Callipyge Genotypes Research Articles

Genotypic characterization of meat-type lambs expressing the callipyge gene in Turkey: II. Effect on body indexes

This study is set out to examine the relationship between the callipyge (CLPG) gene, attributed to sheep affecting growth and meat quality, and body indexes of meat-type sheep breeds to assess its feasibility in using it genomic selection programs. For this purpose, blood samples were collected from 177 lambs (66 male, 111 female) of five different sheep breeds (Kivircik, Karacabey Merino, Ramlic, German Black-Head Mutton × Kivircik, Hampshire Down × Merino) to identify M (mutant) and N (normal) alleles and genotypes. Live weight (LW) and morphometric measurements were taken on the 90th, 180th, and 360th day of the study. The principal component analysis results show that four morphometric measurements (body length, BL; chest circumference, CC; chest width, CW; withers height, WH) and two body indexes (index of body weight, IBF; Baron-Crevat index, BCI) with LW could be used to the morphometric characterization. It was found that the cannon bone perimeter (CBP) was the differentiation point of CLPG genotypes. While a lower to moderate correlation was observed between CBP to CC, LW, CW, chest depth (CD), and rump width (RW) in the NN genotype, no significant correlation was observed in the MN genotype. This investigation also revealed that BCI and IBF were the two body indexes that should be adopted into breeding schemes as a performance and etiological comparison of animals, especially in marker-assisted selection schemes due to well-reflecting animal current status.

Genotypic characterization of meat-type lambs expressing the callipyge gene in Turkey:I. Carcass characteristics and retail yield

This study is set out to evaluate the effects of callipyge (CLPG) gene, attributed to sheep affecting growth and meat quality, on carcass and sensory trait of most preferred meat-type lamb (Kıvırcık, Karacabey Merino, Ramlıç, German Black-Head Mutton × Kıvırcık, Hampshire Down × Merino) in the western part of Turkey. Two datasets were used: (i) 177 lambs (66 males, 111 females) to determine genotype and allele frequencies; (ii) 48 genotypically identified male lambs for the assessment of carcass and meat sensory quality. It was found that homozygous (NN) and heterozygous (MN) conditions of the CLPG gene were observed in all sheep breeds, except Ramlıç which exhibits only NN. No significant differences were observed in CLPG genotypes for slaughter weight, cold carcass weight, and cold dressing percentage (p > 0.05). On the other hand, the effect of genotype on neck percentage was significant (p < 0.01), and lambs expressing CLPG genotype (MN) had a higher neck percentage. Not significantly, but numerically the MN genotype had a higher percentage of the shoulder, rack, and leg than the NN genotype, while the NN genotype had a higher percentage of loin (p > 0.05). Similarly, the effect of genotype on meat quality assessment was not significant (p > 0.05); however, shear force, water holding capacity, and cooking loss of the NN genotype were higher than the MN genotype. Indeed, no interaction between genotype and time was observed on the color parameter of the Longissimus thoracis et lumborum muscle at various storage periods (p > 0.05). The findings suggest that all meat-type sheep breeds (except Ramlıç) were polymorphic, suggesting that heterozygous individuals who received the CLPG mutation from the sire should be adopted in selection programs to improve carcass traits.

Proteomic and metabolomic profiling reveals the involvement of apoptosis in meat quality characteristics of ovine M. longissimus from different callipyge genotypes

Proteome and metabolome changes in muscles from callipyge mutation (+/C) and non-callipyge phenotype (+/+, C/+, and C/C) lambs were profiled to provide insight into the biochemical changes affecting meat quality attributes. M. longissimus thoracis from lambs with all four possible callipyge genotype (n = 4, C/+, C/C, +/C, and +/+) were collected after 3d aging and analyzed using mass-spectrometry based platforms. Among identified proteomes, cytochrome c (pro-apoptotic protein) was detected with significantly lower abundances in +/C. Anti-apoptotic HSP70, BAG3, and PARK7 were over-abundant in +/C, which could result in delayed apoptosis and possibly attributed to tougher meat in callipyge lambs. Eight glycolysis enzymes were overabundant in +/C lambs, whereas 3 enzymes involved in TCA cycle were overabundant in non-callipyge ones (C/C and/or C/+). Twenty-five metabolites were affected by genotypes (P < .05), including metabolic co-factors, polyphenols, and AA/short peptides. Our omics results provided insightful information for revealing the differences in biochemical attributes caused by callipyge mutation.

Callipyge genotypic effects on meat quality attributes and oxidation stability of ovine M. longissimus

The objective of this study was to evaluate the effects of callipyge (C) genotypes on quality attributes and oxidation stability of aged lamb loins. Only lambs possessing a wildtype maternal allele and a paternal callipyge allele (+/C) express a muscle hypertrophy phenotype. Seventeen lambs (7 months), consisting of all four possible callipyge genotypes (C/+, C/C, +/C and +/+), were harvested. Loins (M. longissimus thoracis) were separated from each carcass at 3days postmortem, cut into 3 sections, and randomly assigned into three aging periods (3, 6 and 9days). Genotype had no impact on proximate composition, mineral content, and pH of lamb loins (P>0.05). In terms of water-holding capacity, slightly increased purge/thaw loss of +/C lamb loins was observed (P<0.05), but cooking, drip and display weight losses were unaffected by callipyge genotype (P>0.05). The highest shear force was found at +/C lamb loins (P<0.05). +/C lamb loins showed initially less redness, but greater color stability during 7days of display compared to loins from the other genotypes. Heme iron content of +/C lamb loins was a significantly lower than the other genotypes. Genotype had no impact on lipid oxidation stability (P>0.05). Our results indicate similar quality attributes of lamb loins between non-callipyge phenotypes (C/C, C/+ and +/+) and suggest that callipyge lamb loins have superior color stability, while exhibiting lower tenderness compared to loins from non-callipyge phenotype lambs.

New insights into polar overdominance in callipyge sheep.

The callipyge phenotype in sheep involves substantial postnatal muscle hypertrophy and other changes to carcass composition. A single nucleotide polymorphism in the DLK1-DIO3 imprinted gene cluster alters gene expression of the paternal allele-specific protein-coding genes and several maternal allele-specific long noncoding RNA and microRNA when the mutation is inherited in cis. The inheritance pattern of the callipyge phenotype is polar overdominant because muscle hypertrophy only occurs in heterozygous animals that inherit a normal maternal allele and the callipyge SNP on the paternal allele (+/C). We examined the changes of gene expression of four major transcripts from the DLK1-DIO3 cluster and four myosin isoforms during the development of muscle hypertrophy in the semimembranosus as well as in the supraspinatus that does not undergo hypertrophy. The homozygous (C/C) animals had an intermediate gene expression pattern for the paternal allele-specific genes and two myosin isoforms, indicating a biological activity that was insufficient to change muscle mass. Transcriptome analysis was conducted by RNA sequencing in the four callipyge genotypes. The data show that homozygous animals (C/C) have lower levels of gene expression at many loci relative to the other three genotypes. A number of the downregulated genes are putative targets of the maternal allele-specific microRNA with gene ontology, indicating regulatory and cell signaling functions. These results suggest that the trans-effect of the maternal noncoding RNA and associated miRNA is to stabilize the expression of a number of regulatory genes at a functional, but low level to make the myofibers of homozygous (C/C) lambs less responsive to hypertrophic stimuli of the paternal allele-specific genes.

Evaluation of associations between prion haplotypes and growth, carcass, and meat quality traits in a Dorset × Romanov sheep population1,2

There is concern about potential antagonistic correlated responses due to intensive selection for scrapie-resistant haplotypes of the prion (PRNP) gene in sheep. The objective of the present research was to test for associations of PRNP haplotypes for codons 136, 154, and 171 with growth, carcass, and meat quality traits in an F2 Dorset x Romanov population (n = 415) segregating the 2 callipyge alleles. Haplotypes of the 3 PRNP codons were determined for each sheep, and breed of origin of each gamete was predicted by genotyping 6 microsatellite markers flanking the PRNP locus. Twenty-five growth, carcass, and meat quality traits were evaluated. Data were analyzed using a basic model consisting of fixed effects of year, sex, and callipyge genotype, the random effect of sire, and 7 covariates corresponding to the probability that a lamb inherited a specific PRNP haplotype of either Dorset or Romanov origin. A fixed effect of litter size was added to the model for growth traits. The model for carcass traits contained the linear and quadratic effects of chilled carcass weight and the interactions among callipyge genotype and linear and quadratic terms. For meat quality traits, the model contained chilled carcass weight as a covariate and the interaction between callipyge genotype and chilled carcass weight. A contrast between the resistant ARR haplotype and the average effect of other PRNP haplotypes was tested to investigate the effects of potential selection for ARR within each breed of origin (Dorset, ARR vs. ARQ, VRQ, and AHQ; Romanov, ARR vs. ARQ and VRQ). There was limited evidence that selecting for scrapie resistance would cause correlated responses due to linkage disequilibrium. Associations of only 3 traits with PRNP haplotypes were detected in either breed of origin. In Romanov, the ARR haplotype was associated with longer carcasses (P < 0.013), narrower rumps (P = 0.038), and less marbling (P = 0.022) than the average of ARQ and VRQ haplotypes. No significant contrasts were detected for Dorset. This study is the first to account for breed of origin while investigating haplotype associations in an F2 population. This study provided limited evidence of associations between PRNP haplotypes and growth, carcass, and meat quality traits.

Evaluation of the ovine callipyge locus: IV. Genotypic effects on reproductive traits1,2

A resource population of ewes derived from Dorset and Romanov grandparents was generated over 5 yr to provide genotypic and phenotypic data to estimate effects of each callipyge genotype on component traits of reproduction. Effects on ovulation rate were estimated from data recorded by examination of ovaries from serially slaughtered ewe lambs (n = 174) born in 1994 and 1995. Productivity and longevity through 5 yr of age were recorded on a total of 265 F2 ewes produced in 1996 through 1998. Number of lambs born, birth weights, and weaning weights of lambs were calculated for each ewe. Weaning weight was recorded for each lamb and adjusted for age to 56 and 32 d for dam- and nursery-reared lambs, respectively. Longevity was determined as a binary trait based on the presence or absence of the ewe in the breeding flock at 5 yr of age. Ewes generated 937 records for breeding weight and 925 records for conception rate, which was determined on ewes exposed and still present at the start of each lambing season. Seven hundred seventeen records were analyzed for traits based on the number of ewes lambing. Genotypes for the causative callipyge mutation were determined using a matrix-assisted laser desorption/ionization time-of-flight mass spectrometry assay. Each ewe was classified into 1 of 4 genotypes; NMatNPat, NMatCPat, CMatNPat, or CMatCPat, where N represents wild type and C represents the mutant allele. The mean ovulation rate of the ewe lambs genotyped NMatNPat was greater (P < 0.02) than that exhibited by NMatCPat; however, the estimated difference of 0.25 ovum in the ewe lambs did not affect the number born. The live weight of NMatCPat ewes at breeding was 2.5 kg less than that of other genotypes (P < 0.01). The callipyge genotype class did not significantly affect conception rates, fecundity, maternal ability, or ewe longevity. Maintaining a ewe flock to produce homozygous mutant rams for use in terminal sire mating systems presents no unusual problems from a reproduction standpoint.

Mosaicism of Solid Gold supports the causality of a noncoding A-to-G transition in the determinism of the callipyge phenotype.

To identify the callipyge mutation, we have resequenced 184 kb spanning the DLK1-, GTL2-, PEG11-, and MEG8-imprinted domain and have identified an A-to-G transition in a highly conserved dodecamer motif between DLK1 and GTL2. This was the only difference found between the callipyge (CLPG) allele and a phylogenetically closely related wild-type allele. We report that this SNP is in perfect association with the callipyge genotype. The demonstration that Solid Gold-the alleged founder ram of the callipyge flock-is mosaic for this SNP virtually proves the causality of this SNP in the determinism of the callipyge phenotype.

Feeding high levels of vitamin D3 does not improve tenderness of callipyge lamb loin chops.

The objective of this study was to determine whether feeding high doses of vitamin D3 7 d before slaughter would increase muscle Ca++ levels and result in more tender loin chops. Market lambs (n = 4 callipyge and 4 normal in Exp. 1, and n = 16 calipyge and 16 normal in Exp. 2) were randomly and equally assigned to feeding groups based on callipyge genotype and experimental diet, (vitamin D3 or control). Serum Ca++, muscle Ca++, Warner-Bratzler shear force, and troponin-T degradation data were analyzed. In Exp. 1, vitamin D3 was supplemented at 1 or 2 x 10(6) IU/d. The 2 x 10(6) IU dose resulted in the greatest serum Ca++ reponse and was chosen for Exp. 2. In Exp. 2, serum Ca++ concentration was higher (P < 0.05) for normal and callipyge lambs fed the vitamin D3 diet than for the control diet fed lambs. Muscle Ca++ concentrations, however, were not higher (P = 0.28) for the vitamin D3-fed lambs. Warner-Bratzler shear values were higher (P < 0.05) for callipyge than for normal lambs, but no differences were observed with vitamin D3 supplementation. These data were supported by results from Western blot analysis of troponin-T degradation, in which no differences were observed for vitamin D3 vs control diet lambs at 14 d postmortem. This experiment showed that feeding 2 x 10(6) IU/d of vitamin D3 to market lambs, callipyge or normal, raised serum Ca++ concentration, but did not increase muscle Ca++ concentration. This lack of response in muscle Ca++ was likely the reason that no differences were observed for Warner-Bratzler shear force values or troponin-T degradation data between the vitamin D3 and control loin chops. A higher dose of vitamin D3 may be required to improve tenderness.

High-resolution gametic map of the sheep callipyge region: linkage heterogeneity among rams detected by sperm typing.

The callipyge locus (CLPG) causing muscular hypertrophy in domestic sheep has previously been mapped to the distal part of ovine chromosome 18. In this study, an accurate multipoint linkage map consisting of six microsatellite markers in this chromosomal region was constructed based on the analysis of 1145 single sperm cells. The best supported order of markers was OARHH47-ILSTS54-MCM38-CSSM18-IDVGA30-BM S1561. The log odds against the second most likely order, which has a reversal of the closely linked markers CSSM18 and IDVGA30, was 5.026. Sperm typing can be used to examine a large number of meioses in single individuals, and therefore, was exploited to study individual variability of recombination rate in rams of different callipyge genotypes. The results revealed statistically significant linkage heterogeneity among rams (P < 0.05) for marker interval OARHH47-CSSM18, with individual recombination fractions varying from 0.209 to 0.357.

Evaluation of the ovine callipyge locus: II. Genotypic effects on growth, slaughter, and carcass traits.

A resource flock of 362 F2 lambs provided phenotypic and genotypic data to estimate effects of callipyge (CLPG) genotypes on growth, slaughter, and carcass traits. Lambs were serially slaughtered in six groups at 3-wk intervals starting at 23 wk of age to allow comparisons at different end points. Probabilities of CLPG genotypes were calculated at a position 86 cM from the most centromeric marker of chromosome 18. A contrast of CLPG genotypic effects, based on the paternal polar overdominance model, was used to evaluate callipyge and normal phenotypes. Relationships of traits with slaughter age, carcass weight, or 12th-rib fat depth for callipyge and normal phenotypic groups were estimated by regression. Callipyge and normal lambs did not differ for growth traits measured from birth to slaughter. Callipyge lambs produced 55.9% of live weight as chilled carcass weight compared with 51.7% for normal lambs at the same mean live weight of 48.32 kg. Lighter pelt, kidney-pelvic fat, and liver weights contributed to this advantage of callipyge lambs for dressing percentage (P < .001). Estimated accretion rates of carcass protein at the mean slaughter age were 12.5 and 10.2 g/d for callipyge and normal carcasses, respectively. Corresponding values for carcass fat were 35.2 and 42.1 g/d. Compositional differences in favor of callipyge carcasses were detected at constant values of slaughter age, carcass weight, and 12th-rib fat depth. Callipyge carcasses had 2.56 kg greater fat-free lean and 1.39 kg less fat than normal carcasses at the same mean age of 214.9 d (P < .001). The majority of these differences were established before the initial group was slaughtered and were maintained as age increased. Callipyge carcasses consisted of 24.3% fat and 71.3% fat-free lean, compared with 31.5 and 64.0% for normal carcasses at 25.6 kg of carcass weight. When evaluated at .49 cm of 12th-rib fat depth, callipyge lambs were 15.4 d older and produced 4.1 kg heavier carcasses with 4.3% less fat (P < .001). Effects of CLPG genotypic groups on carcass composition were greater than virtually all reported breed substitution effects. Use of the CLPG mutant allele in structured mating systems can dramatically increase production of lean lamb.

Evaluation of the ovine callipyge locus: I. Relative chromosomal position and gene action.

Genotypic and phenotypic data were collected to estimate chromosomal position of the callipyge (CLPG) gene and to test gene action. Nine Dorset rams of extreme muscling phenotype and 114 Romanov ewes composed the grandparent generation of a resource flock of 362 F2 lambs segregating at the CLPG locus. The parent generation consisted of eight F1 sires and 138 F1 dams. The F2 lambs were serially slaughtered in six groups at 3-wk intervals starting at 23 wk of age to allow comparisons at different end points. A linkage group of 25 marker loci (mean of 708 informative meioses per marker) spanning 87.2 cM was developed and improved the previous known coverage and precision of marker order and interval distance from available maps of ovine chromosome 18. Probabilities of each CLPG genotype were calculated at 1-cM intervals (0 to 107 cM). Statistical models included effects of year, sex, sire, regressions on genotypic probabilities, and genotype-specific linear and quadratic regressions on appropriate covariates. Orthogonal contrasts of CLPG genotypic effects evaluated additive, maternal dominance, and paternally derived polar overdominance models of gene action. The most parsimonious model did not include the additive and maternal dominance genetic contrasts. From analyses of four key traits, a consensus for position of CLPG was obtained at 86 cM relative to the most centromeric marker. An F-test with 3 df representing polar overdominance was maximum at position 86 cM (F = 407.4; P < .00001) with leg score as the dependent variable. These results are consistent with assignment of the CLPG locus to the telomeric region of chromosome 18 and support the polar overdominance model of gene action proposed by Cockett et al. (1996). Furthermore, recombinant individuals with definitive phenotypes confined the position of CLPG to a 3.9-cM interval, facilitating positional cloning experiments.