1. Introduction
Animal blood components reflect their immune system and metabolism of nutrients. Serum is the fluid and solute fraction of blood that lacks erythrocytes, platelets, leukocytes, and clotting factors [
1]. Serum contains a wide range of nutrients, including proteins, electrolytes, antigens, antibodies, hormones, and exogenous elements not needed for clotting. Serum is also required for the body's delivery of nutrients, preservation of the homeostasis of the intracellular environment, and electrolyte and acid-base balance [
2,
3].
Serum biochemical indicators serve as vital proxies that reflect the physiological state and functions of different organs. As molecular phenotypic biomarkers, they are commonly employed as general indicators to assess an organism's immunological status and overall health conditions [4-6]. Many of these parameters appear to have moderate to high heritability in various species including human, pigs, and horses [7-9]. Correspondingly, these traits were expected to be under tighter genetic control compared to the associated diseases and complex traits, since they are directly linked to the biochemical pathways, which might provide valuable information about the underlying biological control [
7,
10,
11]. Therefore, identifying the genetic architecture responsible for their variability may contribute to a better understanding of the biological processes involved in various diseases and complex traits that are linked to these molecular phenotypes.
Animal welfare and health status have paramount importance for all livestock enterprises since any deviation from good health might have adverse effects on the profitability, productivity, and sustainability of production systems [
12]. Mounting evidence suggest that serum biochemical parameters have a wide range of associations with disease resistance, resilience, immune functions, productivity, and feed efficiency in various livestock species [7,13-17]. Ruminant production in particular holds significant relevance in addressing two fundamental global challenges: 1) enhancing the food security and nutrition for an expanding global population and 2) addressing the imperative of climate change mitigation [
18,
19]. However, conventional breeding strategies fall short of meeting expectations, especially for those traits that are difficult and costly to measure directly including traits that are expressed later in life such as disease resistance, immunity, and longevity [
5,
20]. Nonetheless, the economic benefits of prioritizing disease resistance and robustness through genome-based selection are suggested to surpass the potential drawbacks of slower genetic progress in other traits in livestock [
21]. Strong relationships between serum biochemical parameters and other economically important traits in livestock would allow those parameters to be used as indicators of indirect selection on many traits, which is expected to mitigate the limitations of a conventional breeding scheme.
Due to the rapid emergence of high-throughput sequencing and genotyping technologies, GWAS has become a widely used statistical approach to discover QTL related to complex traits in various species including human, pigs, cattle, goat, and sheep [4,7,22-25]. Genome-based selection methods are suggested to speed up the genetic progress in selection schemes by reducing generation interval and increasing accuracy and intensity of selection in livestock production systems [26-30]. Sheep play a significant role in ensuring food security and sustainable production within the livestock species, thanks to their resilient adaptability and robust characteristics [
19]. Furthermore, various studies suggest sheep as a more suitable model than rodents for the investigation and developing treatment for several human clinical conditions [
31,
32]. Therefore, dissection of the genetic basis underlying serum biochemical traits observed in sheep is a potential approach to design a comprehensive marker-assisted selection program to prioritize sustainability, enhance resilience, and support animal model development. To date, various genomic loci were associated with serum biochemical parameters in human as well as in livestock such as pigs, cattle and ducks [
4,
5,
7,
10,
33,
34]. However, only one study has been identified that specifically investigates the genomic heritabilities and QTL associated exclusively with serum protein levels in sheep [
12].
Akkaraman sheep is an adaptive fat-tailed breed representing an extensive share of Turkey’s sheep population and spread through diverse terrain, from harsh, semi-arid regions to the mild climates with comparatively moderate productivity characteristics [
24]. Recently, the genome of the breed has also been characterized against various world-wide sheep breeds to understand genomic relationships [
35]. The large spread of the populations, close genomic relationships with various sheep breeds and its hardy and robust nature indicate potential of the breed’s physiology for sustainable production under increased temperatures and extreme environmental conditions due to global warming. Therefore, the aim of the present study was to identify the genetic architecture and genomic loci underlying measurements of certain serum biochemical indicators in Akkaraman sheep including alanine transaminase, aspartate transferase, lactate dehydrogenase, cholesterol, glucose, phosphorus, calcium, creatinine, urea concentrations and total protein levels. Our findings are expected to profoundly contribute insights into the genomic basis of complex serum biochemical traits that are of clinical and physiological importance.
4. Discussion
Blood measurements known as serum biochemical indicators are widely used biomarkers for monitoring the physiological status of human and animals. These signals are utilized across the entire course of the disease, spanning from diagnosis to prognosis and the recovery, primarily in humans and increasingly in animals that may have exposed to different environmental and genetic triggers of disease susceptibility and adverse conditions. Therefore, these traits are frequently thought of as accurate representations of an animal's health and metabolism. Despite the utmost importance of the serum indicators such as lipids, proteins, enzyme activities, minerals and metabolites to the livestock production systems, exceptionally rare studies have investigated the underlying genetic architecture and mechanisms behind those complex traits [
5,
7,
33,
34]. In this investigation, we measured 10 serum biochemical indicators. Certain serum biochemical indicators demonstrated strong phenotypic and genetic correlations among each other. To the best of our knowledge and according to animal QTL database, no published study has systematically demonstrated the genetic parameters among some or all the 10 serum biochemical indicators and genomic loci using a GWAS of SNPs in lambs [
53]. Additionally, only one study was observed to have focused on the genetic basis of protein levels in sheep [
12]. The aim of the current study was to identify the underlying genetic architecture for blood serum indicators in lambs. ALT and AST averages were in the range with the previous reports for Akkaraman lambs and other breeds such as Ba sheep, Karakul and Tzurcana ewes, Balami ewes, Lori-Bakhtiari and Mehraban sheep and Santa Inês ewes. On the other hand, similarly, low heritability estimate was detected for UREA in Santa Inês sheep [
54,
55].
Genetic variance in serum parameters plays a crucial role in understanding animals' ability to combat infections and stress. This insight can aid in devising better strategies to enhance disease resistance and resilience [
7]. The identified low to moderate genomic heritability estimates for blood serum biochemical traits indicate the potential of genomic selection to result in a gradual improvement in breeding programs in sheep. In the present study, heritabilities were estimated for instance TPRO (0.55±0.14), UREA (0.18±0.11), LDH (0.36±0.14) and ALT (0.14±0.10) (
Table 1), which indicates considerable genetic effects on these protein fractions and probably their potential use as biomarkers for genetic selection. This result differs from the reported studies in Lori-Bakhtiari sheep, where genomic heritability was found as low (0.00 ± 0.29) due to the limited number of animals, causing high standard errors of the heritability estimates [
12]. Similarly, low heritability estimate was detected for UREA in Santa Inês sheep and in Holstein-Friesian cows [
55,
56]. Our study suggests a genomic heritability estimate for serum CA to be 0.27±0.13 in Akkaraman lambs, which is higher than that of described for cattle [
56]. The current study is the first research focusing on the genetic parameters of a wide range of serum biochemical indicators for Akkaraman sheep while one of very first among global sheep populations. However, further research is still required to determine the genetic background of blood serum indicators precisely, as also indicated by the slightly high standard errors of the heritability estimates, which were caused by the relatively low number of animals studied.
Multiple candidate genes were identified in the present study (
Table 2;
Table 3). One of the most striking results of our study is the enrichment of biological processes for the candidate genes that aid disease response and immune system regulation. Many candidate genes suggested by our study are predicted to be part of biological processes such as physiological response to stimulus (GO:0050896), regulation of metabolic process (GO:0019222), immune system process (GO:0002376), regulation of immune system process (GO: 0002682), immune response (GO:0006955), regulation of response to stress (GO:0080134), cell communication (GO:0007154) and regulation of signaling (GO:0023051) in various organisms. Additionally, some of those candidate genes were predicted to have molecular functions such as catalytic and transferase activities as well as ion, small molecule, and enzyme binding.
A genome-wide associated SNP (
rs415766081; p = 1.022×10-06;
Table 2) was located in the intron of Spectrin alpha, erythrocytic 1 (
SPTA1) gene on OAR1 for CHO. Cholesterol is a vital molecule for cellular processes such as membrane fluidity and permeability to gene transcription, growth and development and serving as backbone of steroid hormones and vitamin D analogs [
57]. Spectrins are big, flexible proteins made up of head-to-head connections between α-β dimers, which combine to form the standard heterotetrameric spectrin structure. Functional annotation of
SPTA1 shows that it is involved in fundamental biological processes such as actin cytoskeleton organization (GO:0030036), immune system process (GO:0002376), lymphocyte homeostasis (GO:0002260) and positive regulation of T-cell proliferation (GO:0042102) with molecular functions such as actin filament binding (GO:0051015) and calcium-ion binding (GO:0005509) in various mammals. KEGG enrichment also shows that it is involved in apoptosis. Together with the cytoskeletal network, the spectrin-based membrane skeleton primarily preserves the mechanical characteristics and integrity of the cell membrane [
58]. Both actin filament organization and calcium have long been recognized for their critical role in serum cholesterol levels [
59,
60]. Spectrins are highly conserved across several species and were once thought to be only present in the human erythrocytic membrane [
61]. Orthologues of this gene has been associated with increased B cell number, IgG levels and T cell number in mice [
62].
Another genome-wide associated SNP on OAR17 was found 31 Kb apart of the microsomal glutathione S-transferase 2 (
MGST2) gene for CA (
Table 1). The
MGST2 is a member of the superfamily designated MAPEG (membrane-associated proteins in eicosanoid and glutathione metabolism), and has a role in the interactions between proteins that detoxify foreign and endogenous highly reactive lipophilic substances and proteins involved in the endogenous metabolism of reactive lipophilic intermediates (leukotrienes) [
63]. Functional enrichment showed that the
MGST2 is involved in biological processes such as Eicosanoid metabolic process (GO:0006690), specifically leukotriene metabolic process (GO:0006691) and glutathione biosynthetic process (GO:0006750) as well as response to stress (GO:0006950), defense response (GO:0006952) and inflammatory response (GO:0006954) in various mammals including sheep.
MGST2 has been annotated by KEGG to be involved in the glutathione metabolism, drug metabolism, metabolic pathways, drug resistance, chemical carcinogenesis by receptor activation as well as fluid shear stress and atherosclerosis. Within the functionally varied MAPEG family,
MGST2 is a mainly glutathione-dependent peroxidase and cytoprotective glutathione S-transferase and has high homology with Leukotriene C4 Synthase (
LTC4S) [
63]. Eicosanoid metabolism, in terms of functional coupling of calcium-dependent phospholipase A2 (
cPLA2) plays role in role in the regulation of intracellular Ca2+ concentration in different cells [
64]. It is worth noting that an association between
LTC4S promoter polymorphism and coronary artery calcium thickness was identified in women [
65].
The genome-wide associated SNP for serum creatinine levels is located at 42 Kb upstream of
CACUL1 (CDK2 associated cullin domain 1) on OAR22.
CACUL1 is predicted to engage in a wide range of organic substance metabolic processes (GO: 0071704) such as proteolysis (GO: 0006508), positive regulations of cell population proliferation (GO:0008284) and protein kinase activity (GO: 0045860) with its ubiquitin protein ligase and protein kinase binding activities. Serum creatinine, as a waste product of muscle metabolism, is one of the primary indicators of renal dysfunction or impaired filtration [
66]. Various CDKs (cyclin-dependent kinases) has previously been associated with kidney functions including cell proliferation and filtration in human [
67,
68]. Additionally, a study in mice showed that increased expression of
CDK2 protects podocytes (i.e., a layer of cells around glomerulus where filtration of blood takes place) from apoptosis while reduced expression of
CDK2 leads to increased susceptibility to diabetic nephropathy [
69].
The genome-wide associated SNP for serum glucose (
rs428784360) was intronic to ENSOARG00020040484.1 (
Table 2). This long noncoding RNA has not had much annotated function as yet. The only other gene nearby was LOC121818761, which has RNA evidence but little assigned function as yet. To our knowledge, this study is the first report linking these genes to blood glucose. Further work will be required to investigate their connection to blood glucose and diabetes.
Insulin-like growth factor binding protein 7 (
IGFBP7) on OAR6, as a regulator of insulin-like growth factors (IGFs), was suggested by our study as a genome-wide candidate for serum Lactate dehydrogenase (LDH) levels in sheep. LDH is an enzyme found throughout cells in diverse living organisms, participating in carbohydrate metabolism by facilitating the conversions of lactate and pyruvate using the NAD+/NADH coenzyme system. The
IGFBP7 takes part in diverse biological processes such as the regulation of cell growth (GO:0001558), response to stimulus (GO:0048583), signaling (0023051) as well as the regulation of steroid metabolic process (GO:0019218) and response to corticosteroid (GO:0031969), glucocorticoid (GO:0051384), chemicals (GO:0042221) and steroid hormones (GO:0048545). Its protein shows molecular functions such as insulin-like growth factor binding and structural molecule activity. IGFBPs and IGFs has been consistently documented to play a pivotal role in immune response of animals and human [70-72]. Elevated plasma
IGFBP7 levels was recently found to be correlated with chronic inflammation in human [
73]. Simultaneously, IGFs are known to have a regulatory role in glucose uptake, glycogen and lactate metabolisms, especially in Warburg effect (i.e., increased rates of glucose uptake and preferential breakdown of glucose into lactate, even when mitochondria are operating normally), where LDH is also a key enzyme [
74,
75]. Therefore,
IGFBP7 as a regulator of IGFs can be suggested to have an indirect regulatory role in serum LDH levels.
Finally, a putative QTN (
rs404995480; p = 6.902×10
-07;
Table 2) was detected in the intron of Par-3 Family Cell Polarity Regulator (
PARD3) gene on OAR13 for IP.
PARD3 is known to have significant roles in cytoskeleton organization (GO:0007010), establishment of cell polarity (GO:0030010), organelle organization (GO:0006996) and establishment of the localization in cell (GO:0051649) with its molecular functions such as protein binding and phosphatidylinositol binding. It is annotated by KEGG to have roles in Rap1 and chemokine signaling pathways, endocytosis, Hippo signaling pathway as well as adherens and tight junctions. Inorganic phosphorus plays numerous metabolic roles as a reactant (glycolysis, oxidative phosphorylation, glycogen phosphorolysis, and mineralization) and product (nucleic acid synthesis, ATPases, GTPases, and phosphatases) and recognized as a signaling molecule as well [
76]. Many studies have suggested that
PARD3 is regulated by phosphorylation [
77].
PARD3 is a PDZ-domain-containing scaffold protein that forms a trimetric complex with
PAR6 and atypical protein kinase C (
aPKC) to regulate the initial cell polarity cues. Cell polarity, the asymmetric distribution of proteins, organelles, and cytoskeleton, plays an important role in development, homeostasis, and disease, which might be crucial during many types of asymmetric cell division to set up functional asymmetries between daughter cells [
78].
PARD3, as a member of PAR complex, is one of the cell polarity complexes that can regulate vesicle transport and control the localization of cytoplasmic proteins primarily by regulating the phosphorylation of phospholipids called phosphoinositides [
79]. Phosphoinositides in which one of the isomers is phosphate, serve as docking sites for proteins at the cell membrane, and their state of phosphorylation determines which proteins can bind [
80].
Taken together, our results suggest 6 genome-wide and 17 chromosome-wide SNPs and 19 candidate genes as well as 4 uncharacterized regions to be underlying 10 serum biochemical parameters. The relevant importance of suggested candidate genes to immune system, defense response, cytoskeleton organization and other biological processes are mostly characterized in various species. Simultaneously, our study revealed various genetic parameters and phenotypic correlations for those serum biochemical indicators in sheep. None of the associated SNPs had previously been linked to serum biochemical traits in sheep, mainly because there has been only one study implemented GWAS for only protein levels in sheep. Therefore, results of this study can be used to shed a light on the research of underlying molecular mechanisms behind serum biochemical traits in sheep, which are directly related to welfare and health status of animals and indirectly of high economic importance for sheep production systems. Additionally, since sheep can be observed as a model organism to study welfare and diseases in human, our results also confer significance medical research in human. In any case, further molecular and population-based validation studies are required to prove causality of the associated SNPs and suggested genes for their use in sheep genetic improvement programs, gene editing studies and targeted drug applications that aim better immune system, health, and welfare both in human and sheep.
Author Contributions
Conceptualization, M.K., Y.A., S.N.W. and M.U.C.; methodology, M.K., Y.A., S.N.W. and M.U.C.; software, M.K.; validation, M.K., Y.A. and M.U.C.; formal analysis, M.K.; investigation, M.,K, Y.A., S.B. E.Y. and M.U.C.; resources, M.K., Y.A., S.B., E.Y. and M.U.C.; data curation, M.K. and M.U.C.; writing—original draft preparation, M.K. and M.U.C.; writing—review and editing, M.K., Y.A., S.B., E.Y., S.N.W. and M.U.C.; visualization, M.K.; supervision, M.U.C. and S.N.W; project administration, M.U.C. and M.K.; funding acquisition, M.K. and M.U.C. All authors have read and agreed to the published version of the manuscript.