Indonesia is widely known for chicken domestication (Fumihito et al. 1994; Sulandari et al. 2017) having a diverse breed of chicken. Pelung (Iskandar and Susanti, 2007), Java (Ulfah et al. 2016; Sulandari et al. 2017), Sumatra (Ulfah et al. 2016), Cemani(Daryono et al. 2016; Dharmayanthi et al. 2017), and Nunukan(Tixier-Boichard et al. 1997) are the most popular breeds which have anthropologic and economic benefits. These chicken breeds are rare and expensive; however, they have been massively cultivated. This condition arises due to low productivity of female parent stock. Moreover, in meat-type chicken if an imbalance in demand and supply occurred (Iskandar and Susanti, 2007), it might lead to extinction of chicken breeds. Following the Ethiopian chicken success (Okeno et al. 2012), to improve Indonesian local chicken, several prospective local chickens were crossbred to commercial breeds (Puspita et al. 2016; Saragih et al. 2016; Perdamaian et al. 2017). Despite excellent final stock, the female parent lacks productivity. On the other hand, if large numbers of offspring are produced, their body weights were below the economical threshold at slaughter day. For building Indonesian meat-type chicken, male pelung (promised meat-typed chicken) was mated to female broiler chicken (Retnoaji et al. 2016; Saragih and Daryono, 2016; Utama et al. 2018). Crowing-typed native chicken of Indonesia, pelung which also possess heavy body weight among other local breeds is being crossed to broiler to diminish broody behavior in their progeny but keep the body weight. Prolactin (Prl) was used as a molecular marker to differentiate broody genotype among progeny. Genetics improvement programs are focus to pause the female chicken from laying eggs by selective breeding. Broodiness is not necessary after the introduction of artificial incubation. Chicken Prl is widely known to control avian ability to produce eggs and broodiness (Cui et al. 2006; Jiang et al. 2009). Plasma prolactin inhibits proliferation of follicle stimulating hormone. Prolactin is produced by the anterior pituitary gland (Jiang et al. 2009), and is distributed to the entire body via circulation. Most of research on broodiness focus on Prl(Cui et al. 2006; Bhattacharya et al. 2011; Li et al. 2013). Avian Prl consists of five exons and four introns (Watahiki et al. 1989), and is located on chromosome 2 (Alipanah et al. 2011) whereas Prl receptor (Prl-R) consists of 15 exons and 14 introns (Rashidi et al. 2012). Quantitative traits loci are growth rate and laying performance, which is measured by quantitative genetics assay. Single-strand conformation polymorphism (SSCP) and random fragment length polymorphism (RFLP) were performed as described previously (Kalvatchev and Draganov, 2005; Fathi et al. 2014) targeted specific region contains alternative nucleotide. Single nucleotide polymorphism (SNP) is the most common variation in the genome caused by mutation and is widely used in candidate gene association study. These accumulated or standalone SNPs might later contribute to an emergence of new phenotypes (Patnala et al. 2013). Previous studies reveal a positive correlation of SNPs with growth traits and body measurements, e.g. number, day old chick (DOC) body weight, and age of sexual maturity (Rashidi et al. 2012). Recently, there were several research attempts to identify an association of Prl and Prl-P polymorphism and inbreeding depression to broody and body weight related trait in Indonesian chicken lines.
MATERIALS AND METHODS
Pure genetic lines of male pelung chicken were obtained from a reputable chicken breeder and were confirmed by crowing voice analysis, while female broiler was obtained from a commercial broiler breeder. Chicken mating took place at the breeding facility in the university farm (Pusat Inovasi Agroteknologi Terpadu) Bantul district, Yogyakarta special province of Indonesia. The breeding facility was open-sided pen which accommodated 12-hours of natural light. All chickens were given free access to feed and waterand all produced eggs were artificially incubated using an incubator machine. Progeny were differently mated to produce third backcrossed chicken (BC3) and forth filial (F4) as two chicken lines (Figure 1). Recorded from 2012 to 2018 hatched DOC first filial (F1) and its descent were raised following standard chicken care sheet to 49 days of age and were weighted every week (Fuller, 2009). The individual inbreeding coefficient was assessed as follows:
Fx: inbreeding coefficient.
Fa: inbreeding coefficient of shared ancestor.
n: amount of trace from shared ancestor to desired individual.
Chickens were given free access to accumulate egg and exhibit broody behavior. After laying eggs, female chicken was tagged as non-broody if they ignored to cover the accumulated egg on her nest more than one week and the otherwise for broody traits.
DNA extraction and analysis
The blood sample from each hen was collected by intravenous injection on the wing branchialis artery using 3 Gauge disposable syringes and stored in vacuum (DB VACOTAINER®). Whole genome chicken DNA was extracted according to the manufacture’s protocol (ROCHE®). All DNA quantification was done using spectrophotometer and 1% agarose gel before amplified on thermal cycler (Bio-rad®). Specific primers for Prl were; forward 5′-GCCTCAATTTCCAAACCAGAC-3′ and reverse 5’-TGCCTTCCAACCCCTATGAC-3′ and for Prl-P was: forward 5′-ATAACAATGGCCTGTCTTGC-3′ and reverse 5′-CCACTGATCCTCGAAAACTC-3′according to previous research (Cui et al. 2006). The 25 μL of polymerase chain reaction (PCR) cocktail contained 50 ng of DNA template and 10 μL of PCR mix (Fast Start PCR Kit, ROCHE®), 0.25 mM forward primer, 0.25 mM reverse primer. The PCR protocol was 94 ˚C for 5 min followed by 30 cycles of 94 ˚C for 45 second, 50.6 ˚C (Prl-P) or 57 ˚C (Prl) for 1 min, and 72 ˚C for 1 min and a final extension at 72 ˚C for 10 min. The PCR products were electrophoresed on 1.5% agarose gel.
Figure 1 Representative chicken mating scheme to produce (A) third backcrossed (BC3) and (B) forth filial (F4) as two difference lines
The protocol for SSCP is as follows: 4 μL of PCR product was diluted to 16 μL loading buffer (95% formamide, 0.005% xylene cyanol FF, 0.005% bromphenol blue, 20 nM EDTA, 2 M urea) subsequently heat denatured using water bath then immediately snap chilled using ice block. The mixture was loaded to wells and running on vertical non-denaturing 8% polyacrilamide (37.5:1) gel electrophoresis. Gel was stained by ethidium bromide after electrophoresis. Restriction fragment length polymorphism (RFLP) protocol is as follows: A 10 μL PCR product was mixed to 18 μL H2O, 2 μL 10× buffer R and 2 μL HindIII (Fermentas) and subsequently incubated for 4, 8, 12, 16, 20, and 24 hours at 37 ˚C. After incubation, mixtures were run in 2% agarose gel to visualize enzyme digestion. All acquired molecular data were analyzed using ANOVA one way (SPSS, 2011).
RESULTS AND DISCUSSION
Body weight inheritance in pedigreed chicken
Prolactin (Prl) is a member of growth hormone super family (Brooks, 2012), which has a broad range of effects on the organism. It was well known that prolactin contributes to growth and development (Kulibaba, 2015), female reproduction (Sangeeta Devi and Halperin, 2014), fish osmo-regulation (Seale et al. 2013), and immunomodulation (Costanza et al. 2015). In poultry species, prolactin is reported to be closely associated with growth and biometrical traits in ducks (Mazurowski et al. 2016) turkey (Fathi et al. 2014) and chicken (Zerehdaran et al. 2004). Repeated backcrossing and inbreeding mating were used to generate a fine introgression line. Chicken line which produce eggs continuously without broodiness, derived from repeated backcrossing and inbreeding. Backcrossed to its pelung parent, F1 chicken presumed inherit growth characteristics and morphological characters to its progeny (BC1, BC2, BC3, respectively). Similarly, full-sib mating (F1×F1; F2×F2; F3×F3) were purposed to generate a morphologically uniform generation. It has been shown that F1 gains more than 1300 g, while its descent BC3 and F4 had gained 750 and 850 g at seven weeks of age (Figure 2). Based on our research, weight gain was decreased due to the inbreeding depression caused by individual inbreeding coefficient and allel segregation (Figure 2). Inbreeding coefficient of evaluated chicken was 0.4375 (BC3) and 0.422 (F4). Inbreeding depression might appear within progeny due to the expression of deleterious homozygous alleles (Panetto et al. 2010). Precisely, the weight gain trend through generation was alternately decreased. Not only in chicken, the evidence of inbreeding depression was found in a large number of both domestic (Panetto et al. 2010) and wild species (Christie et al. 2012) and indeed share similar symptoms. Under artificial selection process, major genes are segregated through generation. A long-term genetic selection might result in accumulation of homogeneity and frequencies of unfavorable alleles. The impact of additive gene action which originated from non-additive genes might collaborate in final phenotypes.
Figure 2 Growth rate of pedigreed chicken each generation after genetic selection through five generation
Backcross line (left) and Inbreed line (right)
Broody inheritance in pedigreed chicken
Prolactin plays has an important role in chickens during egg production since the onset of broodiness is initiated by the elevation of plasma prolactin which results in ovarian regression and stops egg production (Li et al. 2013). In our study, breeding facility exploited 8 to 10 hours of natural light to chicken photoperiodicity. While local breeds (pelung, kampong, bantam) used as control exhibit broodiness in the same places in which several pedigreed Ayam progeny not show due to intrinsic factor as shown in Table 1. Based on recent observations, the broody traits were disappeared after three serial backcross mating (F1×BC2) or two serial inbreed mating (F1×F1). Furthermore, in backcross mating, BC1 used as parents which broody traits inherit the behavior to third BC2 progeny. Presumed, F1, BC1, and BC2 were heterozygote and BC3 was homozygote despite less objective due to small population. Similarly, inbreeding lines which use same male F1 as parent, generated F2 and beyond (F3, F4, respectively) were homozygote for broody trait. Limited to our pedigree, a simple Punnet square can be applied to explain broody inheritance. Broody is a recessive trait, and controlled by one gene and one inhibitor gene. Broodiness is when the hen stops laying eggs and stays on nest for a long time. During broodiness, hen will show defensive act when something or someone approach and minimize feed intake (Romanov et al. 2002). Hen also routinely rotates her eggs to distribute heat evenly. Plasma prolactin was detected at highest during broodiness. There are several intrinsic (polymorphism, epigenetic) and extrinsic (humidity, temperature, feed availability, and photoperiodicity) (Geng et al. 2014) factors which affect broodiness. Polymorphism and epigenetic status (DNA methylation, histone acetylation) on regulating genes like prolactin, progesterone, and luteinizing hormone influence body metabolism which definitely alter organism behavior toward broodiness.
Table 1 The occurrence of broodiness within progeny of pedigreed chicken compared to positive control (kampong, pelung, bantamn) and negative control (broiler and Brown Leghorn)
Polymorphism on prolactin and prolactin promtor
The effect of Prl and Prl-P gene polymorphism on growth traits and broodiness were analyzed. The relationship between body weight and broody event with identified genotypes in two chicken lines (backcross and inbreed) were examined. Since only one haplotype was produced during experiment, Table 1 does not contain data concerning the association of this genotype with growth and broody traits in the evaluated chickens. In this study, vertical and horizontal gel electrophoresis failed to differentiate two traits. A specific primer amplified nucleotides 395-576 from a total 4391 bp of chicken Prland Prl-P with presumed variability. According to a previous report by utilizing a sequence primer will be able to detect polymorphism in White Leghorn, Yangshan, Taihe Silke, White Rock, and Nongdahe but monomorphic in Indonesian chicken, it might be Prl and Prl-P conserve region in Indonesian chicken. Current Brown Leghorn, Broiler, and pelung specimen was possess monomorphic sequence but exhibit difference broody traits might be from polymorphism on other region. The inclusion of peppermint and anti-prolactin drug on feed cause diminshed broody behavior (Eltayeb et al. 2010). Other report indicated the plasma level of prolactin may vary on different chicken lines in tropical climate (Banu et al. 2017). These findings show that broodiness trait might be regulated on proteomic level (Banu et al. 2017). Another possibility, recent instrument was not precise to visualize polymorphism. Based on this research, broodiness is a recessive trait which is controlled by a single promoter gene and single inhibitor gene whereas polymorphism on prolactin (Prl) and prolactin promoter (Prl-P) fragment were not detected.
Broodiness is a recessive trait can be annihilated by selective breeding program. Prolactin is importance marker for egg production-related traits in chicken.
The authors wish to acknowledge Pusat Inovasi Agroteknologi Terpadu (PIAT) Gadjah Mada University for providing breeding facility and research funding (Grant No: UGM/KP4/224/C/03/06).