Reproduction characteristics are of profound importance for livestock and poultry both in evolutionary and economic perspectives. Fertility and hatchability are major parameters of reproductive performance for egg-layer type of chickens. The heritability estimates for fertility, embryo mortality, and hatchability of fertile eggs in chickens reportedly range from 0.05 to 0.13, which indicate these important traits bear heavy influence from management measures, nutrient supplies, and surrounding environmental conditions [1] -[5] .

Since the foundations for genetic improvement were laid by Robert Bakewell in the 18^th century [6] , a variety of ways to employ inbreeding in combination with intensive selection to achieve breeding goals has been attempted. The adverse effects of inbreeding, commonly referred to as inbreeding depression, are also richly documented. In poultry, inbreeding depression not only adversely affect production performance, but also reproductive characteristics, including fertility, embryo mortality, and hatchability [7] -[13] .

The adverse consequence resulted from inbreeding, particularly severe in closed small populations of domesticated poultry, leads to partial or complete loss of poultry lines or flocks from time to time [12] . For instance, five out of a series of 24 recombinant congenic strains (RCS) of White Leghorns, initiated and maintained at the USDA, Agriculture Research Service (USDA-ARS) laboratory at East Lansing, Michigan, completely failed to reproduce by the sixth generation of full-sib mating [14] . Inbreeding escalates homozygosity and speeds up fixation, by chance, of deleterious recessive alleles simultaneously at multiple loci. Due to natural or artificial selection, such recessive alleles generally remain with low or very low frequencies at heterozygous state in outbred populations, and their harmful effects are hardly detectable phenotypically. However, when inbreeding takes place, the chance for homozygosity of such deleterious alleles is drastically increased. Especially when inbreeding occurs consecutively over generations, homozygosity of such alleles at multiple loci generally show up at high frequencies, which would have significant negative impact on production, reproduction, and fitness characteristics of the populations [6] [8] [11] [12] [15] [16] .

Despite of the hurdles and barriers along the way in developing inbred animals, inbred, recombinant inbred, and/or recombinant congenic inbred strains have been generated in many organisms including mice, cattle, and chicken [10] [14] [17] . Inbred lines, especially characterized inbred lines of animals are invaluable resources for genetic studies [17] -[21] . In particular, many unique genetic lines of chickens were developed and thirty five of which have been maintained at the USDA-ARS, Avian Disease and Oncology Laboratory (ADOL) at East Lansing, Michigan. Two-thirds of the genetic lines are highly inbred lines or RCS. Chickens from all of the lines have been serving as critical resources for successful completion of important projects in the past and remains being critically vital for new projects that are conducted in the genomic and post-genomic era at ADOL. The ADOL inbred lines as well as outbred lines of chickens also have been one of the important sources for research and diagnostic tests at other research centers and laboratories in the United States and around the world. The National Animal Germplasm Program (NAGP; http://www.ars.usda.gov/Research/docs.htm?docid=22314) approved and collected all chicken lines developed at ADOL, including the thirty five lines that are currently maintained at ADOL. This study was aimed to provide estimates on fertility, embryo mortality, and hatchability of fertile eggs for 21 out of the 35 lines, which include two highly inbred lines of chickens and a series of 19 RCS using sampled reproduction records during a period of eight years.

Inbred lines are invaluable resources for biological research, which provide experimental animals of high constancy within each of individual lines, and of distinctly genetic diversity between lines. The animals with such characteristics enable high detection power of biological experiments for difference in treatment and/or genetic effects on biological traits of interest over time and places with relatively high repeatability [10] [14] [18] [20] [23] .

ADOL at East Lansing, Michigan, currently maintains 35 special experimental lines of White Leghorns. Two- thirds of these lines are highly inbred. These lines are tested annually for purity by blood-typing tests using over 40 antisera. The breeders of all the lines are unique in that they are maintained in a quarantined state and, on the basis of frequent serologic tests for 11 pathogens, are considered free of infection from common poultry pathogens. Many of the lines each year produce either embryos or day-old chickens that are shipped out to meet the research or diagnosis needs of institutes or laboratories in the United States and around the world upon requests, in addition to fulfill the needs for research conducted on the site.

Line 6₃ and line 7₂ were initiated for establishment as inbred lines in 1939 at ADOL with hatching eggs from similar White Leghorn strains. Line 6₃ was one of the lines that were initiated to define genetic resistance to avian virus-induced tumors collectively termed as avian leucosis complex at the time. It was later shown by studies that the avian leucosis complex was consisted of tumors caused by two types of avian viruses commonly co-existed at the time in chicken houses, they are avian leucosis retroviruses (ALV) inducing principally lymphoid leukosis and Marek’s disease herpesviruses (MDV) inducing MD in susceptible chickens [14] [20] [24] [25] . Line 6₃ chickens were selected for susceptibility to both ALV and MDV infection but uniquely resistance to tumor progression. Line 7₂ chickens were selected to define genetic resistance to subgroup A and B exogenous and subgroup E endogenous ALV infection but for susceptibility to MD tumors [14] [20] .

A total of 19 RCS was set up for establishment as described by Bacon et al. [14] and Chang et al. [26] . Briefly, the series of RCS were developed by crossing the MD resistant line 6₃ and susceptible line 7₂ to produce the F₁

followed by two consecutive backcrosses to the background line 6₃. The series of RCS were established from full-sib mating of the backcross-2 progeny [14] . In theory, each of the RCS on average maintains 7/8 of the line 6₃ genome background and received a random sample of 1/8 of the donor line 7₂ genome. All lines, 6₃, 7₂, and RCS, are MHCB*2 haplotype. The series of 19 RCS are the only extensive set of RCS under development in chickens or any other livestock species. The 19 RCS are under development to identify non-MHC genes that influence traits differing between the progenitor lines 6₃ and 7₂. The progenitor lines 6₃ and 7₂ differ in many ways, including resistance to avian virus-induced tumors [14] [26] -[28] , protective efficacy in response to vaccination followed by Marek’s disease virus challenge [26] [29] [30] , behavior and behavior associated characteristics [31] [32] , primary and secondary lymphoid organ sizes [33] , and genetic and epigenetic differences [34] -[40] . This study showed the two lines 6₃ and 7₂ significantly differ in embryo mortality and hatchability of fertile eggs (P > 0.01). When a RCS differs in phenotype of a trait from the rest of RCS, a limited number of responsible non- MHC genes may be identified for further analysis. Each of the RCS is now over 90% inbred, and inbreeding increases each generation.

Fertility in chickens is reported with a very low heritability of about 0.05 - 0.10 [41] [42] , suggesting the fertility of chicken eggs is heavily influenced by factors other than genetics. The fertility of layer-type of chickens in commercial populations is as high as 97% [43] . The progenitor lines 6₃ and 7₂ were observed with relatively lower average fertilities, 82.4% ± 0.6% and 81.5% ± 1.1%, respectively, over the eight years in comparison with either the commercial populations or some of the RCS. This can be in part attributable to the high degree of inbreeding of the lines. The fertility within each of the inbred lines did fluctuate from year to year in the eight years and the both of the year and the line effects on fertility were statistically significant (P < 0.0001). Yet three of the RCS (RCS-B, RCS-M, and RCS-P) maintained fertility consistently over 90% each year in the eight years and had 93.6% ± 0.5% 93.0% ± 0.7%, and 96.8% ± 0.4% average fertility over the year period, respectively (Table 2). Furthermore, although the progenitor lines 6₃ and 7₂ did not differ in fertility during the eight years evaluated, ten of the 19 RCS were observed with average fertility significantly higher than the progenitor lines (P < 0.05) during the eight year period, which strongly suggested the existence of a substantial polygenic component underlying the fertility phenotype despite the known fact that fertility is a trait of low heritability.

Embryo mortality is regarded as a direct fitness trait that could significantly reduce the efficiency of reproduction and increase the costs of production performance in poultry [44] . Like fertility, embryo mortality is also characterized with relatively low heritability, reportedly ranging from 0.06 to 0.12 [5] . The two progenitor lines 6₃ and 7₂ significantly differed (P < 0.01) in embryo mortality with an eight year average of 28.3% ± 0.6% and 15.7% ± 0.6% embryo mortality, respectively. The donor line 7₂ was among the group of the lowest average embryo mortality and was significantly lower in average embryo mortality than 17 out of 19 RCS while the background line 6₃ was only significantly lower than 3 RCS (P < 0.05) over the eight year period (Table 3, Figure 2). The sizable range (14.5% ± 0.7% - 47.0% ± 1.2%) of the average embryo mortality of the inbred lines during the eight year period indicated that the embryo mortality ratios of the inbred lines of chickens followed a polygenic model of inheritance.

Hatchability of fertile eggs is a major component of reproductive fitness. Reported estimates of heritability for hatchability of fertile eggs fall into the low to moderate category [5] [41] . Industry performance target for hatchability is set for greater than 83% [45] . The average hatchability of fertile eggs in the eight year period for the 21 inbred experimental lines ranged from 26.5% ± 1.1% (RCS-M) to 66.8% ± 0.8% (line 7₂), significantly lower than the industrial target, which was highly likely, in a large part, resulted from high inbreeding, since inbreeding is known to have a seriously negative impact on reproductive characteristics including hatchability in chickens [8] . The progenitor donor line 7₂ was one of the lines with the observed lowest average embryo mortality but was the line with the observed highest average hatchability of fertile eggs over the eight year period and significantly different from the background line 6₃ (Table 3 & Table 4, P < 0.01). The large range of hatchability of fertile eggs among the RCS indicated this trait also follow a model of polygenic inheritance.

Earlier studies showed genetic background due to MHC haplotype differences does exert significant effects on production and reproduction characteristics in White Leghorns [18] [46] . The fact that the year to year effect on fertility, embryonic mortality, and hatchability of fertile eggs was very statistically significant (P < 0.0001) suggesting that environmental effects and random variations highly likely predominate. However, since all of the 21 inbred lines of chickens in this study were housed and managed under standardized control conditions on a specific pathogen free farm and all of the lines share the same MHC B*2 haplotype, it should be within reason to assume that the differences in these traits within each year attributable to environmental influence were moderate to minimal. Thus, the statistically significant differences in fertility, embryo mortality, and hatchability of fertile eggs among the inbred lines strongly suggested that polygenic effects play a substantial role influencing the phenotypes of the reproduction characteristics of the inbred lines and the polygenic effects were imposed by non-MHC genes. Furthermore, both of the progenitor lines 6₃ and 7₂ averaged with relatively low fertility in contrast to over half of the RCS, which suggested multiple loci of genes positively affecting fertility are reciprocally fixed in the two lines, respectively. This speculated model for fertility, to some extent, would also be applicable for interpreting the progenitor lines’ ranking positions with the RCS for embryonic mortality. On the contrary, the progenitor line 7₂ was ranked on the very top for hatchability of fertile eggs, and the line 6₃, at the middle, which indicated both of the progenitor lines 6₃ and 7₂ are likely oppositely fixed at multiple loci of genes negatively impacting on hatchability of fertile eggs, in addition to fixations at loci that positively affect hatchability, especially in the line 7₂. Findings from this study paves the way for further investigation on genetic and environmental influence over reproductive performance of inbred lines of chickens, and particularly in understanding and improving the reproduction fitness of invaluable genetic resources like these inbred lines.

Kulkarni and Zhang thank Deborah Ferguson for her supports on keeping and organizing the reproduction records of the genetic lines of chickens at ADOL, from which data for this study were sampled from.