PROLACTIN: CANDIDATE GENE FOR EGG PRODUCTION OR BROODINESS TRAITS IN CHICKEN.
ABSTRACT:: Prolactin (PRL) is one of the pituitary hormones which is important in regulation of different physiological functions like nesting behavior in birds and also implicated in the regulation of broodiness in birds. The present study was conducted to study to find the association of egg production with the insertion/deletion in the promoter region of prolactin hormone. The genetic markers 24bp PRL was used to find out the association of egg production. 24 bp PRL showed significant association with the egg production. Three types of alleles i.e. A B and C were seen with four kinds of genotypes i.e. AA (both alleles at 130 bp) BB (both alleles at 154 bp) AB (one allele at 130 bp and second allele at 154 bp) and CC (greater than 350 bp). AB individuals showed the highest frequency (52 %) than that of CC (24 %) BB (16 %) and AA (8%) individuals. The individual carrying both B genes showed higher egg % (56.072.5) as compared to carrying AA (35.7110) AB (32.943.67) and CC (29.352.23) genotypes.
Higher frequency of homozygous insertion in N.N also signifies the higher egg production as compared to Aseel that is known as poor egg producer with more broody behavior. It may also be concluded from current study that the genotype (greater than 350) might be responsible for more broodiness in Aseel individuals.
KEY WORDS: Prolactin Aseel chicken; Naked neck chicken; egg production; broodiness
Anterior pituitary gland secrets a polypeptide hormone prolactin (PRL) which have diverse range of biological functions and activities in vertebrates. The induction of broodiness and incubation behavior is one of the most important function of prolactin in chickens . It has been observed that prolactin secretions may cause the regression of ovary  which is further elaborated by Shimada et al.  who reported that regression of ovary may lead to reduction in egg production. Removal or reduction of broodiness might have possibility to be due to the inhibition of prolactin expression reducing its secretions or blocking of the receptors binding sites . Sharp et al.  reported that changes of promoter binding site due to the polymorphism in the promoter region results into the influence of mRNA expression due to which egg production and incubation behavior may also be influenced. In avian prolactin is reported to be the candidate gene for broodiness and egg production .
However contrarily findings have also been reported by number of scientists. March et al.  observed non-significant effect of prolactin secretion on egg production. It has been reported that PRL gene transcription has been activated by number of factors including pituitary specific transcription factor (Pit-1)  CCAAT/enhancer binding protein  activine  and vasoactive intestinal peptide . In the 5'flanking region of PRL gene potential Pit-1 binding sites in turkeys and Xinghua chicken has been found . Whereas the molecular mechanism for the activation of Pit-1 mediated PRL gene in unknown.
2. MATERIALS AND METHODS
Total of 105 adult birds 84 from Aseel (21 birds from each variety of Aseel namely Lakha Mushki Peshawari and Mianwali) and 21 from naked neck were maintained for 10 weeks to evaluate their egg production potential. The birds were housed in standard managemetal conditions. In next phase 5 birds from each Aseel variety and 5 from naked neck were selected to evaluate the association of prolactin with the egg production.
Blood sampling and DNA extraction
A total of 25 blood samples were collected from naked neck and four varieties of Aseel each containing 5 birds. Two milliliter blood were collected from brachial vein preserved in falcon tubes containing EDTA (60 micro-litter for each sample). By employing Sambrook and Russel protocol  blood samples were used for subsequent DNA extraction. Primer designing and synthesis Primers were designed from available gene sequence at NCBI data set by using online available Primer-3 software (http:/Frodo.wi.mit.edu/). Designed primer were synthesized from commercially available synthesis facilities.
Temperature optimization of PCR was carried out through gradient PCR methodology. Primers were designed for genotyping of polymorphisms. The primer sequences were as follows forward 5'TTTAATATTGGTGGGTGAAGAGACA-3'; reverse 5'ATGCCACTGATCCTCGAAAACTC-3'. primer pair was used to amplify 130 or 154 bp fragment having 24 bp indel at -358 site. PCR was carried out in the total volume of 20l having 100 ng genomic DNA 0.2 mM deoxynucleoside triphosphate 0.4 M of each primer 1.5 mM Mgcl2 1 U DNA polymerase and 1X reaction buffer with following profile: initial denaturation of 5 min at 94o C; 35 cycles of 94oC for 30 s 62o C for 30s and 72o C for 30 s with final elongation of 5 min at 72oC.
Marker trait association analysis
Association of genetic markers and haplotypes with egg production and egg quality traits were analyzed using GLM procedure of Genstat software. Values were considered signicant at P = 0.05. 3. RESULTSIn the present study significant differences (P less than .0001) were observed in egg production % of naked neck and four varieties of Aseel. Statistical analysis revealed that egg production % of naked neck (47.420.78) is significantly higher (P less than .0001) than Mushki (34.080.73) Lakha (31.430.76) Mianwali (29.590.75) and Peshawari (28.700.58) varieties of Aseel (Figure 1).
Genotyping of 25 individuals five from naked-neck and five from each variety of Aseel i.e. Lakha Mianwali Mushki and Peshawari for these four genetic markers 24-bp indel (insertion-deletion) at the site of -358 of chickens PRL gene was only the one who showed significant affect (P=0.05) on egg production but this marker has no effect on egg quality traits.
A total of 25 individuals were genotyped for detecting the insertion at promoter region of PRL gene. Three types of alleles (A B C) were observed with four kinds of genotypes (AA AB BB CC). AA individuals showed both alleles at 130 bp BB individuals showed both alleles at 154 bp AB individuals showed one allele at 130bp and second allele at 154 bp while CC individuals showed both the alleles at greater than 350 bp (Figure 2). AB individuals showed the highest frequency (52 %) than that of CC (24 %) BB (16 %) and AA (8%) individuals. Association of genotypes with egg production percent showed that the individual carrying both B genes showed higher egg % (56.072.5) as compared to carrying AA (35.7110) AB (32.943.67) and CC (29.352.23) genotypes. BB individuals showed significantly higher egg % as compared to others while there were non-significant variation among egg production of individuals carrying AB AA and CC genotypes.
Naked neck carried the highest frequency (60 %) of BB (homozygous for insertion) while in four varieties of Aseel only Peshawari showed individual carrying genotype homozygous for insertion of 24 bp. Higher frequency of homozygous insertion in Naked-neck also signifies the higher egg production as compared to Aseel that is known as poor egg producer with more broody behavior. Six out of 20 individuals of Aseel population showed another type of genetic makeup. These individuals showed a singular band of greater than 350 bp and they were homozygous for that. It may be concluded from current preliminary study that this Allele might be responsible for more broodiness in Aseel individuals.
Prolactin is one of the pituitary hormone which is important in regulation of different physiological functions like nesting behavior in birds . Prolactin has been implicated in the regulation of broodiness in birds since Riddle et al.  first demonstrated that injections of prolactin induce incubation behavior. Prolactin is reported to be the candidate gene for broodiness and egg production . In the present study only one primer i.e. prolactin at the region of 24-bp indel showed significant effect (P=0.05) on egg production. Similar finding has already been reported by Cui et al.  who observed the association of egg production with the insertion of 24-bp PRL Wong et al.  reported the association of changes in the level of plasma PRL level with the PRL mRNA expression in the anterior pituitary of turkeys. Similarly in another experiment it has been reported that the insertion in the promoter region might be responsible for the inhibition of transcriptional factor binding
site for prolactin thus decrease PRL level which contributes in nonbroodiness. Results of the current study are very similar and in accordance to previous reports indicating the relationship of insertion with production traits especially egg production. A total of 25 individuals were genotyped for detecting the insertion at promoter region of PRL gene. Three types of alleles (A B C) were seen with four kinds of genotypes (AA AB BB CC). AB individuals showed the highest frequency
(52 %) followed by 24 % 16 % 8% of CC BB and AA respectively. Association of genotypes with egg production percent showed that the individual carrying both B genes showed higher egg % (56.072.5) as compared to carrying AA (35.7110) AB (32.943.67) and CC (29.352.23) genotypes. BB individuals showed significantly higher egg % as compared to others while there were non-significant variation among egg production of individuals carrying AB AA and CC genotypes as shown in Table. 2. As in the current study the naked neck individuals showed maximum genotypic frequency of BB genotype (60 %) elaborating the higher egg production rate of naked-neck than the Aseel that is known as poor egg producer with more broody behavior. The higher egg production of BB individuals might be attributed to the negative association of BB genotype with prolactin secretions. Similar findings has been reported that homozygous insertion may cause decrease in prolactin secretion thus increases the egg
production rate and decreases the broody behavior  . Similarly the association of broodiness and egg production is elaborated by number of scientists who reported the induction of broodiness and incubation behavior due to the increase in prolactin secretions thus reducing the egg production rate. It has been reported that prolactin secretions cause the regression of ovary  which leads to the reduction of egg production rate . Removal or reduction of broodiness might have possibility to be due to the inhibition of prolactin expression reducing its secretions or blocking of the receptors binding sites 
Six out of 20 individuals of Aseel population showed another type of genetic makeup. These individuals showed a singular band of greater than 350 bp and they were homozygous for that. It may be concluded from current preliminary study that this Allele might be responsible for more broodiness in Aseel individuals. In the present study the genotypic frequency of BB in Aseel chickens in very less (5%) i.e. one out of twenty. This might also be responsible for the increased broody behavior and poor egg production of Aseel. As it has been reported that homozygous insertion of the 24-bp sequence in the PRL promoter may decrease the expression of PRL leading to non-broodiness . In the previous studies Cui et al.  observed the association of egg production with the insertion of 24-bp PRL. Similarly the absence of insertion in the 24-bp PRL promoter is likely to be associated with broodiness traits in blue shell chickens . Sharp et al.  reported that changes of promoter binding
site due to the polymorphism in the promoter region results into the influence of mRNA expression due to which egg production and incubation behavior may also be influenced. However contrarily findings have also been reported by number of scientists. It has been reported that polymorphism or variation in promoter region that cause the change in promoter binding site mostly influence the expression of mRNA ultimately influence the egg production and incubation behavior  . March et al.  observed nonsignificant effect of prolactin secretion on egg production. Although the current study in insufficient to justify the association of genotypes with poor egg production of Aseel due to the less number of individuals but is helpful in finding the new genotype of CC. Further experiments are required to find out the association of CC genotype with the egg production of Aseel on a large population.
Based upon the findings of present study it may be concluded that Naked neck chickens had better production performance than all four varieties of Aseel. Genetic analysis revealed that the higher egg production of naked neck is likely to be associated with 24 bp insertion while the poor egg production and more broody behavior of Aseel might be associated with genotype of greater than 350 bp.
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|Date:||Sep 30, 2014|
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