Humans have recognized milk and dairy products as a valuable source of sustenance since as early as 4000 BC [8, 95]. Indeed, milk is a source of energy, high quality protein, several key minerals and vitamins [8]. The demand for milk and milk products continues to increase, as does the production capacity of individual cows. For example, from 2005 to 2012, milk production of the Canadian dairy herd increased by 6%, while the number of dairy cows declined by 11% ( Ongoing genetic selection as well as advances in the understanding of the biology of lactation and biosynthesis of milk such as improved understanding of the interrelations between dietary components, digestive processes in the rumen and the regulation of mammary synthesis of milk fat have led to improve in management and substantial increases in milk production and productivity [8, 53]. This knowledge includes advanced understanding of: the biology of lactation in many mammals (such as goat, sheep, guinea pigs, mice, rats and several other species); the relationship between structure and function of mammary epithelial cells; the biochemical pathways for the synthesis of milk components; the role of hormones in the development of mammary gland and the regulation of mammary gland function [8, 55, 72]. Specific genes and gene variants that account for variation in milk production traits have been identified and represent another source of knowledge of the molecular events that can contribute to increases in production. For example, a missense mutation in the DGAT1 gene on chromosome 14 has been identified with major effects on milk composition and fat content in dairy cattle [49]. Two other loci with major effects on milk yield and composition have been identified on chromosomes 6 and 20 and involving the ABCG2 and GHR genes, respectively [18, 29]. The identification of such genes and variants has been guided in large part by existing knowledge of the roles of certain genes in lactation. This review seeks to present information on tissues, pathways and genes that can guide future studies aimed at understanding the underlying genetic differences that contribute to variation in milk production and fertility traits. Consideration of fertility is important because of the unfavourable correlations between milk yield and fertility traits [120]. Many reproductive disorders including late resumption of ovarian activity or poor conception rate are related to negative energy balance at the peak of lactation in dairy cows [148, 161]. The identification of the specific genetic variants responsible for variation in these traits should lead to more accurate approaches to genomic selection that work better across generations and breeds [139], and may help us understand or predict potential negative effects on other traits.

Substantial advances in molecular genetics and genomic tools have made the identification of genes and mutations causing simply inherited Mendelian traits relatively straightforward [34, 46]. However, quantitative traits such as milk production in dairy cattle are polygenic, affected by multiple genes and mutations at many sites in the genome [46, 139]. Many of the mutations that affect these quantitative traits have small effects on the phenotype and explain only a small portion of the genetic variance. Therefore, successfully identifying casual mutations and regions affecting these traits is more difficult compared to simple, highly heritable traits [46].

A major goal of quantitative trait loci (QTL) studies in livestock is to identify regions, genes and markers that can be used in breeding programs. A QTL is a section of DNA (a locus) that is correlated with variations in a given phenotype [77]. Identifying the QTL affecting a trait has previously involved low density markers and the application of linkage mapping [46, 106]. Subsequently, through the discovery of markers within new target regions, the use of fine mapping, and the application of high density SNP (single nucleotide polymorphism) panels, the accuracy of QTL detection increased [73]. Fine mapping relies on linkage disequilibrium (LD) between SNPs and unknown casual variants, which are also called quantitative trait nucleotides or QTN [59, 139]. Recent association analyses using dense genetic markers have detected variants associated with milk production-related traits including mammary gland development, and prolactin signalling and involution pathways [57, 59, 122, 146]. After identifying a QTL region, however, it may still be difficult to determine which variants in the region truly affect the trait. Knowledge of the physiology of the trait and of relevant metabolic pathways can be valuable in this regard, as it can highlight genes in the QTL region of potential importance [164]. One of the aims of this review is to facilitate discovery of the mechanisms underlying QTL associated with milk production and fertility-related traits, through highlighting tissues, pathways and genes that are known to play important roles in lactation.

Physiological and Metabolic Adaptations Early in Lactation in Dairy Cattle

The transition period in dairy cattle presents an enormous metabolic change and challenge to the high-yielding dairy cow [10]. During this time, the energy requirements of the cow increase to accommodate milk production and maintenance [66, 157]. This increase in energy requirements can be partially met by increased feed consumption but is limited due to low dry matter intake and decrease in appetite that tend to occur around this time; the remainder is met by mobilization of body reservoirs [51]. Adipose tissue is the predominant energy reserve in dairy cattle during periods of chronic energy deficit [10, 129]. Through homeostasis mechanisms, adipose tissue optimizes non-esterified fatty acid mobilization to maintain physiological equilibrium and to provide the required energy early in lactation [5, 10, 129]. However, not only is the homeostasis mechanism in adipose tissue important to support changes in a cow’s condition, a coordinated change in lipid metabolism of other body tissues is also necessary to support the physiological state of the animal [5, 129]. This mechanism of regulation is called homeorhesis and applies to nutritionally insensitive (genetically controlled) regulation of lipid metabolism in dairy cattle to support the physiological state of the animal [5, 129]. Liver is the main site for the uptake of serum free fatty acid, increased lipid β-oxidation and increased gluconeogenesis early in lactation in dairy cattle [5, 129]. Another metabolic adaptation associated with negative energy balance and homeostasis in dairy cows is related to increased use of nutrients and milk lipid droplet in the mammary gland [129]. Therefore, the liver and the mammary gland in dairy cattle are also important tissues in homeostasis and homeorhetic control of lipid metabolism during early lactation [37, 42]. The physiological and metabolic pathways as well as the regulatory components in these three tissues (adipose, liver and mammary gland) are described in subsequent sections.

Adipose Tissue

Early in lactation, lipid metabolism characteristics change in adipose cells (adipocytes). Endocrine profile changes and mobilization of fatty acid from adipocytes begin [6]. Subsequently, the abundance of non-esterified fatty acids (NEFA) in the serum albumin increases to allow uptake by various tissues [129]. Two main metabolic pathways optimizing NEFA mobilization to maintain physiological equilibrium are lipolysis and lipogenesis [129].


Major sites where lipogenesis generally occurs are the intestinal mucosal cells, the hepatocytes (liver cells) and the adipose tissue [81]. In ruminants, the predominant sites are adipose tissue and the mammary gland of lactating dairy cows [81]. These tissues are responsible for the uptake of pre-formed fatty acids from lipid circulation and for de novo fatty acid synthesis using acetyl-CoA derived from the catabolism of carbohydrates [81, 129]. Most of the carbohydrates in ruminants are fermented into acetate while butyrate and propionate are produced to a lesser extent. As such, acetate is the predominant lipogenic substrate in adipose tissue and the mammary gland for de novo fatty acid synthesis [81, 153].

Acetate is first transformed into pyruvate and then into acetyl-CoA through oxidation within mitochondria [81]. Fatty acid synthesis (lipogenesis) begins with carboxylation of this acetyl-CoA to malonyl-CoA. This reaction is catalyzed by the rate-limiting enzyme, acetyl-CoA carboxylase (ACC) [129]. Malonyl-CoA is then condensed with acetyl-CoA by Acyl-malonyl ACP condensing enzyme to produce a four-unit substrate and CO2 as a result [12, 129]. The next three steps in fatty acid synthesis are reduction of a keto group at C-3 to a methylene group and formation of butyryl-ACP [12]. With formation of butyryl-ACP, the first cycle of elongation completes. The elongation cycle continues with condensation of butyryl-ACP with malonyl ACP to form C6-β-ketoacyl ACP, and a similar cycle of reactions repeats until C16-acyl ACP is formed; this intermediate is then hydrolyzed by thioesterase to yield palmitate (C16:00) and ACP [12]. In the case of fatty acid uptake from circulating lipids, the second pathway of lipogenesis in the adipocyte starts, which is hydrolysis of plasma triacylglycerides (TAG) by lipoprotein lipase (LPL), producing NEFA and monoacylglycerides [84, 129]. Depending on the availability of glycerol-3-phosphate and monoacylglycerides, TAGs are synthesized through either phosphatidic or monoacylglycerol pathways [84, 129].


The hydrolysis of triacylglycerols (TAG) by lipase is activated by signals from molecules such as catecholamine (epinephrine and norepinephrine) and adrenocorticotropic hormones [129]. These hormones trigger membrane receptors that activate adenylate cyclase (Fig. 1). Increased levels of cyclic adenosine monophosphate (cAMP) then simulate protein kinase A. This simulation leads to activation of lipase (hormone-sensitive lipase or HSL), which hydrolyzes fatty acids at the sn-1 and sn-3 positions [12]. Then, monoacylglycerol lipase hydrolyzes the remaining fatty acids at the sn-2 position and generates 3 fatty acids (NFEA) and glycerol [12]. Following this hydrolysis, NEFA mobilizes into circulation and quickly attaches to serum albumin for transport to various tissues [129].

Fig. 1
figure 1

Proposed steps, genes and pathways controlling energy metabolism in different tissues early in lactation. Solid/thick arrow lines denote processes that are activated. Solid non-arrow lines denote processes that are inhibited. Adapted from Refs. [14, 75, 88, 129]


The liver has a key role in lipid metabolism and maintaining lipid homeostasis in animals [89, 112]. Physiological, metabolic and endocrine adaptations that take place in the liver during early lactation support lipid metabolism in dairy cattle [89]. Many metabolic disorders affecting transition cows, such as fatty liver syndrome and ketosis occur as a result of increased lipid and fatty acid oxidation in the liver during this period of metabolic challenge [33, 47]. The oxidation of long-chain fatty acids occurs in hepatic mitochondria and peroxisomes [33, 129]. Then triglycerides, the end product of liver β-oxidation, are carried by the lipoprotein very low density lipoprotein (VLDL). Lipoproteins are composed of triglycerides, cholesteryl esters, phospholipids and cholesterol [74]. Since cholesterol metabolism early in lactation has been a subject of intense investigation with regard to lipoprotein carriers, lipid metabolism-related disorders, membrane fluidity and steroid hormone synthesis [33, 74], this section will review pathways and genes that are involved in lipid metabolism as well as cholesterol and steroid hormone synthesis in the liver.

Lipid Metabolism in the Liver

NEFA generated through lipid metabolism can be oxidized by liver mitochondria or peroxisomes for use as an energy source or used by the mammary glands as a source of milk fat [37, 129]. β-Oxidation occurring in the mitochondria involves production of acetyl-CoA, and reduction of nicotinamide adenine dinucleotide (NAD+) and flavin adenine dinucleotide (FAD+) in order to produce adenosine triphosphate (ATP) in the citric acid cycle and electron transport chain. The alternative pathway of hepatic fatty acid oxidation is through peroxisomes.

Oxidation of Fatty Acids in Mitochondria

The β-oxidation of fatty acids in liver occurs mainly in the mitochondria. The carnitine palmitoyltransferase (CPT) system is recognized as a component of fuel homeostasis and transport system for these NEFA for β-oxidation [102]. This system is composed of three enzymes, carnitine palmitoyltransferase I (CPT-I), carnitine-acylcarnitine translocase and CPT-II [33, 102]. NEFA are delivered to the liver and converted to fatty acyl-CoA esters by acyl-CoA synthase. Then acyl-CoA is taken up by CPT-I on the outer mitochondrial membrane to be activated in the form of fatty acylcarnitine [12]. This step is believed to be the rate-limiting regulatory step in the metabolism of long-chain fatty acids [102]. Then, fatty acylcarnitine permeates the inner membrane and by the enzyme CPT-II reforms fatty acyl-CoA [102]. The activated fatty acyl then enters the pathway of β-oxidization in the mitochondrial matrix by a recurring sequence of four reactions: oxidation by flavin adenine dinucleotide (FAD), hydration, oxidation by NAD+ and thiolysis by CoA (Table 1) [12, 99]. The nicotinamide adenine dinucleotide hydrogen (NADH) and FADH produced during these reactions generate ATP in the citric acid cycle and electron transport chain, respectively [12]. When fatty acid mobilization increases in adipocytes, excessive acetyl-CoA generated from β-oxidation is converted into acetoacetate and BHBA (beta-hydroxybutyrate), which are ketone bodies [129]. Ketone bodies are an important energy-providing mechanism for vital organs such as the brain in early dairy cow’s lactation [129]. The remaining free fatty acids will be re-esterified to triglycerides (TG) and exported as VLDL to the plasma (Fig. 1) [50, 74]. Van den Top et al. [150] and Kessler et al. [74] showed that plasma VLDL-cholesterol, LPL-cholesterol (lipoprotein lipase) and TG concentrations decrease distinctively after parturition. Limited secretion of VLDL from liver and accumulation of TG in the liver can then lead to fatty liver syndrome [150].

Table 1 Major enzymes and their physiological function early in lactation in dairy cattle

Oxidation of Fatty Acid in Peroxisomes

The oxidative pathway of NEFA in peroxisomes is similar to that in mitochondria. However, one of the products of these reactions is hydrogen peroxide instead of NADH. In addition, peroxisomes do not contain a respiratory chain linked to ATP which results in the capture of less energy and more heat during peroxisomal β-oxidation [37]. Therefore, peroxisomal β-oxidation may be considered as an overflow pathway to oxidize fatty acids (FA) during extensive NEFA mobilization [37].

Cholesterol and Steroid Hormone Metabolism

The transition period not only requires homeorhetic changes in glucose and lipid metabolism but also cholesterol metabolism [5, 74]. Cholesterol is a fundamental lipid in modulating cell membrane fluidity and is the precursor of steroid hormones such as progesterone, testosterone, oestradiol and cortisol [12]. This section describes the biochemical pathways involved in cholesterol and steroid hormone synthesis, as well as regulatory components and interactions mediating cholesterol homeostasis in dairy cows during the transition period.

Cholesterol Synthesis

Cholesterol and fatty acids are synthesized in the liver [61]. Cholesterol has 27 carbon atoms in its structure, all of which are derived from acetyl-CoA. Synthesis of cholesterol starts with the formation of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) from acetyl-CoA and acetoacetyl-CoA. This stage is mediated by 3-hydroxy-3-methylglutaryl-CoA synthase [156]. Then, HMG-CoA is reduced to mevalonate for the synthesis of cholesterol. The synthesis of mevalonate is the main step in cholesterol formation and is catalyzed by 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-CoA reductase) [12, 156]. Mevalonate is converted into 3-isopentenyl pyrophosphate and this molecule condenses in three steps to form farnesyl pyrophosphate [12]. Then, two molecules of farnesyl pyrophosphate reduce to form squalene. This reaction is catalyzed by the endoplasmic reticulum enzyme, squalene synthase. Squalene is cyclized by oxidosqualene cyclase to form lanosterol (Table 1). The final stage of cholesterol synthesis is the conversion of lanosterol to cholesterol in a multistep process [12].

Steroid Hormone Synthesis from Cholesterol

Cholesterol is the building block for all of the five major classes of steroid hormones: progestogens, glucocorticoids, mineralocorticoids, androgens and oestrogens [12]. Steroid hormone synthesis is stimulated and controlled by different peptide hormones in different organs. For example, follicle-stimulating hormone (FSH) controls the progesterone and oestrogen synthesis in ovarian granulosa cells, whereas luteinizing hormone (LH) regulates progesterone synthesis in ovary luteinized granulosa–luteal cells, and androgen production in ovarian theca-interstitial cells [63]. Steroid hormones contain 21 carbon atoms. The first stage in the synthesis of steroid hormones is the removal of a six-carbon unit from cholesterol to form pregnenolone, a reaction catalyzed by the cytochrome P450 side-chain cleavage enzyme (P450scc, CYP11A1) on the inner mitochondrial membrane [63, 82]. Progesterone is then synthesized from pregnenolone in two steps: (1) oxidization of the 3-hydroxyl group of pregnenolone and (2) isomerization of the ∆5 double bond to a ∆4 double bond [12]. This step is catalyzed by the rate-limiting Δ5-3β-hydroxysteroid dehydrogenase–isomerase (3βHSD) enzyme in steroidogenic cells in the ovary [63, 116].

Androgens and oestrogens are synthesized from progesterone in two steps: (1) hydroxylation of progesterone at C-17 and (2) cleavage of the side chain consisting of C-20 and C-21 carbons to yield androstenedione which is an androgen. This reaction is catalyzed by the 17α-hydroxylase enzyme which uses Δ5 as substrate for the lyase activity [116]. Testosterone, which is secreted from theca-interstitial cells of the ovary, is another androgen and is formed by the reduction of the 17-keto group of androstenedione [12, 116]. This reaction is catalyzed by 17β-hydroxysteroid dehydrogenases [116]. The oestrogens, oestrone and oestradiol (E2), are synthesized from androgens by the loss of the C-19 methyl group [12]. Testosterone and androstenedione can be further metabolized to oestradiol and oestrone, respectively, in the ovary in a reaction catalyzed by the aromatase enzyme (CYP19A1) [12, 63]. The ovarian granulosa cells secret progesterone (P4) and oestradiol, and ovarian theca cells predominantly synthesize androgens.

Mammary Gland

The mammary gland synthesizes and secretes a large number of products in the milk including proteins (whey 20% and casein 80%), carbohydrates, coated lipid droplets, water and ions [8]. Milk fat is of major importance to the dairy industry, as it influences the manufacturing properties and other organoleptic qualities of milk and dairy products [8, 14]. Several studies have defined and quantified major metabolic aspects of mammary lipid metabolism. These main lipid-associated metabolic pathways are the ones involved in fatty acid uptake from the blood (through endothelial long-chain fatty acid transport), de novo fatty acid (FA) synthesis (in cytosol), FA synthesis in the mitochondria and milk lipid synthesis, droplet formation and secretion (in the endoplasmic reticulum (ER) membrane) [7, 8, 14]. Fat production and milk FA composition are affected by the stage of lactation and level of production [13, 14, 71]. Transcriptional studies of the bovine mammary gland have highlighted a complex and coordinated set of molecular events that are involved in mammary adaptations to lactation [14, 85]. This section will briefly review these molecular events from endothelial FA uptake to lipid droplet formation in the ER membrane.

Blood Fatty Acid Uptake and De Novo Fatty Acid Synthesis

The mammary gland can use two sources of fatty acids for milk fat synthesis. One source is the de novo synthesized fatty acids produced by mammary epithelial cells; the other source is fatty acids that are obtained from the digestive tract or through mobilization of body reservoirs [96]. Short chain (4–8 carbons), medium chain (10–14 carbons) and a portion of long-chain fatty acids (16 carbons) are synthesized from acetate and β-hydroxybutyrate in the de novo FA synthesis process; the remaining long-chain fatty acids (including the other half of 16 carbon FA and all FA longer than 16 carbons) are taken up from circulation by the mammary gland [7]. In ruminants, fatty acids are derived predominantly from intestinal absorption of dietary and microbial fatty acids [7]. Early in lactation, however, when the animal is in negative energy balance, the contribution from mobilized fatty acids (such as circulating lipoproteins and NEFA) increases [6, 96]. Mammary cells take up albumin-bound FA (or NEFA) and lipoproteins. The VLDL or chylomicrons are also anchored to mammary endothelium by lipoprotein lipase (LPL) which hydrolyzes triacylglycerol (TAG) in the lipoprotein to release the FA [42]. Most of these long-chain fatty acids (LCFA) are then esterified with CoA to LC-acyl-CoA (LCACoA) in the inner face of the plasma membrane before participating in metabolic pathways [14]. This step is regulated by the acyl-CoA synthetase long-chain family member 1 (ACSL1) gene which has been shown to be most predominant among other acyl-CoA synthetase mRNA isoforms in the bovine mammary tissue during lactation [14, 15]. The ACSL1 gene converts free long-chain fatty acids into fatty acyl-CoA esters (Fig. 1). Specific localization of ACSL1 gene product in the plasma membrane, endoplasmic reticulum and the mitochondria-associated membrane supports channelling of LCFA and synthesis of TG from LCFAs [14, 15, 31].

Triacylglycerol (TAG) Synthesis and Formation of Milk Lipid Droplets

The activated long-chain fatty acids (LCACoA) bound to FABP3 (fatty acid-binding protein 3) gene protein are used as substrate for the SCD (stearoyl-CoA desaturase) enzyme, which is located on the ER membrane [14, 15]. SCD adds a double bond to the ∆9 position of unsaturated fatty acids (myristoyl-, palmitoyl- and stearoyl-CoA) and triacylglycerol synthesis (TAG) begins through a series of sequential reactions carried out by the products of the GPAM (glycerol-3-phosphate acyltransferase), LPIN1 (Lipin I) and DGAT1 (diacylglycerol acyltransferase I) genes (Fig. 1) [15]. The first step in TAG synthesis is the acylation of glycerol-3-phosphate to form lysophosphatidic acid (LPA); this step is catalyzed by the glycerol-3-phosphate acyltransferase (GPAT) enzyme [45]. Then a fatty acid is transferred to LPA by LPA acyltransferase (also called AGPAT) enzyme to produce phosphatidate (PA) [147]. The PA is then served as a precursor of diacylglycerol (DAG). Lipin enzyme (an endoplasmic reticulum enzyme) catalyzes this reaction (Table 1) [126]. Finally, DAG converts to TAG by way of the diacylglycerol acyltransferase (DGAT) enzyme [136, 147]. The formed TAGs are enveloped by the ER plasma membrane and gradually move to the apical surface of the cell to the point that they dissociate from the cell [72]. The bovine milk lipid droplet is dependent on the adipose differentiation-related protein (adipophilin, ADFP) for differentiation from the ER membrane and the product of the butyrophilin, subfamily I, member AI (BTN1A1) gene for differentiation from cell membrane [14, 72].

Endocrine Adaptations in Transition Dairy Cows

As a result of negative energy balance (NEB) early in lactation, major changes in hormonal regulation occur in high-yielding dairy cows [35]. This involves changes in the concentrations of key hormones as well as tissue responsiveness. For example, an increase in lipolysis and decrease in lipogenesis occur in order to maintain physiological equilibrium of the body and to satisfy the needs of the mammary gland through nutrient redistribution [5, 6, 35, 129]. Blood hormone concentrations have an important role in mammary gland development and lactogenesis during the periparturient period [5]. Pituitary growth hormone (GH), the thyroid gland hormones, insulin, catecholamines and leptin are some examples of the endocrine factors regulating lipid metabolism [5, 35, 129].

The physiological effects of growth hormone are initiated when it binds to GH receptors (GHR) on target cells. Growth hormone enhances the lipolytic response of adipose tissue to β-adrenergic signals and is reported to have a positive effect on hormone-sensitive lipase (HSL) activity in adipose tissue [39, 129]. Binding of GH to its receptors (GHR-1A) in the liver initiates synthesis and secretion of insulin-like growth factor 1 (IGF-1) [129]. Despite the increase in plasma GH concentrations early in lactation, the abundance of hepatic GH receptors decreases, and as a result plasma IGF-1 also decreases [17, 95, 129]. Decreasing liver GHR abundance initiates lipolysis [129]. Since the concentration of IGF-1 does not fluctuate with feeding activity, it is a good indicator of nutritional status [148]. An optimum concentration of IGF-1 to maintain enough of a pool of circulating IGF-1 and its widespread actions is achieved by six binding proteins (IGFBPs 1–6) and the acid-labile subunit (ALS) [128, 148]. In addition, many members of the somatotropic axis (hypothalamo–pituitary axis) are expressed locally within endometrium [160]. For example, IGF-1 and IGF-2 act through type 1 IGF receptor (IGF1R) and are also expressed in the post-partum uterus [160]. IGF1 and IGF2 are expressed in many organs of the body and have an influence on proliferation, differentiation and metabolic activities. These genes may therefore play a role in uterine involution [93, 160].

Insulin has a regulatory effect on lipogenesis and is an antagonist to the lipolytic actions of GH [153]. Hypoinsulinemia (low concentrations of insulin in the blood) and a decrease in responsiveness of skeletal muscle and adipose tissue to insulin occurs simultaneously in early lactation and leads to an insulin-independent uptake of the available glucose by the mammary gland and greater body lipid mobilization to the liver [11, 129]. This process begins with lower insulin concentration and elevated placental lactogen in the uterus during late pregnancy which stimulates adipose metabolism to provide nutrients for the growing foetus [138].

Leptin, secreted from adipocytes, decreases immediately post-partum as a consequence of energy deficit [87, 129]. This reduction in leptin production matches the plasma insulin profile early in lactation and is consistent with reduced adipose tissue glucose uptake [87, 129]. Leptin hormone secretion is regulated by a complex of different molecules and hormones such as insulin, glucocorticoids and cytokines (tumour necrosis factor (TNF) α), interleukin-1 (IL-1), catecholamines, testosterone and PPARγ [153].

Catecholamines, such as epinephrine and norepinephrine, act as lipolytic stimulators through activating cAMP and then PKA which activates subunits of both HSL and perilipin proteins that subsequently increase lipolysis (Fig. 1) [12, 129]. Perilipin phosphorylation, which occurs through a cAMP-dependent PKA cascade, is essential for translocation of HSL hormone from cytosol to the surface of the lipid droplet [129]. It has been reported that the transcription of the genes producing perilipin, ß-adrenergic receptors and HSL in adipose tissue increases early in lactation in dairy cows [145]. In addition, the responsiveness of bovine adipose tissue to catecholamines increases in early lactation [103].

Thyroid hormones have an important role in the dairy cattle transitional period and in determining cell metabolism intensity, metabolism of lipids and carbohydrates and lactation course in general [36]. These hormones are known for their importance in milk production through stimulation of metabolic rates with other hormones [19]. It has been shown that there is a positive correlation between thyroid hormones in blood and energy metabolism [124]. During negative energy balance and high lipid metabolism, however, the concentrations of T4 (thyroxine) and T3 (triiodothyronine which is four times more active than T4) are reduced (hypothyroidism) in the blood of dairy cows shortly before and after calving [19, 118, 124]. Negative energy balance and an increase in lipid mobilization and hypothyroidism early in lactation in dairy cows are accompanied by metabolic disorders associated with carbohydrate and lipid metabolism such as ketosis and fatty liver [35]. Therefore, thyroid hormones are considered to be important indicators of homeorhetic adaptation to negative energy balance in dairy cows until energy balance is achieved [35, 36, 69].

Reproductive Endocrinology and Hormonal Adaptations in Cows in Negative Energy Balance (NEB) Stage

Reproductive function in dairy cattle is dependent on balanced and coordinated endocrine activity [160]. This includes homeostasis between different reproduction hormones such as gonadotrophin-releasing hormones (GnRH), follicle-stimulating hormone (FSH), luteinizing hormone (LH), prolactin (PRL) and gonadal steroids as well as prostaglandin F2-alpha (PGF) from the uterus [86]. Shortly before parturition, PGF increases and luteolysis of corpus luteum (CL) begins. Progesterone decreases rapidly as a result of CL regression. Plasma oestrogen concentration drops immediately after calving to the values below those found during the normal oestrous cycle [66].

It has been shown that NEB is a reason for poor conception rates in transition dairy cows [41, 161, 162]. The relationship between NEB and fertility stems from the effects of NEB on the resumption of cyclicity and on the quality of oocyte or the corpus luteum which is discussed below.

Ovarian cyclicity and ovulation after calving are necessary for a successful insemination and cows should be able to ovulate approximately 2–3 months post-partum [66]. Gonadotropins play an important role in the onset of this activity. FSH concentrations remain at relatively constant levels throughout the post-pubertal life of a dairy cow, but LH concentrations become pulsatile in cyclic animals [86]. Inhibition of LH pulsatility before puberty leads to decreased ovarian activity [66]. Besides, the onset of puberty has been shown to be associated with the attainment of a critical level of body fat [66, 134]. Therefore, it has been suggested that there is a relationship between the metabolic status of the animal and reproductive endocrine system of dairy cow not only for the onset of puberty in heifers, but also for resumption of ovarian activity in non-adoptive dairy cows early in lactation [134]. In this regard, LH seems to have a more important role than FSH after calving [142]. Low glucose concentrations have also been associated with less amplitude of LH pulses. In addition, endogenous opioid peptides, which are secreted during stress, have negative effects on LH pulsatility and the onset of ovarian activity post-partum [1, 66].

The IGF-1 system is thought to influence the establishment and maintenance of pregnancy of dairy cows through affecting reproductive tract of cows [148]. The IGF-1 protein acts as a co-gonadotroph and amplifies the effects of FSH and LH on the growth and differentiation of ovarian follicles [95, 148]. The IGF-1 system also plays an important role in the survival of the embryo and its plasma concentrations were shown to be associated with longer calving to conception intervals [148, 162]. It has been reported that regulation of IGF1 and IGF2 is in positive manner with ovarian oestradiol production [41, 66, 143]. The concentration of IGF-1 increases notably at a time of increasing oestradiol dominance in the bovine oviduct [143]. Moreover, IGF-1 and insulin have a stimulatory effect on ovarian granulosa cells, increasing proliferation, as well as progesterone and oestradiol production. Insulin and IGF-1 also stimulate androgen production in (ovarian) theca cells [66, 141]. Recent studies have further suggested the role of thyroid hormones in the onset of ovarian activity [66, 150].

The quality of oocytes at the time of insemination is important in non-adopting dairy cows and is dependent on the sufficient number of ovarian cycles and the time that an antral follicle needs to reach its ovulatory size after calving [66]. Several factors and hormones can affect the quality of oocytes. IGF-1 and its binding proteins might affect the quality of oocytes. Follicular development can be inhibited with increased IGFBPs that are known to function as IGF-1 inhibitors [66]. The second is the metabolic status of the dairy cow. As a result of an increase in body fat mobilization and to some extent body protein mobilization, plasma urea concentrations increase early in lactation [66]. The increase in ammonia concentrations may also occur as a result of accumulation of triacylglycerides and inhibition of ureagenesis during the transition period in dairy cows [169]. A high concentration of circulating urea and ammonia in the bloodstream of cows is associated with reduced fertility [83, 128, 160]. Exposure of oocytes in antral follicles to high levels of ammonia concentrations during fertilization may hamper cleavage and blastocyst formation [137]. Increased urea concentration in the blood early in lactation is associated with declined cleavage ratios and blastocyst formation of the fertilized embryo [66, 160]. This increased level of urea concentration in the blood after calving may also influence the expression of endometrial IGF and insulin receptor (INSR) [160]. Wathes et al. [160] reported that expression of IGF1R and INSR was not altered by the energy balance status of the dairy cow early in lactation but was positively correlated with the circulating urea concentration [160].

Regulatory Components and Genes Mediating Metabolic and Endocrine Adaptations

Differences in the success of adaptation early in lactation between cows, under the same conditions and similar production level, suggest that adaptability may have a genetic basis [53, 74, 151]. Many genes, pathways and key candidate metabolites in the plasma have been previously confirmed to be essentially involved in the regulation of metabolic and endocrine adaptations in dairy cow [15, 33, 53, 160]. However, these genes and pathways might be expressed only at a certain point of time in the individual [53]. Some genes, for example those affecting glucose levels, might be expressed in early lactation and others, affecting the abundance of non-esterified fatty acids (NEFA) for example, are expressed 4 weeks before or 13 weeks after calving [53]. Identifying the genes and pathways regulating important biological functions during specific physiological states of dairy cattle may help in the identification of DNA variants that affect milk production and subsequent fertility [139].

Genes and Key Pathways Affecting Multiple Tissues

The onset of lactation in dairy cows is accompanied by an increase in milk synthesis and nutrient requirements, and eventually there is metabolism adaptation to lactation-associated challenges. These adaptations include metabolism adjustments in liver and peripheral tissues (including adipose tissue, mammary gland, skeletal muscle tissues and kidney), and mobilization of body reserves and increased lipid metabolism [163]. The increase in lipid metabolism results in an increase in concentrations of key metabolites NEFA and BHBA in plasma (Fig. 1), and TAG in liver [129, 135]. Several genes and pathways in multiple tissues are involved in regulating these metabolites in lactating dairy cows [14, 15, 23, 53, 90, 98, 135]. A gene-based mapping and pathway analysis indicated that three pathways (steroid hormone biosynthesis, ether lipid metabolism and glycerophospholipid metabolism) jointly affect the concentrations of NEFA, BHBA and glucose in cows during the transition period [53]. The key genes that are involved in regulating energy metabolism in multiple tissues include PPRA, PCK1, PCK, ACACA, FASN, FBP2, FABP3, PPARGC1A, ACSL1, PPARGC1A, AGPAT6, PCCA, LPIN1, ACO, CPT-I, CPT-II and ACSL [4, 15, 27, 94, 131, 163]. These genes are involved in fatty acid uptake (mainly in the liver and mammary gland), mitochondrial and peroxisomal fatty acid oxidation, ketone body metabolism (ketogenesis) and cholesterol metabolism (in liver) early in lactation in dairy cattle [135] and are discussed in the following tissue-specific sections in more detail.

Gene expression studies are revealing the extent to which different genes are involved in different tissues [4, 135, 163, 167]. For example, genes that are involved in carbohydrate metabolism, such as those encoding gluconeogenesis and propionate metabolism enzymes (including PCK1), were expressed more in liver than mammary tissues [4, 167]. However, the related PCK2 gene shows a small difference in expression between mammary gland and liver [4, 163, 167]. Other studies showed that the PCK2 gene might also be active in glyceroneogenesis in lipogenic tissues (adipose tissues) during fasting or restricted feed intake [54, 125] and in the epithelial cells of mammary tissue during lactation [62]. Weikard et al. [163] reported that expression of the PPARGC1A gene was significantly increased in liver, mammary gland and skeletal muscle in lactating cows. The PPARGC1A gene coordinates expression of several proteins and in this way it controls the regulation of several metabolic pathways in response to metabolic challenges [163]. This gene has been reported to have a pivotal role in hepatic glucose synthesis (gluconeogenesis) [121], to be a key gene in mitochondrial oxidative phosphorylation metabolism [109, 115], and to independently regulate the expression of several lipogenic genes after the onset of lactation in dairy cattle [14]. In a study comparing gene expression patterns between liver, mammary gland and skeletal muscle tissues in lactating cows, Weikard et al. [163] indicated that the PPARGC1A and PCCA genes display a significantly altered mRNA abundance between the tissues and across all the cow groups under investigation: cows with different genetic potential for milk performance (high milk performance, medium and low milk performance) and cows with different genetic backgrounds (purebred and combined beef, dairy background). Fatty acid-binding proteins (FABP) are the main transporters of long-chain fatty acids (LCFA) to specific organelles for metabolism [101]. Different isoforms of FABPs have shown unique patterns of tissue-specific gene expression [43] and are most abundant in tissues that are involved in active lipid metabolism [4]. In this regard, FABP3 was shown to have a major role in bovine mammary gland lipid synthesis and is much more abundant in this tissue [4, 14]. FABP1 was shown to be more abundant in liver [27], whereas expression of FABP4 was reported to be greater in mammary and adipose tissues [4, 64]. There are nine isoforms of 1-acylglycerol-3-phosphate O-acyltransferase (AGPAT) in mammals, an enzyme that catalyzes the transfer of fatty acyl-CoA to lysophosphatidic acid [166]. The AGPAT1 isoform is the most abundant one in both liver and mammary tissues [4], but a knockout study in mice suggests an important role for AGPAT6 as well in mammary tissues, in the biosynthesis of milk fat [9].

Genes and Key Pathways Regulating Liver Lipid and Cholesterol Metabolism in Transition Dairy Cow

NEFA, beta-hydroxybutyrate (BHBA) and glucose are key factors in the metabolic status of transition dairy cows [48, 53, 151]. Ha et al. [53] reported that several pathways jointly regulate concentrations of these metabolites, including three highly significant pathways: steroid hormone biosynthesis, ether lipid metabolism and glycerophospholipid metabolism. Several genes are associated with these pathways including CD53, ABCC1, ADCYAP1R1, ZNF551, AHCYL1, WWC1 and MED19 [53]. Ha et al. [53] also reported similar links to pathways and genes associated with cholesterol metabolism and NEFA concentrations in dairy cows. These results are in agreement with Kessler et al. [74] who showed that mRNA abundance of genes involved in cholesterol synthesis (SREBF2, HMGCS1 and HMGCR and ABCG1) markedly increased early in lactation [61, 74].

The high concentration of NEFAs early in lactation can act as signalling molecule, regulating the expression of hepatocyte genes that are involved in lipid metabolism [38, 67, 88]. In this regard, AMP-activated protein kinase (AMPK) signalling pathways have been shown to be a key regulator of hepatic lipid metabolism, responding to hormones and metabolites including NEFAs [88]. AMPK acts as a mediator for expression of transcriptional factors, peroxisome proliferator-activated receptor α (PPARα), sterol regulatory element-binding protein-1c (SREBP-1c) and carbohydrate-responsive element-binding protein (ChREBP) [88, 155]. Activation of these transcriptional factors leads to the expression of lipolytic and lipogenic genes [70, 88, 168]. Activated PPRAα can increase expression of lipolytic genes (ACO, CPT-I, CPT-II, L-FABP and ACSL) and subsequently lipid oxidation [88]. This is while AMPKα inhibits transcription factors (SREBP-1c and ChREBP) which decrease expression of lipogenic genes (ACC1, FAS and SCD-1) and eventually lipid synthesis (Fig. 1) [88]. In addition, CPT-I activity increases by activated AMPKα; activated CPT-I increases downstream hepatic enzymatic activity and lipid metabolism [88].

One of the important genes that control synthesis of sterols is SREBF2 [61]. Kessler et al. [74] showed that there is a significant correlation between SREBF2 mRNA expression and the hepatic gene expression of both 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR) and HMGCS1. In addition, several investigations showed that occurrence of fatty liver syndrome early in lactation is associated with cholesterol carrier lipoproteins such as VLDL [50, 74]. Cholesterol is transported by high density lipoproteins (HDL) from peripheral tissues to the liver [74]. The ABCA1 gene regulates formation of HDL. Furthermore, Viturro et al. [156] reported a maximum increase in the expression levels of two transcription regulatory proteins, SREBP1 and SREBP2, on the week 2 post-partum that was coordinately and significantly correlated with an increase in the expression levels of the enzyme 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR) and 3-hydroxy-3-methylglutaryl-CoA synthesis [156]. These results indicate that there are complex regulatory mechanisms involved in the homeostasis of cholesterol in transition dairy cows.

Genes and Key Pathways Regulating Milk Fat Synthesis in Mammary Gland in Transition Dairy Cow

Early in lactation, fatty acid (FA) uptake from blood predominates relative to de novo FA synthesis in the mammary gland [14]. This change in milk FA synthesis is mediated by up-regulation of genes and pathways that are associated with FA uptake from blood (such as LPL, CD36) and intracellular transport/channelling genes (such as FABP3) [14]. Moreover, early in lactation, significant up-regulation of other genes and pathways has been observed, such as up-regulation of genes responsible in the activation of fatty acids (e.g. ACSL1, ACSS2), de novo FA synthesis (e.g. ACACA, FASN), FA desaturation (e.g. SCD, FADS1), synthesis of TAG (e.g. AGPAT6, GPAM), lipid droplet formation (e.g. BTNA1) and ketone body utilization (e.g. BDH1, OXCT1) [14]. Bionaz and Loor [14] also suggested that expression of SREBF1 is central to milk fatty acid synthesis and that the genes PPARG, LASS2, INSIG1 and OSBP have a pivotal role in regulating lipid synthesis and mammary intracellular equilibrium between cholesterol and sphingolipids in lactating dairy cows.

Genes and Key Pathways Regulating Lipid Metabolism in Adipose Tissue in Transition Dairy Cow

With increasing milk production and onset of a period of negative energy balance, the expression of many genes and enzymes controlling lipid metabolism in adipose tissue changes [75, 88]. These changes favour a decrease in lipogenesis and an increase in lipolysis. Khan et al. [75] reported a decrease in the expression of genes controlling adipogenesis including PCK1, FASN, SCD, DGAT2, PPRAG, WNT10B and SREBF1. These results are in agreement with previous work by Sumner and McNamara [145] and Bionaz and Loor [16] which reported that adipose lipogenesis in cows during early lactation is primarily regulated through control of gene expression. Expression of key lipolytic enzyme genes (LIPE, PNPLA2, MGLL and ADRB2) followed a similar pattern early in lactation, indicating that the control of lipolysis in the adipose tissue is likely controlled by post-transcriptional events [75, 104, 153]. Post-transcriptional activation of HSL through simulation of the β-2-adrenergic receptor and the phosphorylation cascade has been shown to be the first step in the beginning of lipolysis and providing fatty acids to the mammary gland and other tissues [75, 144]. The transcription of other lipolysis genes (LIPE, PLIN1 and ADRB2) increases following an increase in the enzymatic capacity for continued supply of FA to other organs and rebuilding adipose stores [103]. Adipose triglyceride lipase (PNPLA2) has been reported to be a highly expressed lipolytic enzyme in the white adipose tissue of dairy cattle, which is associated with basal and β-2-adrenergic-simulated triacylglycerol hydrolysis [108].

Influence of Energy Balance and Metabolites Early in Lactation on Gene Expression in the Endometrium of Post-partum Dairy Cow

It has been reported that severe negative energy balance (SNEB) in high-producing post-partum dairy cows is associated with subsequent low fertility [160]. Excessive lipid metabolism, increased concentrations of NEFAs and BHB and reduced concentrations of glucose and IGF-1 are associated with reproductive disorders and poor conception rates [7, 161]. The failure of multiparous cows to conceive is correlated with low IGF-1 circulation in the first 2 weeks post-partum [148, 160]. It has been shown that expression of IGF-binding protein 4 (IGFBP4) and inflammatory response genes including matrix metalloproteinases (MMP1, MMP3, MMP9 and MMP13), chemokines, cytokines and calgranulins significantly increase in the endometrium as a result of metritis in cows with SNEB [159, 160]. Wathes et al. [160] also reported that the expression of hormone receptors in the endometrium (IGF1R, IGF2R, INSR, GHR, NR3C1, ESR1 and ESR2) did not change according to the energy balance status and that there is a coordinated expression between hormone receptors IGF1R, IGF2R and INSR as well as GHR with ESR1 and NR3C1 with ESR2 [160]. Furthermore, increased concentrations of blood urea as a result of dietary factors and tissue protein catabolism may influence the expressions of endometrial IGF and INSRs [160].

Candidate Genes Identified in the Key Tissues Through Association Analysis for Production and Fertility Traits

The availability of highly informative marker maps, genome-wide association analysis [34], gene-based mapping (an association approach that tests each gene instead of each SNP separately as described in [53]) and pathway analysis [53] have resulted in the identification of several crucial regulated target genes and metabolic pathways in the mammary gland, liver and blood plasma that are responsible for the regulation of the metabolism early in lactation. For example, a QTL with a major effect on milk yield and composition has been identified on the centromeric end of the chromosome 14, and involves the DGAT1 gene [49]. Figure 2 shows a strong association of SNPs with milk production on chromosome 14 close to the DGAT1 gene in a genome-wide association study done on Canadian Holstein dairy cattle [111]. Similarly, linkage disequilibrium (LD) analysis highlighted a chromosomal region on bovine chromosome 20 harbouring the GHR gene which affects milk yield and composition [2, 18, 44]. Another association analysis revealed highly significant SNPs (false discovery rate at P value ≤10−8) associated with fat and protein percentage on chromosome 19 residing within ACLY, which is a fatty acid biosynthesis gene [123]. Significant associations involving markers within or close to other fat metabolism associated genes such as FASN, SREBPB1 and STAST5A have also been reported for milk production traits in dairy cattle [20]. In a recent study of a German Holstein–Friesian population, two highly significant polymorphisms were found to be associated with milk fat content; one of these variants is located within the promoter region of the EPS8 gene on chromosome 5 and the other variant is located near the GPAT4 gene on chromosome 27 [158].

Fig. 2
figure 2

Genome-wide association analysis of milk production in Canadian dairy Holstein cattle. The −log10 of the P-value for association with SNPs is plotted. Chromosome number is shown on the horizontal axis. The red line is the threshold for significant SNPs at 1% FDR. The green line is the threshold for significant SNPs at 5% FDR. Adapted from Ref. [111]

The product of the EPS8 gene provides a substrate for receptor tyrosine kinases and physically interacts with the epidermal growth factor receptor (EGFR) [40]. Interaction of the EPS8 gene product with EGFR increases the signalling response to epidermal growth factor (EGF) [123]. The promoter SNP reported by [158] in EPS8 may mediate the binding of transcription factor TFAP2A to influence the transcription rate of EPS8. The expression of TFAP2A is correlated with the concentration of NEFA and liver triacylglycerol [158]. It has been demonstrated that sterol regulatory element-binding proteins (SREBPs), which control the expression of genes required for the uptake and synthesis of cholesterol, fatty acid and triglycerides, are regulated by the epidermal growth factors [26]. Therefore, it is plausible that an increased milk fat biosynthesis in the lactating mammary gland is the result of an enhanced transcription rate of EPS8, conferred by the binding of TFAP2A. The GPAT4 gene is near a QTL region reported to contribute to the genetic variation of milk fatty acid composition in the Dutch Holstein population [20, 158]. This gene plays an important role in lipid biosynthesis in mammals. The transcription rate of GPAT4 is highly correlated with concentrations of diacylglycerols and triacylglycerols in milk [9, 15]. In a more recent study, this region on chromosome 27 was reported for associations with milk fat and milk volume, protein and lactose content in Holstein and Jersey crossbreds [92]. Mullen et al. [110] have detected several novel and previously identified associations involving variants within introns of the IGF1 gene associated with milk protein yield, milk fat yield, milk fat concentration, somatic cell score and carcass associated traits in Holstein dairy cattle [110]. The IGF-1 protein stimulates protein synthesis in the epithelial cells of the mammary gland and plays an important role in mammary gland growth and function [22]. A more comprehensive list of major candidate genes associated with milk production traits is shown in Table 2.

Table 2 List of major candidate genes identified through association studies for production traits in dairy cattle

The impact of poor fertility in the dairy industry has led to the inclusion of a female fertility index in some breeding programs and has undoubtedly contributed to interest in identifying the loci affecting fertility [5659]. Association and QTL mapping studies have identified several candidate genes affecting fertility traits including interval from calving to first insemination, days from first to last insemination, 56-day non-return rate and insemination per conception. For example, an investigation of QTL regions affecting female fertility traits in Nordic Holstein cattle identified a strongly associated missense mutation within the multifunctional CD36 gene on chromosome 4 [59]. In two other association studies in Nordic Red dairy cattle using 50 K SNP genotypes imputed into whole-genome sequencing data, several other genes including SLC6A17, SDS5, ADCY1, SLC1A4 and PPM1B associated with cow and heifer non-return rate, calving to first service interval, number of inseminations per conception and days from first to last insemination were identified [57, 60]. The TGFB2, APOH and IGLL1 genes were reported as important candidate genes under significant peaks associated with non-return rate and days to first service in Italian Holstein cattle [107]. The genes TGFB2 and APOH are both involved in the process of the follicular development as they interact with the reproductive hormones LH and FSH [107]. The TGFB isoforms can stimulate FSH receptor expression and amplify progesterone production and LH receptor induction [78]. The IGLL1 gene has also been reported to be up-regulated during the peripartum period in the endometrium of the lactating dairy cow and may play an important role in energy balance by influencing production and fertility traits at the same time [24]. A list of major candidate genes identified through association studies for fertility-associated traits is provided in Table 3.

Table 3 List of major candidate genes identified through association studies for fertility traits in dairy cattle

Genomic Regions and Genes Affecting Multiple Traits in Dairy Cattle

Milk production and fertility traits are polygenic, affected by many genes and variants, each with a small effect on the observed phenotype [111, 139]. GWAS studies of different production and fertility traits in dairy cattle have identified shared quantitative regions and candidate genes—regions that appear to influence multiple traits [27, 56, 111, 127]. In some cases the effects are confined to multiple production traits. For example, the underlying genomic region on chromosome 14 that includes DGAT1 gene has been shown to have a major effect on milk fat content and several other production traits including milk yield, fat percentage and protein percentage [3, 100, 105, 111, 119, 165]. Similarly, several studies have reported associations of SNPs on chromosome 20 surrounding the GHR gene with milk yield, protein yield and protein percentage [18, 25, 105, 111, 123, 154]. More recently, variants close to the MGST1 gene on chromosome 5 have been shown to be associated with increased fat yield, protein percentage and lactose percentage, and a decrease in protein yield, lactose yield and protein volume [91]. Through the collection and analysis of gene expression data the authors demonstrate that a strong MGST1 eQTL (expression QTL) likely underlies these associations, however the specific role of MGST1 in regulating milk composition is not known [91]. The identification of genes that influence multiple production traits is not surprising given the shared underlying molecular mechanisms [140]. For example, it has been shown that the main functional pathways that are regulated by the K232A polymorphism in DGAT1 gene (associated with reduced milk production and increased milk fat yield) were related to cell energy metabolism (lipid biosynthesis, oxidative phosphorylation, electron transport chain), protein degradation and cell signalling [97]. This might reflect the underlying biological pleiotropic effect, where a single casual variant is related to the variations in multiple traits as explained by Solovieff et al. [140].

Regions and candidate genes associated with multiple fertility traits have been described. For example, chromosome 21 was shown to harbour a region overlapping among two fertility traits, calving to first service interval and days open, and a candidate gene in that region has been proposed, FAM181A [111]. In another GWAS study in Nordic Red cattle, a shared significant SNP (rs43271631) on chromosome 1 was associated with multiple fertility traits such as fertility index, 56-day non-return rate, number of insemination per conception and days from first to last insemination [57]. This SNP is located within an intron of the TRPC1 gene, which was shown to regulate osteoblast formation in mice [114]. Cole et al. [30] reported a common SNP (ss86324977) on chromosome 18 in an intronic region of the sialic acid-binding Ig-like lectin-5 (SIGLEC5) gene, associated with sire and daughter calving ease, that was also reported to affect direct calving traits in multiple studies [30, 80, 133, 149]. In humans this gene is expressed in the placenta and has been suggested to have a role in the initiation of parturition [21]. In a GWAS study for calving traits in Danish and Swedish cows, the majority of the identified QTL showed an effect on more than one calving trait (such as birth index, stillbirth, calving ease, calf survival and calving index) [133]. The sharing of regions among fertility traits can reflect the similarity of some of the assessment procedures. For example, the number of inseminations per conception (number of insemination) is related to the days from first to last insemination (which measure time between first and last insemination) [57]. It also likely reflects, as with the overlap among production traits, shared mechanisms.

Perhaps the most interesting and challenging genes and variants are those that affect production and fertility. The success in increasing production in high-producing dairy cows is accompanied by a decline in reproductive performance, first service pregnancy rate and reproductive efficiency [95, 120, 152]. Cows with higher milk production at day 56 post-partum were shown to have significantly a longer commencement of luteal activity post-partum and a shorter first luteal phase [130]. There is an antagonistic relationship between production and fertility traits due to pleiotropic gene effects, linkage or complex physiological associations [68, 152]. For example, a region with effects on both production and fertility traits was reported on chromosome 5 [52, 79, 111, 119, 132]. The significant variants identified in this region (at 87–100 Mb on chromosome 5) were reported to be associated with C22:1 milk fatty acid content, milk fat yield [52, 132], protein yield [28], calving to first service interval [111] and sire conception rate in Angus, Brown Swiss and Holstein cattle [60, 117]. Several candidate genes were reported within this region including ST8SIA1, ABCC9, GABARAPL1 and SLO1C1 [111, 119]. The ABCC9 gene is thought to form ATP-sensitive potassium channels in cardiac, vascular and non-vascular smooth muscles (Gene ID: 10060). This gene is reported as a potential inhibitor of human myometrial contractility [32] through opening ATP-sensitive potassium channels, flowing K+ ion and reducing cellular excitability [76], and was speculated to be a candidate gene in dairy cattle for calving to first service interval [111]. This gene has also been reported to be associated with protein yield in dairy cattle in Holstein cattle [28, 111]. Olsen et al. [113], reported a region on chromosome 12 significant for non-return rate in Norwegian Red cattle previously reported to be associated with several milk production traits (milk, fat and protein yield). They showed that the most significant SNP in this region had a positive effect on milk traits and a negative effect on non-return rate (mainly for cows returning to oestrus after insemination) [113]. A GWAS of fertility and production traits in Italian Holstein cattle revealed one SNP on chromosome 5 (at 199 Mb) associated with protein yield, calving interval, fertility index, angularity and days to first service [107]. This SNP (BTA-27242-no-rs) was reported with a positive effect for protein yield but had negative effects on calving interval, fertility index, days to first service and angularity, and is not located within any gene, however five genes (DUSP6, POC1B, ATP2B1, C12orf12, EPYC) were reported within 1 Mb of the SNP [107]. In another recent GWAS study using imputed whole-genome sequenced data, [65] identified SNPs in five genes (ENSBTAG00000034643, GBF1, TMEM180, ACTR1B and bta-mir-146b) associated with both fertility and milk yield. GBF1 and bta-mir-146b may influence fat yield through a gene network linked to lipid and carbohydrate metabolism and to reproduction through a network connected to inflammatory response and cell-to-cell signalling [65]. The authors suggest that application of whole-genome sequence data in GWAS analysis along with gene network and pathway information may help to better identify candidate genes and variations affecting multiple production and fertility traits and indicates possible pathways that correlate these traits.


Genetic selection in the dairy industry has contributed to impressive gains in productivity that will help address increasing demand for milk and milk products. Knowledge of the biology of lactation including the key tissues, metabolic pathways, hormones and genes that are involved can help researchers identify the underlying variants that contribute to phenotypic differences. Indeed, association studies have highlighted several polymorphisms potentially accounting for variation in production and fertility traits. Continued studies of gene function and expression in the context of lactation and reproduction in cattle and other species will likely improve our ability to identify causative genes and variants for these traits, and may eventually lead to more accurate approaches to genomic selection that work better across generations and breeds.


  • The information provided in this review will facilitate interpretation of the biology underlying QTL associated with milk production and fertility-related traits through highlighting tissues, metabolic pathways and genes known to play important roles in lactation in dairy cows.

  • Studies of gene function in the context of lactation and reproduction in cattle and other species will likely improve our ability to identify causative genes and variants for these traits.

  • Knowledge of possible causative genes and subsequently discovery of new target markers within target regions and genes can be used to increase accuracy of QTL detection and genomic prediction for milk production and fertility traits in dairy cattle, as well as lead to an improved understanding of the process of lactation.

  • Increasing the accuracy of genetic selection in the dairy industry will help address increasing demand for milk and milk products.