele of the Bone Morphogenetic protein 15 gene (BMP15).

Currently, multiple and varied types of procedures for improving the profitability of sheep farming facilities are widely known. Most of them, such as the one described in Spanish Patent 9300049 " Method to increase the production and quality of wool and live weight of lambs " are based solely on certain feeding regimes, which mainly seek to get animals with a good quality of meat and wool, but they do not improve the number of animals of each farm.

One of the most used procedures for try to improve and increase the number of animals in each Livestock exploitation is to try to find a high prolificity in the reproductive specimens, favoring those copies that have a tendency to have multiple births, trying achieve in this way an increase in the number of lambs born by delivery, with the consequent improvement in economic performance, especially when lambs are the primary form of exploitation.

The selection of specimens with a tendency to having multiple births has been done conventionally through a study of sheep over several generations, studying and statistically analyzing the number of lambs in each birth, and selecting those specimens from families that statistically have a clear tendency to do so. Without However, this procedure for selecting specimens is long, tedious, complicated, and with a fairly high error rate, for what its use is not very widespread.

To solve the problem in the news regarding the improvement and increase of the production of lambs in sheep farms have devised the productivity improvement procedure in sheep of the present invention, which he uses for the detection and selection of copies more suitable for reproduction, an allele of Bone Morphogenetic protein 15 (BMP15) gene, which we will call Rasa-oviaragon allele of the BMP15 gene.

This procedure is of special application in the Aragonese Rasa race, in which a deletion of 17 base pairs in an allele of the BMP15 gene in the Rasa race Aragonese Although in the BMP15 gene they have been detected to date other mutations, this mutation is new not having detected the same in sheep, and therefore it is a new allele of the gene. The deletion is located in exon 2 of the gene, and through studies Experimental has been found to be attached to animals of the breed Aragonese Rasa with a high level of prolificacy, which allows ensure that, in case of heterozygous existence of the allele Rasa-oviaragon of the BMP15 gene, females are going to have multiple births with a high probability, well above which can be achieved by a natural selection or by conventional selection procedures.

In this way, through a common analysis genetic of animals, by any of the procedures commonly known and used, those animals are detected endowed with this Rasa-oviaragon allele of the BMP15 gene. These animals are endowed with high prolificacy, so They are ideal for a livestock farm because of its very high tendency to multiple births, getting a lot of lambs In addition, since this feature of the allele Rasa-oviaragon of the BMP15 gene is linked to the footprint genetics of animals, their durability is guaranteed, and that of their associated effects, in following generations.

Homozygous adult females for the allele Rasa-oviaragon have a little reproductive system developed (called infantilism), with hypoplasia of the ovaries and therefore are sterile. In this way the same previous results allow us to detect infertility in Sheep females of the Aragonese Rasa race due to the lack of allele  Rasa-oviaragon of the BMP15 gene.

This productivity improvement procedure in sheep that is presented, it offers multiple advantages over currently available systems being the most important that allows to obtain a safe selection of specimens with a higher prolificity index, avoiding those with infertility problems or with low prolificacy, and promoting genetic improvement that results in an increase in profitability economic exploitation of livestock.

Another important advantage is that the use and detection of this Rasa-oviaragon allele in Prolificity selection schemes are highly profitable due to the speed in the diffusion of the improvement of the prolific nature, as well as for the increase of the profitability of the exploitations by significantly increasing the number of lambs born by Birth.

Another advantage of the present invention is that it they can detect infertility in the population due to this mutation, eliminating these females from reproduction and making the improvement scheme even more profitable.

To better understand the purpose of this invention, an embodiment is shown in the attached information preferential practice of the procedure to improve productivity in sheep.

In that plane figure -1- the situation of the 17 bp deletion produced by the so-called allele Rasa-oviaragon in BMP15. Other figures appear in the figure mutations described so far and already known, indicating the position of the mutation in the protein sequence and the replacement of Amino acids or the appearance of a premature stop codon.

Figure -2- shows the nucleotide sequence of the Rasa-oviaragon allele of the BMP15 gene.

Figure -3- shows the protein sequence of the Rasa-oviaragon allele of the BMP15 gene.

Figure -4- shows the alignment of the sequences between the wild allele of the gene (GenBank Acc. number AH009593) and the Rasa-oviaragon level allele nucleotide

Figure -5- shows the alignment of the BMP15 protein sequence of alleles Rasa-oviaragon and the wild phenotype (GenBank Acc. number AH009593).

Figure -6- shows the location of the primers in the BMP15 gene sequence. The primers appear marked and the place of attachment in the wild allele sequence and the Rasa-oviaragon allele.

Figure -7- shows the amplification specific to a fragment of the BMP15 gene that contains the deletion found in Aragonese rasa. The largest band corresponds to the wild allele (160 base pairs), and the smaller one to the allele Rasa-oviaragon (147 base pairs). Line 1: Size marker, line 2: male with Rasa oviaragon allele, line 3: homozygous female for the Rasa oviaragon allele, lines 4, 6, 8 and 10: heterozygous females for the allele Rasa-oviaragon and the wild allele, lines 5, 7 and 9: homozygous females for the wild allele, line 11: male hemicigotic of the wild allele, line 12: negative control.

Figure -8- shows the microsatellite table advised by FAO and the International Genetic Society Animal (MODAD, Measurement of Domestic Animal Diversity).

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The productivity improvement procedure in sheep subject of the present invention, it comprises basically, as can be seen in the attached information, the use of a Bone Morphogenetic protein 15 gene allele (BMP15), which we will call the Rasa-oviaragon allele BMP15 gene, for the detection and selection of more suitable specimens for reproduction

This procedure is of special application in the Aragonese Rasa race, in which a deletion of 17 base pairs in an allele of the BMP15 gene in the Rasa race Aragonese Although in the BMP15 gene they have been detected to date other mutations, this mutation is new not having detected the same in sheep, and therefore it is a new allele of the gene. The deletion is located in exon 2 of the gene, and through studies Experimental has been found to be attached to animals of the breed Aragonese Rasa with a high level of prolificacy, which allows ensure that, in case of heterozygous existence of the allele Rasa-oviaragon of the BMP15 gene, females are going to have multiple births with a high probability, well above which can be achieved by a natural selection or by conventional selection procedures.

In this way, through a common analysis genetic of animals, by any of the procedures commonly known and used, those animals are detected endowed with this Rasa-oviaragon allele of the BMP15 gene. These animals are endowed with high prolificacy, so They are ideal for a livestock farm because of its very high tendency to multiple births, getting a lot of lambs In addition, since this feature of the allele Rasa-oviaragon of the BMP15 gene is linked to the footprint genetics of animals, their durability is guaranteed, and that of their associated effects, in following generations.

Homozygous adult females for the allele Rasa-oviaragon have a little reproductive system developed (called infantilism), with hypoplasia of the ovaries and therefore are sterile. In this way the same previous results allow us to detect infertility in Sheep females of the Aragonese Rasa race due to the lack of allele  Rasa-oviaragon of the BMP15 gene.

In Figure -2- and -3-, you can see the 1118 base pair nucleotide sequence and protein allele Oviaragon Rasa, respectively. In the protein sequence you can note that deletion entails a change in the reading pattern of the protein and the appearance of a premature stop codon in the region that is preprotein. In this way, this allele will produce that The protein is not functional.

In figure -4- and -5- you can see the sequence alignment between the wild allele of the gene (GenBank Acc. Number AH009593) and the allele Rasa-oviaragon both nucleotide and protein The wild allele is the one that has no mutations in its sequence, and presents a normal phenotype for the character prolificity It is important to highlight the loss of 17 pairs of bases, which translates into a change in the reading pattern of the protein, producing a different protein, as well as the appearance of a stop codon 55 amino acids ahead of the deletion.

BMP15 is a gene located on the X chromosome (OARX), so that the mode of inheritance is linked to X. The gene is has shown that it acts on the ovulation rate that is highly correlated with the prolificity or number of lambs by birth in females. The females that carry this mutation, is that is, they carry the Rasa-oviaragon allele, that is heterozygous for it, have a prolificity superior to normal females that do not carry the mutation. In total it has been estimated that on average 0.6 more lambs per delivery and an ovulation rate 1 point higher than the population average. The homozygous adult females for the allele Rasa-oviaragon have a little reproductive system developed, called infantilism, with a hypoplasia of the ovaries and therefore are sterile. Therefore, the use and detection of this Rasa oviaragon allele, in the selection schemes for prolificity it is highly profitable due to the rapidity in the dissemination of the improvement of prolificacy character, as well as by the increased profitability of farms by increasing importantly the number of lambs born by birth. For other In part, infertility can be detected in the population due to this mutation, eliminating these females from reproduction and making the improvement scheme even more profitable.

Males have no homozygosis or heterozygosis for possible alleles, since the gene is located on the X chromosome but absent in the Y, and then it Talk about hemicigosis. Hemicigotic males for allele Rasa-oviaragon have a normal phenotype with normal reproductive and productive characteristics. Thus, the males will pass their daughters the allele Rasa-oviaragon or the savage while their children the hemicigotic genotype for this gene will be determined by the mother, since the father will pass the Y chromosome, which is not Present this gene.

This procedure has been obtained and verified. experimentally. For the detection of this mutation, they selected males that had a high genetic value (VG; the genetic value is calculated using BLUP type methodology, and that correct the animal's value based on different effects, called fixed, which can influence an animal even better genetically, phenotypically worse. For example, good or bad food, handling on the farm, and others). Be studied the genetic value of the prolificity of male daughters with a high VG, observing that all the daughters were very good, and No bad daughter appeared. This type of segregation is typical of genes linked to the X chromosome, since males always they transmit to the daughters the Rasa-oviaragon allele and by Therefore they are all good. Because the frequency of this allele in the population is very low no sterilities have been observed due to crosses with female carriers of this allele. In these males, the entire gene was sequenced by finding this deletion that He had been described so far. The choice of this gene was because other mutations had already been described therein, such and as shown in figure 1 that they had associated with a increase in the ovulation rate and therefore of the prolificity, and the inheritance model conformed to what was observed in our population

The mutations described in this gene show that females in heterozygosis have an ovulation rate higher than the homozygous for the wild / non-mutated allele and so both greater prolificity. The homozygous for the mutation are sterile

To confirm, the association of the allele Rasa-oviaragon with prolificity was performed a selective genotyping design by creating two tails of Aragonese rasa females with extreme values for character prolificity In total 110 females were genotyped with a value genetic of -10 or less and 97 females with a genetic value of +10 or plus. The selected females had at least 5 deliveries each. He genotyping was performed by amplifying a fragment of DNA containing the deletion region of the Rasa oviaragon allele (Figure 4), and visualization on 4% agarose gel and staining with ethidium bromide. The amplification was performed in a final volume 25 µl containing 5 pmol of each primer: F 5 '- CTC TGA GAC CAA ACC GGG TA -3, R 5 '- TTT GAG GAG CCT CTT CCT GA-3 ', 200 nM dNTPs, 2mM MgCl2, 50 mM KCl, 10 mM Tris-HCl, 0.1% Triton X-100 and 0.3 U Taq polymerase (Biotools). 35 cycles of amplification were performed with a denaturation step at 94 ° C for 30 s, hybridization at 56 ° C for 30 s, and extension at 72 ° C for 30 s. In the Figure -6- you can see an amplification, in which the band of larger size corresponds to the wild allele (160 base pairs), and the smaller in the Rasa-oviaragon allele (147 pairs of bases). Heterozygous animals had a genetic value for high prolificity character while females homozygous for the Rasa-oviaragon allele were sterile

The results showed that all animals of the low prolific tail showed the wild allele in homozygosis The animals of the high tail 32 animals presented the Rasa oviaragon allele in heterozygosis. Of these females 16 were the females that had the highest genetic value from 24 to 39. Statistical analysis was performed using an ANOVA test a through the GLM procedure of SAS. Differences were found significant between allelic and genotypic frequencies of two tails studied (p <0.0001). Likewise, a statistical analysis between the genetic value of the character prolificity and genotype within the high prolific tail, showing significant differences (p <0.0001), among the genotypes and genetic value, that is, a genetic value was associated higher with females who had the heterozygous mutation. Subsequently 145 more females have been analyzed with a value genetic below -10, and in no case was the Rasa allele detected Oviaragon

In order to know the frequency of the population different type populations were analyzed, finding a heterozygous female over 60 females, that is 1.6% of the population. In other populations analyzed that have not been inseminated with the males that have the mutation, belonging to the scheme of genetic improvement by prolificity of UPRA-oviaragón, the Rasa-oviaragon allele has not appeared. Equally in order to observe if the homozygous females for the allele Rasa-oviaragon were sterile two were selected females who were sisters of father and mother, and who had been produced within a MOET (Embryo Transfer) program. The two females had a laparoscopy following all Animal welfare standards. A female was heterozygous for the Rasa-oviaragon allele and presented an apparatus normal reproductive, with ovaries with follicles in different states and Two luteal bodies. The other was homozygous for the rasa allele oviaragón and presented a child reproductive system with hypoplasia of the ovaries and with total absence of follicles, showing that the female was sterile. On the other hand, another was detected homozygous female for the Rasa-oviaragon allele that It also presented a infantilism of the reproductive system.

On the other hand, 8 males have been found hemicigoto for the Rasa-oviaragon allele that have a normal phenotype, and reproductive characteristics and normal production, within the scheme of genetic improvement by prolificity of UPRA-oviaragon.

Finally, it is important to highlight that the mutation that defines the Rasa oviaragon allele, is a mutation, which is a deletion of 17 base pairs, and the probability that it occurred by chance in two individuals of The same race. It is described that the probability of a mutation Occur by gene, generation and base is between 10-7 and 10-9, therefore the probability that there was a deletion of 17 base pairs is very low, and that has occurred in two animals Different is virtually impossible. This shows us that animals that have this mutation come from the same ancestor common and therefore if this mutation is not very far in the time, it is possible to detect animals with a high degree of kinship that come from other animals that present the deletion For this purpose, 31 microsatellites can be genotyped distributed throughout the sheep genome advised by FAO and the International Society of Animal Genetics (MODAD, Measurement of Domestic Animal Diversity), according to figure -8-. The typing with microsatellites have proven to be the most effective and sensitive method as an indicator of genetic drift, migration and introgression of populations, and at this time it is the accepted method internationally as a measure to genetically characterize the populations, as well as for paternity studies, and detection of fraudulent uses of semen or other biological material available in animal populations. In this regard, and due to that this gene is located on the X chromosome, markers located on the X chromosome and close to the BMP15 gene will be highly informative when building haplotypes and plotting from which animals come animals that have the Rasa allele Oviaragon In this sense, 8 microsatellite markers will be tested located on the X chromosome: INRA 30, MAF 45, HEL 14, MCM551, MP1, UCD045, BMS1008.

<110> Meat OVIARAGÓN SCL

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Centro de Investigación y Tecnología Agroalimentaria de Aragón (CITA)

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Aragon Agrifood Research and Technology Center (CITA)

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Instituto Nacional de Investigación y Tecnología Agrícola y Alimentaria (INIA)

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National Institute of Agricultural and Food Research and Technology (INIA)

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Claims (6)

1. Procedure for improving productivity in sheep, of the type used in livestock farms, especially in the case of the Aragonese Rasa breed, for the improvement of prolificacy and the detection of infertility, characterized in that it includes the detection of existence of an allele of the Bone Morphogenetic protein 15 (BMP15) gene, formed by the deletion of 17 base pairs in exon 2 of the BMP15 gene from position 459 according to the sequence list shown in SEQ ID No. 1. 2. Procedure for improving productivity in sheep, according to the preceding claim, characterized in that the detection of the existence of the Bone Morphogenetic protein 15 allele, formed by the deletion of 17 base pairs in exon 2 of the BMP15 gene a from position 459 according to the sequence list shown in SEQ ID No. 1, it is carried out using any of the methodologies currently known and used in the laboratory for analysis of genetic information, prepared with the appropriate reference chains or standards for the detection of said allele 3. Procedure for improving productivity in sheep, according to the preceding claims, characterized in that the preferred methodology for detecting the existence of the Bone Morphogenetic protein 15 gene allele, formed by the deletion of 17 base pairs in exon 2 of the gene BMP15 from position 459 according to the sequence list shown in SEQ ID No. 1, is the use of Polymerase chain reaction (PCR), FISH, biochips, DNA microarrays or microarrays of genetic material prepared with the appropriate chains Reference or patterns. 4. Procedure for improving productivity in sheep, according to the preceding claims, characterized in that, once the existence of the Bone Morphogenetic protein 15 gene allele is detected, formed by the deletion of 17 base pairs in exon 2 of the BMP15 gene from position 459 according to the sequence list shown in SEQ ID No. 1, these females are selected, heterozygous for the same, for their tendency to have multiple deliveries with a very high probability. 5. Procedure for improving productivity in sheep, according to the preceding claims, characterized in that, in case of homozygous presence of the Bone Morphogenetic protein 15 gene allele, formed by the deletion of 17 base pairs in exon 2 of the gene BMP15 from position 459 according to the sequence list shown in SEQ ID No. 1, these females are discarded for infertility. 6. Use of the procedure expressed in any of the preceding claims in the improvement of prolifility and the detection of infertility in the Aragonese Rasa sheep.