Genes Involved Litter Size in Olkuska Sheep

Major genes increasing litter size were identified in certain sheep breeds. These genes include BMPR‐1B , BMP15 , GDF9 and B4GALNT2 , FecX2 . Polish Olkuska sheep is a high‐ fecundity sheep breed; while some animals might give birth to just one or two lambs, there are Olkuska ewes which have six or even seven lambs/lambing. Fertility of this breed is caused by mutation in the major gene FecX O ( BMP15 gene), but analysis of polymorphism at the locus GDF9 revealed presence of four polymorphisms: G447A (L159L), A978G (G326G), G994A (V332I) and G1111A (V371M). Substitutions V371 and V332I are missense mutations found in the sequence encoding active GDF9 protein. V371 polymorphism has also an effect on litter size in Olkuska breed ewes. Study of genes associated with litter size in Olkuska sheep is of high importance, as they could be used in breeding programmes as selection markers for increasing production efficiency.


Introduction
Currently, more than 1200 sheep breeds differing in many production features, including fertility, are known globally. Ewes usually give birth to one or two lambs/litter. However, there are certain highly prolific breeds-such as inter alia Cambridge, Thoka, Javanese, Belclare, Lacaune, Woodland, Booroola, Aragonesa, Romney (Inverdale and Hanna), Garole (Bengal), Belle-ile, small-tailed Han, Hu and Kendrapada-whose litter size ranges from three to six lambs. Genetically conditioned differences in number of maturing and ovulating follicles in different breeds of sheep have been researched already since 1980 [1], when an attempt was made to explain the genetic basis for multiple births within a litter in various breeds around the world. It has been shown that sheep fertility depending on breed may be determined either polygenic or by a major segregating gene named the Fec gene.
Most mutations increasing ovulation levels and the number of lambs born to an ewe were identified in genes encoding proteins belonging to the family of transforming growth factors TGF-β: BMPR1B (bone morphogenetic protein receptor-1B), BMP15 (bone morphogenetic protein 15) and GDF9 (growth differentiation factor 9). In addition, mutations FecL L in Lacaune breed on chromosome 11 and FecX2W (fecundity gene X2) in Woodland sheep were identified. FecX L locus contains two genes IGF2BP1 (insulin-like growth factor 2 mRNA binding protein1) and B4GALNT2 (beta-1, 4-N-acetyl-galactosaminyl transferase 2). It has been shown that B4GALNT2 is responsible for high prolificacy of Lacaune sheep [2]. The mechanism of action of the gene FecX2 on X chromosome in Woodland sheep is not yet exactly understood [3].

FecB mutation in the bone morphogenetic protein receptor 1B (BMPR1-B) gene
Booroola was the first major gene (FecB) significantly influencing level of ovulation and litter size in sheep to be identified [4]. The effect of FecB mutation (Q249R) identified in Booroola Merino breed in the gene of bone morphogenetic protein receptor BMPR1-B on chromosome 6 is an increase in ovulation level and litter size in carriers of one or two copies of the gene FecB. This mutation was also identified in Asian breeds: Garole, Javanese, thin-tail Hu and Han. With regard to level of ovulation, the observed increase is additive in nature, while as concerns litter size-it is partly dominant. In ewes with heterozygous FecB B+ ovulation level increased by 1.3 CL (corpus luteum), while in ewes with FecB BB , the observed increase was up to 3.6 CL. The effect on litter size between homozygous++ and BB was +0.8 [6].
Research results in Indian sheep of Muzaffarnagari breed engendered some discussion, as presence of ewes with FecB gene that still had single births was proven, and share of sheep with genotypes FecB BB and FecB B+ was identified as 3.47 and 41.73%, respectively [9].
In antral follicles of FecB B+ ewes, a decrease in granulosa cell proliferation and premature expression of LH receptors were observed [10]. Appearance of FecB mutation in sheep compared with ewes devoid of mutation is associated with higher number of growing small cavitary (antral) follicles on the ovaries. Both total number of granulosa cells and total secretion of estradiol and inhibins in the ovaries of FecB BB ewes still remain the same as in ewes with FecB ++ genotype. Significant impact of gene FecB on development of ovaries during foetal life is also observed, including, inter alia, a reduced number of oocytes from the 35th to the 50th day of gestation, a smaller number of class 1 follicles from 75th to 90th day of gestation and class 2 follicles (on 135th day). A small number of class 2 follicles were also observed in early neonatal life of FecB BB sheep [10,11]. Significant effect of FecB gene on function of the hypothalamic-pituitary system has been proven as well. It has been found that it may lead to increased secretion of follicle-stimulating hormone (FSH) in FecB BB homozygotes, although it has not been confirmed in all of the animals investigated. No FecB influence on release of luteinizing hormone (LH) has been found [12].

Protein BMP15
BMP15 protein together with growth factor 9 (GDF9) plays an important role in folliculogenesis [13,14]. Its paracrine action affects granulosa cells, theca cells and the oocyte itself [15]. In vitro cultures indicate that recombinant protein BMP15 is a potent stimulator of granulosa cell proliferation, suggesting that both cell growth and their subsequent division and differentiation require BMP15 action [16]. The protein inhibits expression of FSH and LH receptors, as decrease in activity of BMP15 increases cell sensitivity to action of gonadotropins [17]. The conducted experiments showed that BMP15 inhibits expression of StAR protein, responsible for transport of cholesterol-specifically low density lipoprotein (LDL)-into cells. LDL is the substrate for production of steroid hormones. In rats, BMP may modulate the process of steroidogenesis in granulosa cells, modifies biological effects of FSH by inhibition of progesterone-induced FSH secretion, without affecting production of estradiol [16]. BMP15 mRNA transcripts are observed from the primary follicle stage, in secondary, tertiary and dominant follicles stages, ending with atretic antral follicles [18]. In sheep, BMP15 affects proliferation rate and inhibits baseline and FSH-mediated secretion of progesterone by granulosa cells of small antral follicles [11]. Presence of BMP15 mRNA was determined in the oocytes (class 1, 2, 3, 4 and 5 follicles), but it has not been demonstrated in granulosa cells and follicle theca [19].

Polymorphism in the BMP15 gene
In mammals, formation of BMP15 protein is determined by action of a single gene on chromosome X [20]. An encoded sequence with a length of 1179 bp is contained in two exons separated by an intron of 5400 bp in length. mRNA translation produces a peptide built of 393 amino acids, and the resulting mature protein has a length of 125 amino acids [21]. Mutations identified in the BMP15 locus (FecX I , FecX H , FecX L , FecX G , FecX B and FecX R ) result in appearance of the same phenotype in different sheep breeds, and have different molecular basis. Heterozygous ewes give birth to more lambs, while homozygotes with two copies of a gene are infertile. Action of mutations marked FecX O and FecX Gr identified in Olkuska and Grivette breeds differs from the above-mentioned, as in the case of ewes with both one and two copies of the gene, an increase in prolificacy is observed ( Table 1).
Substitution of FecX H detected in Hanna sheep causes introduction of a stop codon in place of glutamine in position 23 of amino acid residue of mature protein. Transition C>T observed in the case of FecX G in Galoway and Cambridge breeds, also introduces a premature stop codon in place of glutamine at position 31 of the polypeptide chain. As concerns carriers of these mutations, heterozygotes show increased levels of ovulation, while homozygotes with two copies of the gene are infertile [17].
A similar phenotype is observed for FecX I mutation, where hydrophobic valine is substituted for asparagine at position 31 of mature protein. This leads to changes in electrostatic potential of the region involved in formation of dimers-this, in turn, interferes with dimerization and consequently leads to the elimination of biological effects of BMP15. Homozygous ewes are infertile due to folliculogenesis being arrested at initial stage of the process [10].
FecX B mutation found in BMP15 gene locus in Belclare sheep is a result of polar serine being substituted for non-polar isoleucine at position 99. This substitution has a significant impact on the ability of a protein to bind to type II membrane receptor; it determines the structure of the ligand-receptor complex with BMP2, BMP7, activin A proteins. This polymorphism abolishes biological activity of BMP15, most likely by prejudicing the binding of protein and receptor [21]. Homozygous ewes are infertile due to hypoplasia of ovaries. Admittedly, presence of antral follicles was observed, but with oocytes of anomalous size, surrounded by a single layer of abnormal granulosa cells. Morphology of ovaries of such sheep is similar to image of ovaries of FecX I Inverdale ewes.
One copy of the allele FecX I (Inverdale) or FecX H (Hanna) results in ovulation level increase of +0.8-1.0 CL, significantly affecting size of the litter by +0.6 lamb. Homozygous FecX II and FecX HH Hanna ewes are sterile; in their small and poorly developed ovaries, follicles are provided with a single layer of granulosa cells [22]. In FecX I+ sheep, follicles were determined to be of a smaller diameter than those produced in ovaries of homozygotes devoid of mutation. However, FecX I+ ewes present a greater number of mature preovulatory dominant follicles of smaller diameter, and therefore do not differ in the level of estradiol and inhibin from nonmutated ones [23]. Preovulatory follicles are also characterized by early maturation, which determines greater secretion of FSH and early formation of LH receptors [12].
Studies by Bodin et al. [24] on prolific sheep of Lacaune breed showed presence of FecX L mutation, leading to substitution of cysteine for tyrosine at position 53 of the BMP15 chain. Just as observed in the case of polymorphisms (FecX I , FecX H , FecX G and FecX B ), also this change in gene structure causes increased level of ovulation in heterozygous ewes and infertility in homozygotes. In vitro studies revealed that the mutation FecX L is responsible for defective secretion of both precursors and active forms of BMP15. It is believed that this may be caused by damage to structure of one of the disulphide bonds whose formation involves cysteine, and that play an important role in stabilizing the structure of BMP15 [24]. In the case of presence of one copy of FecX L in ewes, ovulation level increase of +1.9-2.17 CL is observed [2,25].
Lacaune sheep are, next to Hanna, Inverdale, Cambridge, Belclare and small-tail Han breeds, an example where increase in litter size is determined by the presence of two major genes: FecX L and FecL on autosomal chromosome 11 [2].
In Aragonesa FecX R ewes, a 17 bp deletion results in a frameshift and appearance of a stop codon, even before the coding region of mature protein. The consequence of this mutation is an 85% modification in the propeptide sequence, specifically limitation of its size to 45 amino acids out of 245 present in the original propeptide. Heterozygous ewes are very highly fecund, while the presence of two copies of the mutated gene leads to a blockage of follicular development at initial growth state and female infertility. FecX R+ ewes have an average of 2.66 lamb/ litter compared to flock average of 1.36. Presence of one copy of the gene resulted in litter size increase of +1.3 lamb [26].
A mutation in the gene BMP15 changing the amino acid sequence in the amino acid chain p.T317I was identified in French Grivette sheep. Ewes of FecX GrGr genotype have 2.5 lamb/litter, while FecX ++ and FecX Gr+ give birth to 1.83 and 1.93 lamb in a litter, respectively. Sterilization of homozygotes with the FecX GrGr mutation has not been identified in the studied population [27].
Recent research in phenomenon of high fertility of ewes of the African breed Barbarine (167% annual lamb crop) showed presence of another substitution in the gene encoding BMP15, resulting in A119T mutation in the coding sequence of mature protein. Frequency of the mutated allele in the population of these sheep, or its impact of prolificacy are unknown [28].

Protein GDF9
Growth differentiation factor 9 (GDF9) is another protein belonging to TGF-β family. This peptide regulates development of ovarian follicles in rodents and ruminants (sheep), as well as humans [12,29]. It has been shown that its synthesis, similarly as in the case of BMP15, occurs in the oocyte [30]. Mice lacking functional protein GDF9 (GDF9KO) were infertile, their follicle growth was arrested at primary follicle stage, with granulosa cells layer not properly formed, and changes in the zona pellucida. Oocytes were excessively enlarged and surrounded by a single layer of deformed granulosa cells, or oocyte was not observed in the follicle whatsoever [30,31]. Growth differentiation factor 9 as a multi-functional protein is responsible for follicular growth from its early stage-it initiates and regulates folliculogenesis and oocyte development. It has autocrine effects on oocytes, plays a role in their development and maturation, and paracrine effects on somatic cells, inhibits expression of luteinizing hormone receptor gene and stimulating synthesis of hyaluronic acid [32,33]. GDF9 gene expression in sheep occurs in oocytes, with the presence of the transcript and protein demonstrated in the oocyte during formation of the follicle, in class 1 primary follicles as well as in follicles in the phase of intensive growth. In vitro experiments demonstrated that GDF9 is also found in degraded oocytes and in malformed follicular structures [34,35]. In humans, GDF9 plays an important role in pathogenesis of polycystic ovary syndrome and premature ovarian failure [36].

Polymorphism in the GDF9 gene
The gene encoding protein GDF9 was found to be located in sheep on chromosome 5 [37]. The gene consists of two exons, with length of 397 and 968 bp, respectively. Its total length is 5644 bp, with coding sequence comprised of 1359 nucleotides [35].
Mutation FecG H (G8) occurs within the sequence responsible for coupling a protein to the receptor and is a missense mutation. As a result, synthesized protein exhibits less affinity for the cell surface receptor. Ewes with one copy of the gene with FecG H mutation have higher ovulation level. Early maturation of small secondary follicles, inhibition of their growth and earlier ovulation of a larger number of oocytes were noted [21]. Most likely, early maturation of the developing follicles is associated with inhibition of FSH receptor expression at mRNA level due to the absence of biologically active GDF9 [19]. N337H mutation) had a frequency of 0.55 and ewes with one (AC) and two (CC) copies of the gene constituted 56 (AC) and 27% (CC) of the animals, respectively [43].

Effects of N337H mutation on litter size of Olkuska sheep
Analysis of effects of mutation N337H on litter size of ewes showed a significant impact of polymorphism on prolificacy, which in sheep of genotype FecX ++ was 1.74 ± 0.55 lamb/litter, with 2.47 ± 0.77 and 2.98 ± 1.50 lamb/litter for FecX +O and FecX OO genotypes, respectively [43]. Very similar results showed Demars et al. [27] for genotypes FecX ++ , FecX +O and FecX OO namely, 1.84, 2.46 and 3.05, respectively.
Changes in litter size in subsequent lambings of Olkuska ewes show an increase in fecundity correlated with increasing age of a mother. The maximum size of litter in FecX OO ewes was noted in their third lambing, with ewes giving birth to an average of 3 lambs/litter. However, for mothers with genotype FecX ++ , litter size continued to increase up to their fourth lambing, when the litter size reached 2.25 lambs [43]. Increase in the number of lambs in the first three consecutive lambings, and then subsequent decrease in litter size has been demonstrated in studies on other highly prolific sheep breeds. Liu et al. [44] showed that average litter size for FecB BB homozygotes in small-tail Han sheep was 2.47 in the first lambing of an ewe, and 3.17 for older mothers. Increase in litter size as ewes were aging was also observed in Chinese Hu breed. Carriers of FecB gene bore an average of 1.92 lamb/litter in their first lambing, compared to 2.56 lamb in the third lambing [45]. The number of lambs in a litter was also positively affected by the number of prior lambings, an ewe has undergone in hybrids Garole × Marpura; heterozygous mothers gave birth into 1.51 ± 0.06, 1.55 ± 0.07, 1.70 ± 0.09 lamb/litter in their first, second and third lambing, respectively [46].

Litter size in Olkuska sheep population
In all herds of sheep breeds with a segregating major gene, distribution of litter size similar to the one determined in Olkuska sheep, that is, with high proportion of twin births and large share of triplets and larger litters, was determined. Distribution analysis of litters of Olkuska ewes showed that 29% of FecX OO mothers gave birth to four or more lambs in a litter, including sextuplets and septuplets. In the most numerous group of FecX +O ewes, only 14% of animals showed similar litter size, and among sheep of genotype FecX ++ no litters of such size were observed ( Table 4).
In the case of FecX ++ , the proportion of triplets was also only 11.2% [43]. A much smaller share of quadruplet and lager litters was found in Javanese FecB BB ewes (16%) [8]. The most common litter sizes in Garole FecB B+ sheep were twins, single births and triplets; accounting for, respectively, 65, 21 and 5% of the total [47]. In turn, share of quadruplet and triplet litters in FecB BB ewes of Javanese breed was, 34 and 20%, respectively, with an average litter size of 2.5 lamb/litter [48]. Interestingly, in the flock of Olkuska sheep, for mothers with genotypes FecX OO and FecX +O singleton births were twice less frequent than for FecX ++ ewes (16 vs. 33%). Share of twin births decreased along with appearance of additional alleles with the mutation; among FecX ++ ewes they accounted for 55% of litters, and for genotype FecX OO only for 27%. In the population of Olkuska sheep with average annual lamb production of 218%, the distribution of litters of various sizes was: 21.7% of singletons, 41% of twins, 24.6% of triplets and 12.7% of quadruplets and larger. Thus, the share of litters larger than triplet is as high as 37.1%. In Garole sheep, characterized by slightly lower prolificacy (168-187%), the proportion of mothers with twins, triplets and quadruplets was 65, 21 and 5%, respectively [6]. Similar differences in distribution of litter size were observed in a herd of Chevoit-Thoka sheep [49]. With an average litter size of 2.23 lamb/litter, the authors found similar share of twin litters (56.5%) in this population, but noted a much smaller share of births with quadruplet and larger litters (3.1%). High frequency of twin births, reaching 47% of the studied population, was demonstrated in Thoka-Chevoit sheep; this breed was also characterized by a high share of singleton births (35%) [50]. A much smaller share of multiple births in comparison with data collected for Olkuska sheep was demonstrated in Aragonese breed with 120-150% fecundity: 66.4% of singleton litters, 28.4% of twin ones, but only 1.9% of triplets. It should be noted that among more than 2000 ewes studied, only three gave birth to quadruplets and only one to quintuplets. For sheep lacking the FecX R mutation, only singleton births were noted [26].

Effect of mutation N337H on litter size
In studies on effects of mutation N337H (FecX O ) on litter size, it was shown that the effect on ovulation levels in O+ and OO ewes was an increase of +2 and +3.3 CL [27]. Effect of the O+ copy of the gene was measured at +0.73 lamb, with the effect of two copies of the gene estimated at +1.07 lamb/litter. Analysing litter size over three first lambings of an ewe, the effect on O+ ewes was +0.62 lamb/litter, and +1.07 lamb for the OO genotype ( Table 5) [43].

Mutations in the gene GDF9
Identification of variations in the gene sequence of GDF9 revealed presence of point mutations G3, G5, G6 and G7-detected in 2004 in Belclare and Cambridge sheep by a team led by Hanrahan et al. [21]-also in Olkuska sheep ( Table 6).
Presence of FecG H (G8) mutation was excluded [43]. Thus, the Polish Olkuska breed can be classified as one of the few breeds in the world, where presence of polymorphisms has been confirmed both in the gene BMP15 and the autosomal gene. Next to the N337H mutation in BMP15 gene, missense mutations V371M (G7) and V332I (G6) have been identified in the region encoding mature protein GDF9. It should be noted that in Olkuska sheep, the allele with mutation V371M occurs with very low frequency of 0.06, but GA heterozygotes demonstrate high annual lamb production of 346%, with the same for GG ewes at 236% ( Table 7) [43].
Analysis of litter size distribution in sheep with GG and GA genotypes in the locus V371M (G7) revealed that the most frequently occurring litter size in GG ewes was twins (40.6%), followed by singletons (20.8%). Litters of quadruplets and larger accounted for 11.8% of all births. Among mothers with identified one allele with the GA mutation, the share of twin litters was twice smaller (20%), while litters with four and more lambs accounted for almost 50%. The share of single births was only 8.3% [43].
Also mutation V332I (G6) was found to be present in Olkuska sheep; with two alleles (G and A) and three genotypes (GG, AG and AA) found ( Table 7).
The G allele appeared with a very high frequency of 0.84, and ewes of genotype GG accounted for 70% of all animals. Mothers with the GG genotype did not differ in average litter size from GA sheep, both over total duration of their productive life (2.24 ± 0.87 vs. 2.13 ± 0.91 lamb), and in the first three lambings (2.39 ± 0.90 vs. 2.30 ± 0.87) [43].

Effect of mutation V371M (G7) on litter size
Ewes with one allele with V371M substitution showed an increase in litter size of +0.55 lamb, while those with the V332I mutation showed a decrease of 0.18 lamb/litter [43]. Thus, only in the presence of the A allele, the mutation V371M resulted in an increase in litter size. Mutations described earlier by Hanrahan et al. [21], namely: G3, G4, G5 and G6 were also detected in the case of Olkuska breed. However, impact of these mutations on prolificacy of ewes has not been studied.
In ewes of the Brazilian breed Santa Ines, a mutation that does not cause infertility has been identified in gene GDF9. Melo  Genes Involved Litter Size in Olkuska Sheep http://dx.doi.org/10.5772/intechopen.69205 was 82% higher than that observed for the FecG ++ genotype. No statistically significant difference was identified between FecG +E heterozygotes and FecG ++ ewes (1.34 ± 0.08 vs. 1.22 ± 0.11). As concerns litter size distribution, share of twin litters for ewes with the identified three genotypes was 44 and 12% for FecG EE and FecG +E ewes, respectively, while no twin litters were noted for FecG ++ sheep [41].
Presence of the mutation G1 was also confirmed in Iranian Moghani and Ghezel breeds. Presence of three possible genotypes was identified, and all the ewes were fertile. Infertility was found in only one sheep, carrying also an additional copy of the gene FecX G in gene BMP15. Ewes with heterozygous genotype were more prolific compared to those with wild sheep genotype. Share of twin litters was small (6.3%) also among animals with the wild sheep genotype, while among heterozygotes twin births amounted to as much as 53.8%. Four homozygotic mothers with two copies of the gene G1 were fertile, but gave birth to singletons [56]. Mutation G1 was als identified in Indian Garole sheep, and frequency of the wild allele and of that carrying G1 mutation was, G-0.82 and A-0.18, respectively, with respective genotype frequency of 0.64 for GG and 0.36 for AG ewes [57]. Also in this case, presence of genotype with two copies of the AA gene was not detected. Mutation in the gene GDF9 was accompanied by the presence of FecB gene. In Chinese breeds, similar to the case of Olkuska sheep, several mutations in several different genes have been identified. In addition to gene FecB, also presence of mutations G2 (C471T) [58], and G3 (G477A) was revealed. Entirely new mutations G729T (Q243H) and T692C [59] were identified. The authors, however, found no impact of G3 on the number of lambs bore by ewes. While identifying mutation G729T outside the region coding mature protein, the researchers have found the allele T (0.091) as well as genotypes GG and GT (respective frequency of 0.817 and 0.183). In GT ewes, estimated litter size was 2.88 ± 0.19, while for GG sheep, it was only 2.11 ± 0.11. Effect on litter size was also recorded for the T692C mutation, where litters of CC mothers were larger by 0.63 lamb than litters of CG ewes [59]. In Hu sheep, A154G mutation at position 51 of the amino acid located outside the region coding mature protein GDF9 (Asn51Asp) was identified [60].

Effect of N337H and V371M (G7) mutations on litter size
Determining effect of simultaneous presence of N337H (A1009C) and V371M (G7) mutations on fertility of Olkuska breed ewes showed that the largest number of lambs was born to FecX OO mothers that were carriers of V371M mutation (3.32 ± 0.26 lamb/litter) ( Table 8).
Effect of presence of both the allele N337H and V371M in ewes was similar to how presence of two copies of gene N337H affected the sheep, and amounted to +0.92 lambs/litter [43].
Studies conducted so far on interaction and potential interdependencies between different mutations within the loci of genes encoding transforming growth factors TGF-β have shown that presence of one copy of the gene with a mutation in the locus of BMP15 led to an increase in litter size; the result was sterilization in the case of homozygous genotype with two copies of the gene. This issue has thus far been researched only within a very narrow range. Presence of several alleles of various genes in the same animal was demonstrated by Hanrahan et al. [21] and Davis et al. [42], sheep carrying a copy of both FecX B and FecX G or FecX H and FecX I were infertile. Sheep carrying one copy of any of the above mutations on chromosome X and the mutation FecB (BMPR-1B) had fully functional ovaries and ovulation levels higher than observed when these factors were occurring independently. The effect of simultaneous presence of one copy of FecX in BMP15 and one copy of FecG H (GDF9) was varied. Most observations showed that the effect of these two genes was additive, but in some cases the demonstrated impact of GDF9 was weakened when the FecX mutation was present alongside it. Estimated effect on level of ovulation due to the presence of FecX G in Belclare and Cambridge sheep amounted to 0.77 and 1.18 CL, respectively, while presence of FecX B in Belclare sheep resulted in 2.38 CL. The effect of carrying one copy of FecG H in Belclare and Cambridge ewes was much higher, and amounted to 1.79 and 2.35 CL, respectively. Generally, it can be assumed that the average stimulating effect on level of ovulation in case of mutations FecX G , FecX B and FecG H was 0.70, 0.97 and 1.39 CL, respectively. Simultaneous appearance of different copies of a gene with a mutation in one animal resulted in a much greater increase in the observed number of corpora lutea in the ovaries. In sheep with genotypes FecG H /FecX G and FecG H /FecX B , the number of ovulatory follicles was 5.8 and 6.09, respectively, while for carriers of only one copy of the gene FecX G it was estimated at 2.69 CL, with 3.26 CL for FecX B ewes, and 2.67 CL for FecG H genotype [21]. Presence of FecX I and FecB genes in ewes resulted in high level of ovulation (4.4 CL), suggesting multiplicative effect of these mutations resulting from interactions between the genes BMP15 and BMPR-1B [20].

Effect of N337H, G6 and G7 mutations on body weight
Mutations N337H, G6 and G7 detected in Olkuska sheep have no effect on body weight at 2, 28 and 56 days after birth [43]. These results are consistent with observations conducted for sheep breeds with the major gene FecB, such as hybrids Rambouillet × Booroola [61]. No effect of FecB on body weight at birth and weaning was identified for this population. Also, Kleemann et al. [62] and Abella et al. [63] found no impact of FecB on body weight and daily gains in the initial period of a lamb's life. Visscher et al. [64] identified the weak effect of the gene FecB on initial and final body weight of lambs from 7 to 12 weeks of age in Booroola × Texel hybrids. In contrast to these observations, in nine breeds of Chinese sheep with FecB gene, its significant impact on litter size, body weight and body size has  Table 8. Estimated effect of N337H mutation in the BMP15 gene and V371M mutation in the GDF9 gene on number of lambs born to Olkuska ewes (combined genotype) [43].
been identified. On day 90 after birth, the body weight of lambs with genotypes FecB BB and FecB B+ was higher than those of lambs with the genotype FecB ++ . However, these differences were age dependent and have not been detected with respect to weight measured at 2 and 120 days of age [45]. Gootwine et al. [65] have demonstrated lower body weight in lambs that were carriers of FecB B+ gene compared to FecB ++ lambs that did not carry the mutation. Body weight at birth for lambs with various genotypes, but from the same birth type differed significantly; FecB BB lambs compared to FecB ++ and FecB B+ ones had lower body weight. Comparing weight of Assaf breed sheep with different genotypes, lower body weight was identified in the case of lambs with two copies of the gene FecB [66]. Studies on Garole-Malpura sheep aimed at assessing the impact of FecB genotype on body weight have shown that this gene significantly affects body weight at birth and at 12 months of age, as well as weight gains up to 3 months of life of a lamb. There was no effect on body weight at 3 and 9 months of age [46].

G617A polymorphism in inhibin-α gene (INHA)
Proteins encoded by the genes GDF9 and BMP15, necessary for an oocyte to gain cytoplasmic maturity, are a group of more than 35 proteins in the transforming growth factor β family. Inhibin A and B is one of these proteins. A missense-type substitution 617G > A-not causing amino acid substitution of proline-was identified in Olkuska sheep in exon 2 of the inhibin-α gene (INHA) [43]. The highest frequency was found for allele G (0.86) and genotype GG (0.71), and no ewes with genotype AA were identified. No effects of the mutation on litter size were determined; while the difference between mothers of GG and AG genotypes did indeed amount to +0.3 lamb/litter, it was not statistically significant. The impact of the mutation was dependent on the age of the animals, and for an ewe's first litter was only +0.1 lamb. This result confirms conclusions from other studies, which demonstrated the association among INHA, INHBA and INHBB and litter size of sheep [67]. Influence of variation in the inhibin gene on litter size of Merino and Friesian sheep at the level of, respectively, +0.04 and +0.09 lamb had been demonstrated previously [68]. The effect of TaqI/INHA polymorphism on the number of offspring has also been confirmed for Merino, East-Friesian and Romanowska breeds [69]. After comparing frequency of allele A in sheep with varying prolificacy, the above authors found that with increasing fertility, an increase in the frequency of allele A could be observed as well. Ovis ammon and Ovis vignei presented only allele B, while high frequency of allele A (0.65) has been determined in romanowska sheep. Studies in fecund small-tail Han and Hu breeds, as well as in low fecundity breeds Dorset, Texel and German Mountain Merino showed presence of polymorphism in the locus of inhibin βB A276G, in the untranslated region 3′-UTR, only in Hu sheep. Presence of genotypes AA, AB and BB with respective frequencies of 0.636, 0.046 and 0.318, as well as alleles A (0.659) and B (0.341), has been identified. Evaluation of genotype influence on litter size of Hu ewes showed that sheep with genotype BB gave birth to +0.58 lambs more than sheep with AA genotype [59].

Conclusions
To summarize, Olkuska sheep are among the few breeds with significant polymorphisms in genes coding proteins of the TDF-beta family. Fecundity of this breed is determined not only by the presence of major gene FecXO (N337H,1009 > C) in BMP15 locus, but also by the presence of a missense mutation V371M (1111G>A) in the gene locus of GDF9. Considerable polymorphism in GDF9 locus -where three other, low-frequency mutations G3, G5 and G6 can be found-is particularly noteworthy. Ewes of Olkuska breed are highly prolific, both in the case of carriers as well as FecX OO animals, in contrast to sterilization observed in the majority of known homozygotes with mutations in the BMP15 gene. Research in genes determining prolificacy is an important factor for increasing fecundity of ewes and thus profitability of sheep production as well.