In the present work, the technique of the tetra-primer ARMS-PCR (T-ARMS-PCR) was successfully applied to type a total of five known mutation points (G1, G4, G6, G7, and G8) of the GDF9 gene that had been associated with fecundity traits in sheep and goats. Also, in previous studies on Indian prolific black Bengal goats  and prolife Garole sheep , the T-ARMS-PCR method was successfully utilized for a description of the same five mutation points (G1, G4, G6, G7, and G8) of GDF9 gene. This technique is considered to be an efficient procedure for genotyping single nucleotide polymorphisms (SNPs) . It can achieve and able to produce many gene markers (SNPs) in a single PCR without prior sequence knowledge of the genomes of interest, where the mutant and wild-type alleles as well as the control fragment can be amplified in a single tube PCR reaction [24, 36]. This because the designing of primers in this technique has been made to amplify fragments of different sizes for each allele band. Therefore, the size of PCR product with different length can easily be separated by simple agarose gel electrophoresis [1, 24, 28, 36]. So, T-ARMS-PCR is an economical method for SNP scoring and a very useful tool for large-scale SNP analysis [27, 36]. In the present study, T-ARMS-PCR analysis clarified genetic polymorphisms of the GDF9 gene in Egyptian sheep and goats, where polymorphic types including heterozygous and wild-type homozygous genotypes were together observed in sheep at G1, G4, and G6 mutation points and goat at G4 and G7 mutation points in mothers. Also, the genetic polymorphisms were detected in young females at age of sexual maturation (FSH) at G1 and G4 of sheep breeds and at G4 of goat breeds. Similarly, in several studies on sheep and goats, the GDF9 gene was found to display abundant genetic polymorphisms: Concerning sheep, in Cambridge and Belclare sheep breeds, Hanrahan, et al.  and Khodabakhshzadeh et al.  detected by PCR-SSCP, eight variants (G1 to G8) of GDF9 gene. Also, in four sheep breeds including Small Tail Han. White Suffolk, Texel, and Tibetan sheep, Chang et al.  determined by PCR-SSCP, G2 mutation of GDF9 gene. Moreover, in five sheep breeds including Tail Han, Poll Dorest, Suffolk, German Mutton Merino, and Chinese Merino, Chen et al.  identified by PCR-SSCP, G4 mutation of GDF9 gene. In Iran, Barzegari et al.  revealed G1 mutation of GDF9 gene in Moghani and Ghezel sheep breeds. In India, Polley et al.  also observed G1 mutation of the GDF9 gene in the Garole sheep breed. Polley et al.  clarified five mutation points (G1, G4, G6, G7, and G8) of the GDF9 gene by using the T-ARMS-PCR technique in prolific Garole sheep. Chu et al. [7, 9] displayed G3 mutation of GDF9 gene in Small Tail Han and Dorset sheep. Concerning goats, Wu et al.  and Feng et al.  reported three mutations, c. 423G ≥ A, c.959A ≥ C, and c. 1189C ≥ A of the GDF9 gene in Jining Grey, Liaoning Cashmere, and Boer goats. Zhang et al.  detected one mutation (c.959A > C) in Yangtse River Delta White and Huanghuai goats. Moreover, Chu et al. [7, 9] identified two mutations (c.183A > C and c.336C > T) with three genotypes AA, AB, and BB in five goat breeds including high prolificacy breed (Jining Grey “JG”) and low prolificacy breeds (Boer “BO,” Wendeng Dairy “WD,” Liaoning cashmere Lc and Beijing native goats “BN”).
The effect of variants of GDF9 gene on reproduction (or fecundity traits) in sheep and goats
The present results revealed that the heterozygous genotype (AG) at G4 mutation point of sheep mothers and G4 and G7 mutation points of goat mothers was discriminated with high rates of fecundity traits as compared to wild type homozygous genotype (AA in G4 or GG in G7), where the best findings of MNL and MNTP were identified of heterozygous mothers. Similarly in a previous study, Hanrahan et al.  observed that heterozygous of G8 mutation point of GDF9 gene was associated with increased prolificacy in Belclare and Cambridge sheep. Their results found that heterozygous carrier mothers exhibited one to two additional ovulations when compared to non-carrier, where in Belclare ewes, the ovulation rates of heterozygous mutation and wild type were 2.67 ± 0.89 and 1.92 ± 0.28, respectively. Also, in Cambridge ewes, the ovulation rates of heterozygous mutation and wild type were 4.28 ± 0.31 and 2.27 ± 0.49, respectively.
In Guizhou white goats, Du et al.  detected heterozygous genotype (g.1189G > A mutation) of GDF9 gene in eight of 33 high prolificacy mothers, where these eight mothers were found to give 3 kids per litter rather than in 112 low prolificacy goats with wild type genotype. Also, in Guizhou black goats, Huang et al.  revealed a heterozygous genotype (c.1133C < T mutation) of the GDF9 gene that could increase the prolificacy in goats. Their results pointed that the two heterozygous mothers were found to give 3 kids per litter as compared to 12 of low prolificacy goats with wild-type genotype. In Thoka sheep, Nicol et al.  pointed in Fec TT mutation of GDF9 gene that mothers with heterozygous genotype produced 0.68 more lambs per ewe lambing than wild type animals. Moreover, in Small Tail Han Sheep, Chu et al. [7, 9] determined two genotypes, heterozygous (CD) and wild type (CC) of the GDF9 gene, and discovered that the mothers with heterozygous genotype (CD) had 0.77 (P < 0.05) lambs more than the mothers with wild-type homozygote genotype (CC). Our findings were also similar with that of Chu et al. [7, 9] who detected in Jining Grey goat does, genetic polymorphisms of the GDF9 gene and revealed that the mothers with heterozygous genotype (AB) had 0.56 (P < 0.01) kids more than those with wild type homozygous genotype (BB). Also, Feng et al.  observed in Jining Grey goats that heterozygous genotype (AC) of the GDF9 gene had been associated with high litter size in relation to wild-type genotype (AA), where their results proved that the mothers with heterozygous genotype (AC) had 0.63 (P < 0.01) kids more than mothers with wild-type homozygous genotype (AA). In a previous study on Moghani and Ghezel sheep, Barzegari et al.  found the obvious major effect of G1 mutation heterozygous of GDF9 gene on fecundity traits as compared to wild type or homozygous genotypes. Their findings clarified that seven ewes out of thirteen (53.8%) with G1 heterozygous gave twin birth, while five of 79 ewes (6.3) with wild-type gave twin birth as well as four ewes with homozygous genotype were observed to be fertile and all gave single birth.
The present results of T-ARMS-PCR analysis observed that the sheep mothers with heterozygous genotype (AG) of G1 and G6 mutation points had given a reduction of MNL and MNTP as compared to mothers with wild-type homozygous genotype (GG). This reduction was found to be due to the very little number of mothers (n = 2) with heterozygous genotype (AG) as compared to a high number of mothers (n = 81) with wild-type homozygous genotype (GG). However, in approximate similarity, Silva et al.  found a genetic variant of GDF9 in Brazilian Santa Ines sheep gene and detected that the homozygous ewes (EE) were fertile and had increases of ovulation rate (82%), twin pregnancy (44%) and prolificacy (58%) as compared to heterozygous mothers (+ E). Also in a previous study, Chu et al.  detected G3 mutation point of the GDF9 gene in Small Tail Han sheep and found that the litter size in the first and second parity of the mothers with homozygous genotype (AA) were 0.30 (P < 0.05) and 0.77 (P < 0.0001), respectively more than those of mothers with heterozygous (AB) genotype.
Furthermore, in Small Tail Han sheep, Gao  revealed genetic polymorphism of GDF9 gene and clarified that ewes with homozygous genotype (CC) had 0.63 (P< 0.05) lambs more than mothers with heterozygous genotype (CD). Melo et al.  reported in Brazilian Santa Ines sheep, that the mean average number of corpus luteum in the ewes with homozygous genotype (Fec G SI of mutation of GDF9 gene) was more than those of females with heterozygous genotype (2.4 ± 0.2 vs. 1.3 ± 0.1) after estrus synchronization.
Based on the present findings and above discussion, it must be selected the young sheep females (FSH) of heterozygous genotype in G4 mutation point as well as young goat females (FSH) of heterozygous genotype in G4 and G7 mutation points for participating in a successful breeding program, because these young animals will have potential high fecundity traits.