Effects of nonglycosylated and glycosylated prolactin on basal and gonadotropin-stimulated steroidogenesis in chicken ovarian follicles.

In galliformes, the circulating isoform of prolactin (PRL) significantly changes during different reproductive states. However, the role of the major isoform (glycosylated PRL [G-PRL]) in ovarian steroidogenesis is unknown. The present study aimed to compare the effects of nonglycosylated (NG-) and G-PRL on basal and gonadotropin-stimulated estradiol (E2) and progesterone (P4) production in granulosa cells or follicular walls of chicken of different size class follicles. In the initial experiment, granulosa cells of preovulatory F3-F1 and prehierarchical 6- to 8-mm follicles were incubated for 24 h with different concentrations of NG- or G-PRL (0, 1, 10, 100, or 1,000 ng/mL). In the subsequent experiments, these categorized granulosa cells and follicular walls of prehierarchical 4-6, 2-4, and <2-mm follicles were incubated for 24 h in the absence and presence of 10-ng/mL FSH or LH, or in combination with different concentrations of NG- or G-PRL (10, 100, or 1,000 ng/mL). We observed that lower levels of NG-PRL induced (P < 0.05) E2 and P4 secretion in granulosa cells of either preovulatory or prehierarchical follicles, but at higher levels, this effect was reduced. In contrast, G-PRL promoted (P < 0.05) basal E2 and P4 secretion in preovulatory granulosa cells but was inhibitory (P < 0.05) in prehierarchical granulosa cells. Results obtained by real-time quantitative PCR (qPCR) demonstrated that these effects were mediated through modulation of the expression of StAR, CYP11A1, CYP19A1, and 3ß-HSD. Furthermore, G-PRL was less potent than NG-PRL in inhibiting FSH- or LH-stimulated E2 and P4 production in granulosa cells of preovulatory follicles, whereas NG-PRL enhanced (P < 0.05) but G-PRL reduced (P < 0.05) FSH-induced P4 production in those of prehierarchical follicles. In follicular walls from each group of prehierarchical 4-6, 2-4, and <2-mm follicles, NG- and G-PRL had both stimulatory and inhibitory influences on the actions of FSH on E2 and P4 secretion, but both suppressed (P < 0.05) LH-induced E2 and P4 secretion except for the synergistic effects of LH and G-PRL on P4 secretion by follicular walls of the follicles of 4-6 mm. Taken together, these results suggest that both NG- and G-PRL are biologically active in regulating basal and gonadotropin-stimulated E2 and P4 production in chicken ovarian follicles. However, their effects are different depending on the concentration, the type of gonadotropin (FSH or LH), and the stage of follicle development.

Association of a marker in the vitamin D receptor gene with Marek's disease resistance in poultry.

Vitamin D is an important immunomodulator that mediates its effect via a nuclear receptor. In this study, we analyzed 3 uncorrelated genetic markers (tag single nucleotide polymorphisms) in the vitamin D receptor gene for association with Marek's disease (MD) resistance. The database consisted of 400 commercial White Leghorn chickens that had been vaccinated with herpes turkey virus and challenged by intraperitoneal injection of the virulent MD virus RB1B. Viral titers in feather tips were determined at weekly intervals for 8 wk, mortality was recorded, and necropsy analyses preformed on all chickens. The 3 genotypes defined by 1 of the markers were associated with significant differences in the viral load (integration of the viral titer over time; P = 3 x 10(-4)). The effect was additive, with the 2 homozygotes differing by a factor of 2. The ranking of the genotypes by viral load, frequency of MD lesions, mortality, and bursal atrophy were consistent. There was no effect on the tissue distribution of MD lesions. The degree of MD resistance in the 9 genotypes defined by the 3 tag single nucleotide polymorphisms was proportional to the frequency of major histocompatibility complex class II-positive peripheral blood leukocytes that had been previously measured in uninfected chickens in a different database.

Expression patterns of the prolactin receptor gene in chicken lymphoid tissues during embryogenesis and posthatch period.

Prolactin (PRL) is a pituitary hormone with multiple homeostatic roles among vertebrates. Although it has mainly been studied in relation to its role during the initiation and maintenance of incubation behavior in avian species, it has also been shown to act on the immune system. In this study, levels of PRL receptor (PRLR) mRNA were quantified by real-time PCR, and tissue expression was localized by in situ hybridization in primary and secondary lymphoid organs. Prolactin receptor was shown to be expressed in the bursa follicles, thymus lobules, and splenic pulp at all stages of development examined. Levels of PRLR expression were consistently higher in the bursa of Fabricius when compared with other lymphoid organs, suggesting that PRL acts primarily on bursal development. Furthermore, levels of PRLR mRNA appeared to fluctuate during embryogenesis, with a significant increase observed at embryonic day 19 in the bursa, at 7 d of age in the thymus, and on hatching day in the spleen. Thus, PRL might play an important role during the development of the immune system in chickens.

Ontogenesis of the expression of prolactin receptor messenger ribonucleic acid during late embryogenesis in turkeys and chickens.

Changes in circulating levels of prolactin (PRL) and tissue content of PRL receptor (PRLR) messenger RNA (mRNA) in the liver, pancreas, kidney, and gonads (testis/ovary) were measured in turkey and chicken embryos from embryonic day (ED) 21 or ED15, respectively, to 1 d after hatch by real-time PCR. There were no differences between the sexes in chickens or turkeys. Both species had very similar patterns of PRL release and expression of PRLR mRNA, and no major differences were observed between turkey or chicken embryos. Plasma levels of PRL increased from low levels during the last week of embryonic development and were at significantly higher levels (about 4-fold) by 1 d after hatch. Similarly, in all tissues the content of PRLR mRNA was minimal at the outset and increased to reach maxima about the time of hatch. In both species, the highest levels of transcript were observed in the kidney followed by the gonad, liver, and pancreas. The tissue content of PRLR was correlated (0.6 to 0.8 dependent on the tissue) to circulating levels of PRL, which suggested that PRL may be associated with an increase in its receptor number around the time of hatch. Because levels of PRL and tissue content of PRLR mRNA increased around the time of hatch, this suggests that these tissues may be targets for PRL and may be involved in the physiologic changes occurring in embryos around the time of hatching.

Development of a real-time (Q) PCR assay to measure variation in expression of prolactin receptor mRNA in the hypothalamus and pituitary gland during late embryogenesis in turkeys and chickens.

Changes in levels of PRLR mRNA in the pituitary gland and hypothalamus of chickens and turkeys from embryonic day (ED) 15 and ED21 to 1 day post-hatch, respectively, were measured by real-time PCR. In both species, PRLR mRNA increased from low levels during the last week of ED to reach maxima at the peri-hatch period. Similarly, circulating levels of PRL also increased during this interval and were highly correlated with levels of the PRLR mRNA in both the pituitary gland and hypothalamus. This suggests that PRL was up-regulating its receptor. In support of this, stimulation of the turkey pituitary gland with VIP on ED24 resulted in a 4- and 3-fold increase in PRL and PRLR, respectively. Since VIP had no direct effect on the levels of PRLR transcript in the hypothalamus, it is likely that VIP is acting indirectly through increased PRL to up-regulate the number of receptors.

Ornithine decarboxylase: haplotype structure and trait associations in White Leghorn chickens.

Sequence analysis of 4,230 bp of the 3' end of the ornithine decarboxylase gene of 20 chickens from a noninbred White Leghorn strain revealed a total of 62 polymorphisms. Of these 61% were transitions, 24% were transversions, and 16% were deletions or insertions. Despite the high number of polymorphisms, only 3 haplotypes were present among the 40 alleles analyzed. Based on the genetic distances between haplotypes they segregated early during domestication of the chicken. Ornithine decarboxylase is a pivotal enzyme in regulating the synthesis of polyamines, cations that are important regulators of cell division, differentiation, and apoptosis. Variants of ornithine decarboxylase are, therefore, expected to affect many different traits. Association analyses between genotypes and the major egg production traits in female chickens of the same strain revealed significant effects on the onset of sexual maturity, BW at sexual maturity, eggshell thickness (a measure of calcium deposition), and residual feed consumption (a measure of the metabolic rate). Further, comparisons of the genotypes indicated that the 3 haplotypes differ in their phenotypic properties. Our results show that variations in a gene that is ubiquitously expressed in all cells of an organism may nevertheless contribute to distinct phenotypic properties of the organism as a whole.

Alleles of cytosolic phosphoenolpyruvate carboxykinase (PEPCK): trait association and interaction with mitochondrial PEPCK in a strain of White Leghorn chickens.

White Leghorn chickens from a nonselected closed population were typed for two RFLP located in the 3' end of the gene coding for cytosolic phosphoenol-pyruvate carboxykinase (PEPCK-C), a major control gene of gluconeogenesis. The two RFLP gave rise to three alleles (or haplotype classes), thus defining six genotypes. Feed efficiency (FE) and residual feed consumption (RFC) varied significantly among the genotypes and indicated that all three haplotypes differed from each other. FE is the ratio between feed consumption and egg mass produced, whereas RFC is the feed consumption after correcting for BW and egg production. There was significant interaction between PEPCK-C genotypes and mitochondrial PEPCK (PEPCK-M) genotypes defined by a single RFLP. The latter enzyme catalyzes the same reaction but is located in the matrix of the mitochondria and is encoded by a different nuclear gene. Interaction was evident from an analysis of the egg weight and egg specific gravity in the early phase of egg laying. It was such that the effect of the variation in one gene depended entirely on the genotype of the second gene. In addition, significant genotypic disequilibria were observed between two of the three alleles of PEPCK-C and between one of these alleles and the two RFLP alleles of PEPCK-M. This finding indicates variations of genes in the gluconeogenesis pathway may affect feed utilization and egg production traits, as well as reproductive fitness.

The dynamics of the genotype-phenotype association.

The integrity of an organism is maintained by networks of interacting genes. Such networks predict that genetic variants affect phenotypes in a nonadditive fashion. That is, the effect of an allelic variation in one gene is dependent on the variations in other genes. We summarize the analyses of a series of genes in a White Leghorn strain that support the existence of such gene networks: 1) genes are pleiotropic, 2) genes affect trait correlations, 3) genes affect trait distributions in a nonadditive fashion, 4) genes interact with each other, and 5) genes are at linkage disequilibrium, even when located on different chromosomes. The latter observation indicated that certain gene combinations lead to reduced reproductive fitness. Each candidate genes we analyzed segregated for multiple alleles that affected production traits. This finding was surprising, even for a strain with a large effective population size. The shapes of trait distributions appear to be a better descriptor of gene effects than measures of central tendency. Despite this complexity, it is feasible to conduct DNA-based selection, starting from any of several different genes that affect a trait. Gene networks may be altered in many different ways to improve a particular phenotype, but networks may differ in their effects on other traits.

Genetic variability of the cytosolic phosphoenolpyruvate carboxykinase gene in white leghorn chickens.

Phosphoenolpyruvate carboxykinase (PEPCK) is a key regulatory enzyme of gluconeogenesis. Genetic variations in this gene may therefore affect a wide variety of traits, including tumor growth that is heavily dependent on glucose metabolism. We have previously shown the gene coding for the mitochondrial form of PEPCK (PEPCK-M) segregates for markers that are collected with resistance to Marek's disease. In this communication we analyze the genetic variability of PEPCK-C, the gene which codes for the cytosolic form of PEPCK. A 3,792-bp segment of 5'-region of the PEPCK-C gene (position -1723 to 2069) was sequenced in four individuals from eight different strains of White Leghorn chickens (a total of 64 genomes). A total of 19 single nucleotide polymorphisms (SNP) were identified. Neither deletions nor insertions were present. The most frequent SNP were transitions (79%), and in most cases the ancestral allele coincided with CpG dinucleotides (10-fold excess after correcting for dinucleotide frequencies). A gene tree was constructed assuming maximal parsimony. It led to the delineation of 6 haplotypes (combination of alleles). Two of the SNP coincided with RFLP detectable by the restriction enzymes AciI and BstEII, respectively. Based on this analysis we can now identify individuals with the evolutionary most distant PEPCK-C haplotypes, establish strains of these haplotypes, and analyze trait associations and epistasis with other genes.

Molecular cloning of chicken vasoactive intestinal polypeptide receptor complementary DNA, tissue distribution and chromosomal localization.

Chicken vasoactive intestinal polypeptide receptor (VIPR) cDNA was cloned by the reverse transcription-polymerase chain reaction method using primers designed on the basis of other species of VIPR cDNA. The cDNA obtained was sequenced by the dideoxy-mediated chain-termination method. Of the 2227 nucleotides that were sequenced, 84, 855, and 1338 bases represent the 5'-untranslated region (UTR), the 3'-UTR, and the open reading frame that predicts a peptide of 446 amino acids. The cDNA of the chicken VIPR shows 65% and 60% homologies to human cDNA of VIP1 and VIP2 receptors, respectively. The clone had the expected similarity to highly conserved features of the other G protein-coupled receptors (GPCRs) such as six cysteine residues that are functionally important in the VIPR subfamily. In addition, the seven potential membrane-spanning domains characteristic of the family B group III GPCR superfamily and highly conserved motif within the third cellular loop between transmembrane regions 5 and 6. Northern blot hybridization analysis in this study indicated mRNA expression of VIPRs in the various tissues of the chicken. Strong signal was detected in the brain and anterior pituitary gland. High levels of VIPR mRNA in the brain was consistent with VIP-binding experiments and with the function of VIP in the brain as a neuroendocrine factor or neurotransmitter. Expression of VIPR was detected in the anterior pituitary gland of chick embryos. The expression of VIPR mRNA in the chick anterior pituitary gland may indicate a regulatory function of VIP on prolactin (PRL) production or PRL cell proliferation during embryogenesis. Chicken VIPR shows high homology with mammalian type I VIPR but, in some part, possesses similarity of amino acid sequence. Expression of VIPR in various tissues supports diverse functions for VIP in the chicken.

Trait association of a genetic marker near the IGF-I gene in egg-laying chickens.

The insulin-like growth factor I (IGF-I) gene was screened for genetic variants associated with trait means and trait correlations. Analysis of an unselected randomly mated White Leghorn population revealed a PstI restriction fragment length polymorphism (RFLP) in the 5' region of the gene which segregated at a frequency of 0.83 for the PstI(+) allele (presence of a PstI restriction site). A comparison of the three genotypic classes revealed that the PstI(-/-) genotype was associated with a significantly lower egg weight measured in three different time periods, while the PstI(+/-) genotype was significantly associated with a higher eggshell weight estimated from the egg weight and egg specific gravity. For eggshell weight, the effect was age dependent and significant only for the last two periods of egg laying. No genotype associations were found for body weight, feed consumption, and egg laying rates. Significant dominance effects of the IGF-I genotype were observed for two of the egg weight measurements and three of the eggshell weight estimates. Partial correlation analyses in the two most frequent genotypic classes, PstI(+/+) and PstI(+/-), revealed the presence of a regulatory loop between feed consumption, body weight, egg weight, and the rate of egg laying. Several aspects of this regulatory loop were different between the two genotypic classes. In particular, for the PstI(+/+) genotype, feed consumption was positively associated with egg weight, while there was no significant association for the PstI(+/-) genotype. Further, the degree of association of body weight with egg weight decreased with age in the genotypic class PstI(+/-), while it was constant for the PstI(+/+) genotype. The results indicated that the marker in the IGF-I gene was not only associated with changes in some trait means, but also with changes in the stability of the coordination between feed intake, body weight, and egg production traits.

Quantification of prolactin messenger ribonucleic acid, pituitary content and plasma levels of prolactin, and detection of immunoreactive isoforms of prolactin in pituitaries from turkey embryos during ontogeny.

The content of prolactin mRNA as well as total prolactin content and type of isoforms of prolactin were measured in single pituitary glands from turkey embryos and poults. Levels of mRNA and pituitary content of prolactin remained low until 5 days before hatching, while plasma concentrations remained low until 2 days before hatching. Levels of prolactin mRNA then increased until the day of hatch, stayed stable during the 3 first days of age, and significantly increased until 2 wk of age. Similar changes were observed in pituitary content and plasma levels of prolactin. Two immunoreactive bands of apparent molecular masses of 24 and 27 kDa, corresponding to the nonglycosylated and glycosylated form of prolactin, respectively, were visualized on Western blots. In pituitary glands from embryos at 22 days of incubation, 31.5% of the protein was glycosylated, whereas in embryos at 27 days of incubation and poults at 1 and 7 days of age, 48.6%, 48.0%, and 56. 0% of prolactin was glycosylated, respectively. The results indicate that the increases in the synthesis and the release of prolactin occur mainly around and after the time of hatching in the turkey embryo. Higher percentages of glycosylated isoforms were associated with increasing levels of total prolactin in the pituitary gland. Thus, the synthesis of prolactin and its post-translational modifications may be important factors involved in the physiologic changes occurring around the time of hatching.

Markers within the regulatory region of the growth hormone receptor gene and their association with milk-related traits in Holsteins.

We studied sequence variations in the regulatory region of the bovine growth hormone receptor gene. A polymerase chain reaction (PCR)-based method for detecting AluI, AccI, and StuI restriction fragment length polymorphisms (RFLPs) in the 5' flanking region of the bovine growth hormone receptor gene was developed and tested for association with milk-related traits in Holstein bulls. Allele frequencies of the polymorphisms in two groups of Holstein progeny-tested bulls born from 1950 to 1970 and in the 1980s, respectively, were estimated. The allele frequency of the AluI(-) allele was 0.63 and 0.42 in the bulls from 1950 to 1970 and in the 1980s, respectively. The frequency of the StuI(-) allele was 0.14 and 0.07 in the two respective bull groups. Allele frequency for AccI(-) allele was about 0.22 in both bull groups. The differences in allele frequencies for the AluI polymorphism in the two bull groups were significantly different (P < or = .005). The AluI(+/+) bulls had a higher estimated breeding value (EBV) for fat (P < or = .016) than AluI(-/-) bulls. The average effect of allele substitution for the AluI polymorphism was +/- 8 for fat EBV. The AluI polymorphism could be further evaluated for use in marker-assisted selection in dairy cattle.

Changes in levels of immunoreactive prolactin isoforms during a reproductive cycle in turkey hens.

Changes in the ratio between immunoreactive isoforms of prolactin using Western blotting and in the total prolactin content using radioimmunoassay were measured in pituitary glands from turkey hens at different physiological stages. The type of glycosylation (N- or O-linked carbohydrates) was determined using endoglycosidase digestion (N-glycosidase F, O-glycosidase, and neuraminidase). Low levels of prolactin were observed in pituitary glands from sexually immature, out-of-lay, and molting hens. Higher levels were present during the egg-laying period and the highest levels were detected in hens which expressed incubation behavior. Two immunoreactive bands of apparent molecular weights of 24 and 27 kDa were visualized on Western blots, corresponding to the nonglycosylated and glycosylated forms of prolactin, respectively. In pituitary glands from incubating turkey hens, about 70% of the prolactin was glycosylated (27-kDa isoforms), whereas about 60% was glycosylated in immature and in hens during the first egg-laying period. In pituitaries from out-of-lay and molting hens the percentage of glycosylated prolactin was 38 and 33%, respectively. Thus, higher percentages of glycosylated isoforms (27 kDa) were associated with high levels of total prolactin and lower percentages were associated with low levels of prolactin content in the pituitary gland. Digestion of the isoforms with N-glycosidase F resulted in a single band with an apparent molecular weight of 24 kDa. Partial deglycosylation was achieved using neuraminidase, whereas digestion with O-glycosidase had no apparent effect on the isoforms. Thus it appears that the glycosylated isoforms of prolactin have N-linked carbohydrates containing sialic acid.

In vitro release of isoforms of prolactin from pituitary glands of turkey hens at different physiological stages.

To study the in vitro release of PRL isoforms, anterior pituitary glands from medium white turkeys at various physiological stages were stimulated by cVIP in a perifusion system. Pituitaries were cut into hemi-pituitaries after collection and placed into separate perifusion chambers. Medium (M199) was continuously perifused through the system and pituitaries were stimulated with cVIP (10(-7) M). Total PRL content was monitored by RIA and, the ratio of immunoreactive PRL isoforms in the perifusate was estimated by Western blotting. After exposure to X-ray film for autoradiography, the relative intensity of the bands was analyzed by densitometry. All the perifused pituitaries responded to cVIP stimulation by increasing the release of PRL. Two immunoreactive bands with relative molecular weights of 24 and 27 kDa were detected by Western blotting. The immunoreactive band corresponding to the glycosylated isoforms of PRL (27 kDa) was predominant in samples from egg-laying and incubating hens and the band corresponding to the nonglycosylated isoform (24 kDa) was predominant in samples from out-of-lay and molting stages. No changes in the ratio of isoform released were detected during cVIP stimulation. Our data clearly show that glycosylated and nonglycosylated PRL isoforms are released by the pituitary gland in vitro in the same relative proportion that was previously observed in pituitary extracts and thus are likely to reflect the secreted forms of PRL in the blood during various physiological stages. In addition, the PRL-releasing activity of VIP does not affect the ratio of isoforms secreted by the pituitary gland in vitro.

Comparative FISH mapping on Z chromosomes of chicken and Japanese quail.

Using direct R-banding fluorescence in situ hybridization, we assigned five functional genes-growth hormone receptor (GHR), prolactin receptor (PRLR), spleen tyrosine kinase (SYK), aldolase B (ALDOB), and muscle skeletal receptor tyrosine kinase (MUSK)-to the chicken Z chromosome. SYK and MUSK were newly localized to the chicken Z chromosome in this study. GHR and PRLR were situated close to each other on the short arm of the chicken Z chromosome, as are their counterparts on human chromosome 5. SYK, MUSK, and ALDOB, which have been mapped to human chromosome 9, were localized to the long arm of the chicken Z chromosome. Thus, the present results indicate the presence of conserved synteny between the chicken Z chromosome and human chromosomes 5 and 9. Using the same method, four of the genes (GHR, PRLR, ALDOB, and MUSK) were assigned to the Japanese quail Z chromosome. The locations of these four Z-linked genes were conserved between chicken and Japanese quail. The results support the notion that the avian Z chromosome and the mammalian X chromosome did not evolve from a common ancestral linkage group.

Cytogenetic mapping of 31 functional genes on chicken chromosomes by direct R-banding FISH.

Using direct R-banding fluorescence in situ hybridization, we determined the location of 31 functional genes on chicken chromosomes. Replication R-banded chromosomes were obtained by synchronizing splenocyte cultures with excessive thymidine, followed by BrdU treatment. Thirty-one functional genes were directly localized to banded chicken chromosomes using genomic DNA and cDNA fragments as probes. The possibility of conserved linkage homology between chicken and human chromosomes was demonstrated for seven chicken chromosome regions (1p, 1q, 2q, 4p, 4q, and 5q).

Mitochondrial PEPCK: a highly polymorphic gene with alleles co-selected with Marek's disease resistance in chickens.

The gene coding for the mitochondrial phosphoenolpyruvate carboxykinase (PEPCK-M), a pivotal component in gluconeogenesis from lactate via the Cori cycle, was highly polymorphic in strains of egg-type chickens (White Leghorn) of different origins. Based on MspI restriction fragment polymorphisms a total of seven alleles could be distinguished. The allele frequencies were determined in six pairs of strains derived from different genetic base populations. Each pair consisted of two strains which differed in their susceptibility to Marek's disease (MD), a virus-induced neoplastic disease. The frequency of the most common haplotype (M2) was consistently higher in the susceptible strains than in the corresponding resistant strains (P < 0.05, Wilcoxon signed-ranks test), indicating that the observed differences were not due to random genetic drift. This result suggests that PEPCK-M may be a candidate gene which contains genetic variants affecting MD susceptibility. Variations in gluconeogenesis may affect the interplay between proliferation of neoplasia and host metabolism.

A genetic marker in the growth hormone receptor gene associated with body weight in chickens.

A genomic clone spanning 16 kb of the GH receptor gene was mapped and used as a probe for identifying restriction fragment length polymorphisms (RFLPs) in chickens. Several strains of meat-type and egg laying chickens were found to segregate for an HindIII RFLP located in the intron preceding exon 4. The polymorphic HindIII site overlapped with a poly(A) signal. Association of the HindIII RFLP with traits was analyzed in a random-bred White Leghorn strain in three generations using either selective or random genotyping. Both methods revealed significant association of the HindIII+ allele (presence of the poly(A) signal) with an increased juvenile body weight (130 days of age). In two meat-type strains divergently selected for size of the abdominal fat pad, the HindIII+ allele was coselected with leanness. The results indicate the presence of a genetic variant of the GH receptor gene which affects growth and abdominal fat deposition and which is relatively frequent in egg laying as well as in meat-type chickens.

Evidence for a genetic variation in the mitochondrial genome affecting traits in White Leghorn chickens.

A mitochondrial Mspl RFLP which was coselected with Marek's disease (MD) resistance in White Leghorn chickens was mapped to the NADH subunit IV. The RFLP was due to a transition, resulting in the change of the low-usage threonine triplet ACT (Mspl- allele) to the high usage triplet ACC (Mspl+ allele). Trait association studies within an unselected strain revealed that the Mspl- allele whose frequency was reduced in MD resistant strains was associated with high body weight and high egg specific gravity (a measure of eggshell thickness). Analysis at three different time points indicated a significant interaction between the mitochondrial genotype and the growth hormone genotype in early but not in late adulthood. The analysis indicates that mitochondrial variants may contribute to phenotypic variation in chickens and that such contributions may be dependent on the genetic background.