Genetic Diversity of the Melanocortin 4 Receptor (MC4R) Gene and its Association with Slaughter Traits in the Landes Goose

Melanocortin 4 receptor (MC4R) plays a crucial part in regulating feeding behavior in humans and rodents. We detected two single nucleotide polymorphisms (SNPs; c.108G → A and c.627C → T) in the goose MC4R gene and genotyped 94 Landes geese for association analysis with several carcass traits. Significant associations (P < 0.05) were obtained for c.108G → A with carcass weight, breast muscle percentage, and leg muscle percentage, and for c.627C → T with body weight, carcass weight, semi-eviscerated weight, and eviscerated weight. We re-constructed haplotypes based on the two SNPs and analyzed diplotypes in association with carcass traits, obtaining significant associations with several of the traits. These results suggest that polymorphisms in the MC4R gene could have effects on carcass traits in Landes geese. More study is required to confirm these results.     

Associations of polymorphism within the GHSR gene with growth traits in Nanyang cattle

GH secretagogue receptor (ghrelin receptor, GHSR) is known to be involved in the control of GH release by mediating the strong stimulatory effect of the endogenous ligand, ghrelin, on GH secretion. Associations between the GHSR gene polymorphism and the growth traits were revealed in Nanyang cattle. The mutations at nt456(G > A) and nt667(C > T) were complete linkage and located in exon 1 of the coding region of the GHSR gene. Least squares analysis revealed a significant statistical effect (P < 0.05) of the GHSR gene different genotypes on body weight and average daily gain at 6 months of age in Nanyang cattle. Individuals with GHSR-MM genotype showed higher body weight and average daily gain than individuals with GHSR-MN genotype.     

SLCO2B1 genetic polymorphisms in a Korean population: pyrosequencing analyses and comprehensive comparison with other populations

SLCO2B1, also known as OATP2B1 (Organic Anion Transporter) or OATP-B or SLC21A9, is an organic anion uptake transporter that is encoded by the SLCO2B1 gene. In this study we assessed the frequencies of SLCO2B1 polymorphisms in a Korean population using newly developed pyrosequencing methods and compared their frequencies with those in other ethnic groups. We developed pyrosequencing methods to identify the following six SLCO2B1 non-synonymous polymorphisms: c.1175C > T (rs1621378), c.1457C > T (rs2306168), c.43C > T (rs56837383), c.935G > A (rs12422149), c.601G > A (rs35199625) and c.644A > T (rs72559740). The allele frequencies of these polymorphisms were analyzed in 227 Korean subjects. The allele frequencies of SLCO2B1 polymorphisms in the population tested were as follows: 0.0 for c.1175C > T, c.43C > T and c.644A > T; 0.2687 for c.1457C > T; 0.4273 for c.935G > A; and 0.0727 for c. 601G > A. Even though the allele frequencies of the c.1175C > T and c.1457C > T polymorphisms were comparable to those in Japanese subjects, the frequencies in this Korean population differed from those in other ethnic groups. The developed pyrosequencing methods are rapid and reliable for detecting non-synonymous SLCO2B1 polymorphisms. Large ethnic differences in the frequency of SLCO2B1 genetic polymorphisms were noted among ethnic groups. The SLCO2B1 polymorphisms at c.1175C > T, c.43C > T and c.644A > T were not found in the Korean population while c.1457C > T, c.935G > A and c.601G > A exhibited mostly higher frequencies in Koreans compared with Finnish, Caucasian and African-American populations.     

Analysis of the associations between polymorphisms in GNAS complex locus and growth, carcass and meat quality traits in pigs

Imprinted genes are interesting candidates for marker assisted selection in farm animals. One of them—GNAS complex locus is engaged in obesity pathogenesis in humans and mice. In our study, we identified new polymorphism in porcine GNAS gene (variable number of CT repeats, accession number: rs196952953) and found that this polymorphism is in linkage disequilibrum with GNAS AM490165:g.324C>T. Statistical analysis (GLM procedure), performed on 552 animals (Large White n = 258 and Landrace n = 269), revealed that deduced haplotypes and GNAS AM490165:g.324C>T are associated with growth performance and a few carcass traits, but not with feed intake. We observed significant additive effects of GNAS AM490165:g.324C>T genotype and haplotype 2 (C/278 bp) on test daily gain (TDG), average daily gain (ADG), number of days on test, age of the slaughter (P < 0.01) and FCR ratio (P < 0.05). Animals with two copies of C/278 haplotype had significantly higher: TDG, ADG, lower feed:gain ratio and faster reached the weight of 100 kg. When carcass traits were considered, significant associations between GNAS AM490165:g.324C>T polymorphism, haplotype 2 (C/278) and weight of ham with and without backfat and skin (WH) (WH2), length of the carcass, height and the width of the loin, meat percentage, weight of the main cuts were identified. The significant dominance effects of GNAS AM490165:g.324C>T polymorphism and haplotype 2 on WH and WH2 were observed (P < 0.05). When the two breeds were analyzed separately significant associations were observed for most of the traits in Landrace while in Large White the same trends were present but the differences were mostly not significant. Among meat quality traits we found significant association between haplotype and IMF content in Landrace (P < 0.03). Our results show for the first time that GNAS complex locus may modulate economically important traits in pigs.     

Developmental changes and effect on intramuscular fat content of H-FABP and A-FABP mRNA expression in pigs

H-FABP and A-FABP genes are considered as candidates for intramuscular fat (IMF) accretion. The aim of the present study was to assess the expression of H-FABP and A-FABP genes in m. longissimus dorsi (LD) and liver tissues of Laiwu and Lulai Black pig populations of different body weight (BW). Eighty-four barrows at different BW (30, 40, 50, 60, 70, 80, 90 and 100 kg, n?=?6 per group) of Laiwu Black pig (no 100 kg group) and Lulai Black pig (no 30 kg group) were used to study the development changes of A-FABP and H-FABP mRNA expression and their relationships to IMF content. The results showed that, in both breeds, the IMF content increased continuously with growing (P?<?0.05). The expression of H-FABP and A-FABP genes also increased with growing in LD tissue (P?<?0.05), and reached a peak at 50 and 70 kg BW in Laiwu and Lulai Black pig, respectively. However, this regularity was not observed in liver tissue in both breeds. A positive correlation was just found between the A-FABP mRNA expression level in LD tissue and IMF content and BW in both breeds (P?<?0.05). In conclusion, the A-FABP gene is strongly related to the development and function of IMF accretion in pigs.     

A novel 18-bp deletion mutation of the AMPD1 gene affects carcass traits in Qinchuan cattle

The objectives of the present study were focused on detecting deletion mutation in bovine AMPD1 gene, and analyzing its effect on body measurement and carcass traits in Qinchuan cattle by using DNA sequencing and agarose electrophoresis methods. The 198-bp PCR products of AMPD1 gene exhibited three genotypes and two alleles were revealed: A and B. The frequencies of genotype AA/AB/BB in Qinchuan populations was 0.7163, 0.2233 and 0.0605. The χ²-test analysis demonstrated that the breed was not in agreement with Hardy–Weinberg equilibrium (P < 0.05). The association of the 18-bp deletion mutation of AMPD1 gene with body measurement and carcass traits of Qinchuan cattle were analyzed. The cattle with AA genotype had slaughter weight and carcass weight than those with genotype AB (P < 0.01 or P < 0.05). These results suggest that the 18-bp deletion mutation may influence the carcass traits in Qinchuan cattle.     

中国美利奴羊H-FABP基因多态性及其与部分肉质性状的相关性

利用聚合酶链式反应-单链构象多态（PCR-SSCP）技术检测中国美利奴羊（Ovis aries var. Merino）心型脂肪酸结合蛋白基因（H-FABP）外显子2的单核苷酸多态性（SNPs）和遗传多态性,分析其与肌内脂肪（IMF）含量、肌纤维直径和肌纤维密度的相互关系,为该品种绵羊的分子标记辅助选择提供理论依据。结果显示,H-FABP基因外显子2有AA、AB和BB 3种基因型,AA型和BB型在778位均发生了C缺失,939位均发生了A→G转换,BB型还在789位发生了T→C转换,该突变导致所编码氨基酸发生了缬氨酸→丙氨酸的替换;BB型为IMF的优势基因型,与AB型相比差异显著（P<0.05）,与AA型相比差异极显著（P<0.01）;BB型对肌纤维直径存在负相关。结果提示,中国美利奴羊H-FABP基因外显子2具有多态性,该基因可能是中国美利奴羊肉质性状的主效基因,或者与控制肉质性状的主效基因相连锁。     

Investigation of four candidate genes (IGF2, JHDM1A, COPB1 and TEF1) for growth rate and backfat thickness traits on SSC2q in Large White pigs

As important quantitative traits, the growth rate and backfat thickness are controlled by multiple genes. The aim of this investigation was to evaluate the effect of the single and multiple SNPs of four candidate genes (IGF2, JHDM1A, COPB1 and TEF-1) on growth rate and backfat thickness. The four candidate genes were mapped on the p arm of SSC 2, and there are several QTLs, such as average daily gain, backfat thickness, an imprinted QTLs affecting muscle mass and fat deposition have been reported in this region. The polymorphisms of these genes were detected using PCR–RFLP methods, mixed procedure was used to analyze the single marker association with the growth and backfat thickness traits, and the gene–gene combination was investigated using multiple-markers analysis. The single marker association analysis indicated that the IGF2 intron-3 g.3072G > A and the substitution g.93G > A of TEF-1 gene were significantly associated with the age at 100 kg (P < 0.05). The JHDM1A 3′UTR g.224C > G, the c.3096C > T polymorphism of COPB1 gene and the substitution g.93G > A of TEF-1 gene were all significantly associated with the backfat at the shoulder (P < 0.05), backfat at the last rib, backfat at the lumbar, and the average backfat thickness, respectively. The multiple-markers analysis indicated that IGF2 and TEF-1 integrated gene networks for the age at 100 kg. Therefore, we can suggest that the polymorphism of IGF2 and TEF-1 gene could be used in marker-assisted selection for the age at 100 kg in Large White pigs.     

Three Novel SNPs in the Coding Region of the Bovine MC3R Gene and Their Associations with Growth Traits

The involvement of melanocortin-3 receptor (MC3R) is well recognized in the regulation of feeding efficiency, body weight, and energy homeostasis. The objective of this study was to investigate the associations between MC3R gene polymorphisms and growth traits. Three novel SNPs (c.24C→T, c.220T→A, c.734G→C) and five haplotypes were identified in 234 Xiangxi cattle. The associations between MC3R gene polymorphisms and growth traits indicated that the individuals with TT and AT genotypes maintained higher body weight than those with the AA genotype at the c.220T→A locus (P < 0.05). The animals with GG and CG genotypes had higher heart girth and body weight than those with the CC genotype at c.734G→C (P < 0.05). The animals with H3H3 and H2H3 haplotype combinations had higher body weight than those with other haplotype combinations (P < 0.05). The results suggest that these SNPs in the MC3R gene might be useful genetic markers for marker-assisted selection and cattle breeding.     

Association between clinical expression and molecular heterogeneity in β-thalassemia Tunisian patients

Beta-thalassemia is the most frequent hereditary blood disorder in Tunisia because of its geographic localization and history. This pathology is characterized by a complex multisystem process with genetic and biochemical interactions. The aim of this work was to establish phenotype/genotype association through studying the distribution and the relationship between β-thalassemia and α-thalassemia mutations and three polymorphic markers: the C → T polymorphism at ?158 of the Gγ gene, the RFLP haplotype and the repeated sequence (AT)_xT_y in the β globin silencer, in two groups of β-thalassemia major and β-thalassemia intermedia (TI) patients. Statistical analysis has shown that moderate expression seen in TI patients was significantly associated to β⁺ ?87 (C → G), ?30 (T → A) and IVSI-6 (T → C) mutations, haplotypes VIII, IX and Nb and to XmnI polymorphism. The regression analysis of combined genotypes (mutation/XmnI/RFLP haplotype) revealed that they contribute to justify 17.1 % of clinical expression diversity (p < 0.05). Among the studied genotypes the XmnI polymorphism seems to be the most determinant modulating factor, followed by the β-thalassemia mutation and RFLP haplotype. Our findings highlight the heterogeneity of molecular background of β-thalassemia that would be responsible of clinical variability.     

Association between <Emphasis Type="Italic">TNF,IL1B,IL6, IL10</Emphasis> and <Emphasis Type="Italic">IFNG</Emphasis> polymorphisms and recurrent miscarriage: a case control study

Background

Approximately half of recurrent miscarriages have unexplained etiology. Recent evidences suggest that cytokines are important determinants in pregnancy maintenance and as such, cytokine gene polymorphisms, which can affect cytokine production and/or functionality, could play a role in the disorder. Thus, we aimed to investigate the association of selected cytokine gene polymorphisms with risk of recurrent miscarriage among Chinese.

Methods

TNF -238G > A, TNF -308G > A, IL1B -511 T > C, IL1B 3954C > T, IL6 -174G > C, IL6 -634C > G, IL10 -1082A > G and IFNG 874A > T polymorphisms were genotyped on 775 women with idiopathic recurrent miscarriage and 805 healthy parous control women. Logistic regression analysis was performed to determine the odds ratios (ORs) of the association between the polymorphisms and recurrent miscarriage risk.

Results

Among the eight polymorphisms studied, only the IL1B -511 T > C and IL6 -634C > G polymorphisms showed statistically significant associations with recurrent miscarriage risk. For the former, a significantly increased risk of recurrent miscarriage was observed for the mutant (CC) genotype (OR: 1.377; 95% CI: 1.039–1.824; P?=?0.026). However, for the IL6 -634C?>?G polymorphism, a decreased recurrent miscarriage risk was observed for the heterozygous (CG) genotype (OR: 0.614; 95% CI: 0.493–0.765; P < 0.001) and the mutant (GG) genotype (OR: 0.414; 95% CI: 0.251–0.684; P?=?0.001).

Conclusions

The IL1B -511 T > C polymorphism may serve as important risk factor for recurrent miscarriage while the IL6 -634C > G polymorphism may protect against the risk of recurrent miscarriage.

     

Physical,chemical and organoleptic properties of ram lamb carcasses from four fat-tailed genotypes

Physical, chemical and organoleptic characteristics of 20 ram lambs representing four fat-tailed genotypes Awassi, Red Karaman, Tushin and Awassi×Tushin were compared. After slaughter, carcasses were chilled at 5°C for 24 h, graded and evaluated. Then, the carcasses were dissected into wholesale cuts and longissimus dorsi (LD) and semimembranosus (SM) muscles were excised. The muscles were subjected to moisture, protein, fat and ash analyses and they were cooked to evaluate palatability. Cold carcass weight of RK and A×T lambs were heavier (P<0.01) than those of A and T lambs. A×T crossbreds had higher (P<0.05) cold dressing percentage and larger LD area than T lambs. However, the influence of genotype on the proportion of boneless retail cuts and marbling were not significant. Most of the proportions of wholesale cuts except for sirloin and flank were not affected by the genotype. The sensory panel found the meat from all genotypes acceptable (tenderness, juiciness, flavor and acceptability). Chemical composition of meat from the different genotype was not significantly different. The study reveals generally insignificant differences in chemical, physical and organoleptic properties between fat-tailed lambs with different genetic backgrounds.     

Associations of A-FABP and H-FABP Markers with the Content of Intramuscular Fat in Beijing-You Chicken

This study has assessed the association of single nucleotide polymorphisms (SNP) identified in the adipocyte fatty acid binding protein (A-FABP) and heart-type fatty acid binding protein (H-FABP) genes with the content of intramuscular fat (IMF) in a population of male Beijing-You chickens. A previously described SNP in the chicken A-FABP gene had a significant (P < 0.05) effect on IMF content. Chickens inheriting the homozygous BB genotype at A-FABP had a significantly higher content of IMF in thigh muscles and breast muscles than did those inheriting the AA and AB genotypes. A novel SNP, identified here, in the H-FABP gene was also significantly (P < 0.05) associated with IMF content in thigh and breast muscle. Chickens inheriting the genotypes of DD and CD had much higher content of IMF than those inheriting the homozygous genotype of CC. Markers at the A-FABP and H-FABP genes were associated with IMF content in the studied population. Chickens inheriting the BB genotype at A-FABP, along with the CD genotype at H-FABP, produced muscles with a much higher content of IMF when compared with all other genotypes. A weak interaction between A-FABP and H-FABP was detected (P < 0.09) for IMF content in the tested population. The statistical significance of interaction is tentative because of the limited number of observations for some genotypic combinations. Markers identified within the A-FABP and H-FABP genes are suitable for future use in identifying chickens with the genetic potential to produce more desirable muscle with higher IMF content, at least in the population of Beijing-You male chickens.     

Ovine insulin induced-gene-2: molecular characterization,polymorphisms and association with milk traits

The aim was to characterize the INSIG-2 gene in Sarda sheep and to highlight associations between polymorphisms and milk traits. Two-hundred ewes, in their third or fourth lactation who lambed a single lamb between 20th and 30th of November, were chosen. Monthly individual milk yield was recorded and from each ewe a sample of milk was taken to analyze fat and protein content. PCR–RFLP and DNA sequencing were carried out to detect polymorphisms. Five exons have been characterized and five mutations have been found G88A, 436TCAGdel, A471G, C1071T and T1737G all in the intronic regions. The ovine sequence and related variations were deposited in GenBank with accession number JX843812.1. The animals carrying AA genotype at position 88 showed a lower milk fat concentration than those with the AG or GG genotype (P < 0.05). A lower milk fat concentration was registered also in the animals with the TCAG deletion in position 436 (P < 0.05) and in the animals carrying AA genotype at position 471 compared to those with the AG or GG genotype (P < 0.05). Moreover, the animals carrying CC genotype at position 1071 had a greater milk yield than those with CT or TT genotype (P < 0.05) while ewes with TT genotype showed a higher milk protein concentration compared to the others (P < 0.05). A total of 11 haplotypes were detected but no significant associations with milk traits were found. In conclusion for the first time the complete coding sequence of INSIG-2 gene and its association with milk trait has been reported in this study.     

Effect of metabolic syndrome risk factors and MMP-2 genetic variations on circulating MMP-2 levels in childhood obesity

Matrix metalloproteinase-2 is involved in the development of the adipose tissue, and associated with cardiovascular diseases. Metabolic risk factors (MRFs) and functional polymorphisms in the MMP-2 gene may affect its expression and activity. We investigated whether traditional MRFs and two MMP-2 gene polymorphisms (C^?1306T; rs243865, and C^?735T; rs2285053) affect circulating MMP-2 levels in children and adolescents, and whether MMP-2 polymorphisms and/or haplotype are associated with susceptibility to childhood obesity. We studied 114 healthy controls, 43 obese, and 83 obese with ≥3 MRFs children and adolescents. Genotypes were determined by Taqman allele discrimination assay and real-time PCR. Plasma MMP-2 was measured using zymography. We found positive correlations between MMP-2 concentrations and mean blood pressure in all children and adolescents group (r = 0.132; P < 0.05) and in obese children and adolescents (r = 0.247; P < 0.01). We found that the CC genotype for the C^?1306T polymorphism was more common in subjects with higher MMP-2 concentrations in controls (P = 0.003) and in the obese group (P = 0.013). The CT genotype (OR = 0.40; P < 0.01) and the T allele (OR = 0.48; P < 0.01) for the C^?735T polymorphism were less common in obese children and adolescents than in controls. The haplotypes distribution did not show significant differences between control and obese (P > 0.05). Ours findings show that blood pressure is associated with circulating MMP-2 concentrations, and that the CC genotype for the C^?1306T polymorphism was more common subjects (controls and obese) with higher MMP-2 concentrations, whereas the CT genotype and the T allele for the C^?735T polymorphism are less common in obesity.     

Maternal backfat depth in gestating sows has a greater influence on offspring growth and carcass lean yield than maternal feed allocation during gestation

A commercial pig spends nearly half of its life in utero and its nutrition during this time can influence birth weight and postnatal growth. We hypothesised that postnatal growth is increased in pigs raised by sows with a high backfat depth and high level of energy intake during gestation compared with sows with a low backfat depth and low level of energy intake during gestation. This was tested in a 2×3 factorial design experiment with 2 factors for gilt backfat depth (Thin and Fat) and 3 factors for gestation feed allowance (Restricted, Control and High). Between d 25 and d 90 of gestation, Thin gilts (n=68; 12±0.6 mm P2 backfat) and Fat gilts (n=72; 19±0.6 mm P2 backfat) were randomly allocated, as individuals, to a gestation diet (6.19 g/kg lysine, 13.0 MJ DE/kg) at the following feed allowances: 1.8 kg/day (Restricted); 2.5 kg/day (Control) and 3.5 kg/day (High). For the remainder of gestation and during lactation all gilts were treated similarly. At weaning (day 28), 155 piglets were sacrificed and 272 were individually housed and followed through to slaughter (day 158). At day 80 of gestation, fasted Thin Restricted gilts had lower serum IGF-1 concentrations than Thin High or Thin Control fed gilts (P<0.001). Pigs born from Fat gilts had greater backfat depths (P<0.05), a lower lean meat yield (P<0.05) and were heavier (P<0.05) at slaughter than pigs born from Thin gilts. Gilt gestation feed allowance had only transitory effects on average daily gain and feed conversion efficiency and had no effect on pig weight at slaughter (P>0.05) or lean meat yield (P>0.05). In conclusion, gilts with a backfat depth of ~19 mm at insemination produced pigs that were heavier and fatter at ~158 days of age than those born from gilts with ~12 mm backfat depth at insemination. Maternal body condition during gestation had a more predominant influence on growth parameters of the offspring, such as weight at slaughter and backfat depth, than did feed level during gestation.     

Relationships of LDLR genetic polymorphisms with cerebral infarction: a meta-analysis

This meta-analysis was undertaken to identify the relationships between genetic polymorphisms in the LDLR gene and the risk of cerebral infarction. The Web of Science (1945–2013), the Cochrane Library Database (Issue 12, 2013), PubMed (1966–2013), EMBASE (1980–2013), CINAHL (1982–2013) and the Chinese Biomedical Database (CBM) (1982–2013) were searched for relevant articles without language restrictions. Meta-analysis was conducted using the STATA 12.0 software. Crude odds ratios (OR) with their corresponding 95 % confidence interval (CI) were calculated. Eight case–control studies with a total of 4,655 patients with cerebral infarction and 15,920 healthy control subjects were included in our meta-analysis. Five common polymorphisms in the LDLR gene were evaluated, including rs11669576 A > T, rs1433099 C > T, rs5925 C > T, rs688 C > T, rs1122608 T > G in the LDLR gene. The results of this meta-analysis revealed that cerebral infarction patients had a higher frequency of LDLR genetic polymorphisms than that of healthy controls (allele model: OR 1.17, 95 % CI 1.05–1.30, P = 0.004; dominant model: OR 1.18, 95 % CI 1.05–1.33, P = 0.007; homozygous model: OR 1.50, 95 % CI 1.03–2.16, P = 0.032; respectively), especially for the rs11669576 A > T, rs1433099 C > T and rs5925 C > T polymorphisms. Among different ethnic subgroups, the results demonstrated positive correlations between LDLR genetic polymorphisms and an increased risk of cerebral infarction among both Asians and Caucasians under the allele and dominant models (all P < 0.05). Our findings indicate that LDLR genetic polymorphisms may be strongly involved in the pathogenesis of cerebral infarction, especially the rs11669576 A > T, rs1433099 C > T, rs5925 C > T polymorphisms.     

Effects of age on slaughter performance and meat quality of Binlangjang male buffalo

Twelve representative buffalo were selected from 22 suckling calves, 41 weaned calves, 57 reserve bulls and 20 adult bulls for slaughter. The study aims to assess the effect of age on dressing percentage, meat percentage and carcass meat yield and physico-chemical properties of longissimus dorsi and biceps femoris, and to evaluate the correlation between live weight and marbling, backfat thickness, rib eye area. The results showed that the slaughter performance and meat quality of Binlangjang male buffalo showed an obvious change with age. The dressing percentage decreased from 54.93% to 51.22% with the increase of age, while meat percentage and carcass meat yield increased gradually with age, which were 34.58–38.59%, 62.95–75.34%; Marbling, backfat thickness and rib eye area increased with age, and there was significant difference between the situation before 3 months and after 12 months of age (P < 0.05). The moisture content was maximum at birth, which then gradually decreased, but the difference was insignificant (P > 0.05). The levels of fat, protein, cholesterol and inosine acid were significantly different before 3 months of age from those after 12 months (P < 0.05). Cholesterol content was negatively correlated with age, the minimum was 80.25 mg/100 g; Inosine acid content increased with age, reaching 133.11 mg/100 g. Marbling, backfat thickness, rib eye area had a high correlation with live weight, with correlation coefficients respectively at 0.9096, 0.9291, 0.9551. Based on the prediction model of live weight, Buffaloes was suitable for slaughtering for superior slaughter performance and meat quality after 24 months of age.