**1. Introduction**

The increasing adoption of molecular biology techniques and bioinformatics in nutrition and physiology in ruminant research has provided a wealth of knowledge on regulatory mechanisms of major biological processes related to milk synthesis and meat quality and marbling at a cellular level. This body of knowledge has prompted a compelling case for a change in the paradigm in ruminant nutrition, where nutrients in ruminant diets can act as bioactive molecules and exert alterations in molecular mechanisms depending on the animal physiological state. Such alterations can be carried out through gene regulation mechanisms, also known as nutrigenomics. The continuous accumulation of nutrient-gene interactions in ruminant research will eventually lead to practical applications where nutritional interventions may be made in order to improve performance and efficiency in milk yield or skeletal muscle.

#### **1.1 Ruminant model for nutrient-gene interactions**

The adoption of advanced molecular technologies in basic nutritional research in ruminants has led to a more robust notion of how nutrients can affect the animal at the cellular level. Then, this body of knowledge has led toward a general notion in ruminant nutrition, where nutrients in the diet can no longer be considered only as: (1) the building blocks for cells, tissue, and organs or (2) energy source for cell metabolism and basic cell function, but rather a new alternative concept for nutrient is that they (3) act as bioactive molecules that can regulate fundamental molecular mechanisms depending on the animal physiological stage.

Because of the inherited gastrointestinal differences between ruminants and monogastrics, the final effect of a nutrient at the molecular level will differ primarily based on how susceptible such a nutrient is to rumen fermentation. Therefore, rumen fermentation and kinetics play an important role in the context of nutrientgene interaction in ruminants (**Figure 1**). Then, from a nutrigenomic standpoint, a given nutrient in a ruminant diet will likely be fermented or bypass the rumen. If fermented in the rumen, this nutrient will become either part of the microbial biomass or an intermediate metabolite such as volatile fatty acids (VFA) which can be absorbed through the rumen wall and enter the metabolism of ruminants. In the case of nutrients bypassing the rumen, these can be converted to intermediate metabolites [1], produce a signal transduction cascade [2, 3], or directly bind and activate specialized cellular proteins called transcription factors (or nuclear receptors) [4, 5] which are responsible for carrying out the final change in gene expression by binding to specific sections in the DNA upstream of the target gene. Some transcription factors can create a secondary wave of change in gene expression by upregulating the transcription of subsequent transcription factor [6], and previously, it has been proposed that transcription factors may work in an orchestrated fashion creating a network of transcription factors that respond to dietary effects [7]. An alternative effect from intermediate metabolites is the production of DNA or histone modifications by changing the available information in the DNA [8], also known as epigenetic effects. A potential epigenetic mechanism mediated by transcription factors is the increased transcription of noncoding RNAs such as microRNAs [9], which upon transcription these small RNAs will target coding RNA prior to their translation into proteins.

#### **Figure 1.**

*Proposed ruminant model for gene expression regulation of dietary nutrients through transcription factor activation and epigenetic mechanism (i.e., DNA or histone methylation and noncoding RNA).*

**123**

ruminant diets.

*Gene Regulation in Ruminants: A Nutritional Perspective*

**1.2 Mediators of nutrient-gene interactions in ruminants**

fatty acid oxidation by PPARα in transition dairy goats [19].

retinoids such as 9-cis-retinoic acid through RXRα.

Similar to the PPARs, the liver X receptor (LXR) belongs to the LdNR family and has a prominent role in controlling cholesterol synthesis [15]. The LXR is known to be activated by oxysterols and derivatives from cholesterol metabolism, and fatty acids [15]. From the two known isoforms of LXR (e.g., α and β), the LXRα presents interesting characteristics including the potential control of sterol regulatory element-binding transcription factor 1 (*SREBF1*) gene expression [20], which is a major transcription factor associated with the regulation of milk fat synthesis [21]. The ability of LXRα to regulate *SRBEF1* expression confers this TF a strong potential to enhance milk fat synthesis in ruminants; however, most of the current data on LXRα activity have been conducted with synthetic agonist [20, 22]. Therefore, a stronger case for the nutrigenomic potential of this TF could be made by future research including its activation by common fatty acids present in

Retinoids are metabolites derived from vitamin A, and they can regulate gene expression through two classes of receptors: retinoic acid receptors and retinoid X receptors (RXR). The latter can form homodimers and be activated in the presence of the retinoid 9-cis-retinoic acid and consequently activating specific target genes [23]. From the two isoforms (i.e., α and β) of RXR, the RXRα has been the most evaluated in ruminants, primarily because it can form heterodimers with most LdNR including PPAR, LXR, and VDR [24]. Although the latter confers RXRα a tremendous biological significance, there are limited data in ruminants on the potential nutrigenomic effects of vitamin A and derivative

Nutrients and bioactive compounds in regular ruminant diets will mainly interact indirectly with the genome through mediators in the form of specialized molecular proteins such as transcription factors, DNA methyltransferases, histone methyltransferases, among others. Here, we provide a brief overview of the major mediators of nutrient-gene interactions in ruminants known to date. Based on the ruminant model for nutrient-gene interactions (**Figure 1**), the specific transcription factors, enzymes, or cellular mechanisms with *in vivo* or *in vitro* data in ruminants are presented in **Table 1**. Among the known transcription factors with nutrigenomic potential, the peroxisome proliferator-activated receptors (PPARs) have been well studied in ruminants [10]. These transcription factors belong to the ligand-dependent nuclear receptors (LdNR) family [11], and their importance for nutrigenomic interventions in ruminants relies on their ability to bind and be activated by long-chained fatty acids (LCFA) commonly present in ruminant diets. The PPAR isotypes (e.g., α, γ, and δ) play multiple roles across several tissues in mammals, for instance, PPARγ has been observed to regulate adipogenesis and insulin sensitivity [12, 13], while PPARα has a crucial role in hepatic fatty acid catabolism [14]. In contrast to PPARγ and PPARα, PPARδ has been studied to a lesser extent; however, it is known for its role in fatty acid catabolism in skeletal muscle [15] and regulation of glucose uptake [7]. Additionally, Bionaz and collaborators [10] proposed a model for the concomitant and orchestrated regulation of major physiological adaptations by the three isotypes of PPARs in dairy cows going from late pregnancy into lactation. These effects are exerted across several tissues (e.g., liver, skeletal muscle, mammary gland, adipose, immune cells, etc.) where PPARs have a strong effect, and their ability to be activated by dietary fatty acids makes them a strong candidate for nutrigenomic effects in ruminants. The PPARs exert similar effects as observed in dairy cows in other ruminants, for instance, PPARγ has been associated with adipogenic effects in beef, cows [16, 17], and goats [18] as well as

*DOI: http://dx.doi.org/10.5772/intechopen.82193*

*Gene Expression and Control*

prior to their translation into proteins.

Because of the inherited gastrointestinal differences between ruminants and monogastrics, the final effect of a nutrient at the molecular level will differ primarily based on how susceptible such a nutrient is to rumen fermentation. Therefore, rumen fermentation and kinetics play an important role in the context of nutrientgene interaction in ruminants (**Figure 1**). Then, from a nutrigenomic standpoint, a given nutrient in a ruminant diet will likely be fermented or bypass the rumen. If fermented in the rumen, this nutrient will become either part of the microbial biomass or an intermediate metabolite such as volatile fatty acids (VFA) which can be absorbed through the rumen wall and enter the metabolism of ruminants. In the case of nutrients bypassing the rumen, these can be converted to intermediate metabolites [1], produce a signal transduction cascade [2, 3], or directly bind and activate specialized cellular proteins called transcription factors (or nuclear receptors) [4, 5] which are responsible for carrying out the final change in gene expression by binding to specific sections in the DNA upstream of the target gene. Some transcription factors can create a secondary wave of change in gene expression by upregulating the transcription of subsequent transcription factor [6], and previously, it has been proposed that transcription factors may work in an orchestrated fashion creating a network of transcription factors that respond to dietary effects [7]. An alternative effect from intermediate metabolites is the production of DNA or histone modifications by changing the available information in the DNA [8], also known as epigenetic effects. A potential epigenetic mechanism mediated by transcription factors is the increased transcription of noncoding RNAs such as microRNAs [9], which upon transcription these small RNAs will target coding RNA

*Proposed ruminant model for gene expression regulation of dietary nutrients through transcription factor* 

*activation and epigenetic mechanism (i.e., DNA or histone methylation and noncoding RNA).*

**122**

**Figure 1.**

## **1.2 Mediators of nutrient-gene interactions in ruminants**

Nutrients and bioactive compounds in regular ruminant diets will mainly interact indirectly with the genome through mediators in the form of specialized molecular proteins such as transcription factors, DNA methyltransferases, histone methyltransferases, among others. Here, we provide a brief overview of the major mediators of nutrient-gene interactions in ruminants known to date. Based on the ruminant model for nutrient-gene interactions (**Figure 1**), the specific transcription factors, enzymes, or cellular mechanisms with *in vivo* or *in vitro* data in ruminants are presented in **Table 1**. Among the known transcription factors with nutrigenomic potential, the peroxisome proliferator-activated receptors (PPARs) have been well studied in ruminants [10]. These transcription factors belong to the ligand-dependent nuclear receptors (LdNR) family [11], and their importance for nutrigenomic interventions in ruminants relies on their ability to bind and be activated by long-chained fatty acids (LCFA) commonly present in ruminant diets. The PPAR isotypes (e.g., α, γ, and δ) play multiple roles across several tissues in mammals, for instance, PPARγ has been observed to regulate adipogenesis and insulin sensitivity [12, 13], while PPARα has a crucial role in hepatic fatty acid catabolism [14]. In contrast to PPARγ and PPARα, PPARδ has been studied to a lesser extent; however, it is known for its role in fatty acid catabolism in skeletal muscle [15] and regulation of glucose uptake [7]. Additionally, Bionaz and collaborators [10] proposed a model for the concomitant and orchestrated regulation of major physiological adaptations by the three isotypes of PPARs in dairy cows going from late pregnancy into lactation. These effects are exerted across several tissues (e.g., liver, skeletal muscle, mammary gland, adipose, immune cells, etc.) where PPARs have a strong effect, and their ability to be activated by dietary fatty acids makes them a strong candidate for nutrigenomic effects in ruminants. The PPARs exert similar effects as observed in dairy cows in other ruminants, for instance, PPARγ has been associated with adipogenic effects in beef, cows [16, 17], and goats [18] as well as fatty acid oxidation by PPARα in transition dairy goats [19].

Similar to the PPARs, the liver X receptor (LXR) belongs to the LdNR family and has a prominent role in controlling cholesterol synthesis [15]. The LXR is known to be activated by oxysterols and derivatives from cholesterol metabolism, and fatty acids [15]. From the two known isoforms of LXR (e.g., α and β), the LXRα presents interesting characteristics including the potential control of sterol regulatory element-binding transcription factor 1 (*SREBF1*) gene expression [20], which is a major transcription factor associated with the regulation of milk fat synthesis [21]. The ability of LXRα to regulate *SRBEF1* expression confers this TF a strong potential to enhance milk fat synthesis in ruminants; however, most of the current data on LXRα activity have been conducted with synthetic agonist [20, 22]. Therefore, a stronger case for the nutrigenomic potential of this TF could be made by future research including its activation by common fatty acids present in ruminant diets.

Retinoids are metabolites derived from vitamin A, and they can regulate gene expression through two classes of receptors: retinoic acid receptors and retinoid X receptors (RXR). The latter can form homodimers and be activated in the presence of the retinoid 9-cis-retinoic acid and consequently activating specific target genes [23]. From the two isoforms (i.e., α and β) of RXR, the RXRα has been the most evaluated in ruminants, primarily because it can form heterodimers with most LdNR including PPAR, LXR, and VDR [24]. Although the latter confers RXRα a tremendous biological significance, there are limited data in ruminants on the potential nutrigenomic effects of vitamin A and derivative retinoids such as 9-cis-retinoic acid through RXRα.


*Important mediators associated with nutrient-gene interactions in ruminants via transcriptional regulatory factors (transcription factors) and epigenetic factors (DNA methyltransferases and* 

*noncoding RNAs).*

**125**

*Gene Regulation in Ruminants: A Nutritional Perspective*

fat synthesis [25], which resulted in a deep understanding how

The study of milk fat depression revealed the importance of SREBP1 in milk

mary tissue [21]. Consequently, this effect downregulates the expression of genes associated with milk fat synthesis such as fatty acid synthase (*FASN*), lipoprotein lipase (*LPL*), and insulin-induced gene 1 (*INSIG1*) [21]. The importance of this TF for nutrigenomics in ruminants cannot be overstated since this was the first nutrigenomic effect documented. And the importance of this TF in the regulation of fat synthesis has also been observed in beef cattle and translated in marbling and meat quality [26]. A section on this commonality between dairy and beef cattle is

Epigenetic mechanisms play a significant role as mediators of nutrient-gene interactions in ruminants and the ramifications of these effects in ruminant nutri

tion and physiology are only beginning to be uncovered, and they add another layer of complexity to our model (**Figure 1**). From a nutrigenomic standpoint, methyl donors present in common diets fed to ruminants such as folate, vitamin B (e.g., 2, 6, 12), choline, and methionine can regulate epigenetic modifications through the one-carbon metabolism where the intermediate s-adenosylmethionine (SAM) is produced and subsequently used as the universal methyl donor for DNA and his

tone methylations [27]. These effects are carried out through specialized enzymes such as DNA and histone methyltransferases. While the effect of dietary methyl donors on DNA methyltransferases has been evaluated in ruminants, the effect of methyl donors on histone methyltransferases in ruminants remains unknown. Similarly, other histone modifications such as acetylation and phosphorylation have

Among the various epigenetic mechanisms, noncoding RNA and specifically microRNAs have received a lot of notoriety in recent years [28], and, in contrast, long noncoding RNAs (LncRNAs) are only beginning to be evaluated in ruminants [29]. Examples of microRNAs with a potential application to improving milk and meat quality are the miR33b and miR192 (**Table 1**). The former has been previously associated with lipogenesis in the mammary gland of dairy cows as well as having the greatest upregulation from pregnancy into lactation [30]. In the case of miR192, it has been observed to influence muscle development through myogenesis in sheep. Since the interactions between microRNAs and coding mRNAs are one-to-many, meaning that a single microRNA can regulate the translation of several coding mRNAs, special caution should be applied when interpreting this type of data. The LncRNAs are relatively new in the context of ruminant nutrition and physiology and are commonly characterized by containing >200 nt that are not translated into proteins [31]. This work provided nuances on the role of LncRNAs in the mammary

The importance of understanding these multiple mediators of nutrient-gene interactions cannot be overstated. The authors envision that the continuous accu

mulation of this wealth of knowledge will lead to accurate and consistent manipula

tion of the ruminant genome to access or unlock the full genetic potential with the aim to produce ruminant products more efficiently, with a targeted effect on human

Milk is one of the most nutritious foods known to man, and milk from dairy cattle has been part of the human diet from approximately 9000 years ago [32]. And, currently, the consumption of milk and milk-derived products around the world is expected to

not been investigated within the context of nutrigenomics in ruminants.

gland in terms of mastitis and milk quality and production.

health, and with a lesser cost for the environment.

**2. Gene regulation in dairy cattle**

a milk fat-depressing diet consistently downregulate *SREBF1* in bovine mam

*t*10,

*c*12 CLA, and






*DOI: http://dx.doi.org/10.5772/intechopen.82193*

dedicated at the end of this chapter.

#### *Gene Expression and Control*

*Gene Expression and Control*

[10, 24], [126, 127]

B, D, G, and S

**124**

**Common name**

Transcription factors

Peroxisome proliferatoractivated receptor α

PPARα

*PPARA*

Fatty acids

Fatty acid metabolism, inflammation, and tissue regeneration Adipogenesis, insulin sensitivity, and lipogenesis

Fatty acid metabolism, tissue regeneration, and glucose uptake in mammary tissue

Cholesterol homeostasis, macrophage functions, and

inflammation

Forming heterodimers with other LdNR and neutrophil

differentiation

Cholesterol and fatty acid synthesis

B, D, G, and S

[25, 26], [135]

B, D, G, and S B, D, G, and S B, D, and G

B and D

[132–134]

[22, 62], [131]

[10], [127–130]

[10, 16–18, 24], [126]

Peroxisome proliferatoractivated receptor γ

PPARγ

*PPARG*

Fatty acids

Peroxisome proliferatoractivated receptor β

PPARβ

*PPARD*

Fatty acids

Liver X receptor α

Retinoic X receptor α

Sterol regulatory elementbinding protein 1

SREBP1

*SREBF1*

N/A

DNA methyltransferases

DNA methyltransferase 1

DNA methyltransferase 3 α

DNA methyltransferase 3 β

Noncoding RNA

MicroRNA 33 MicroRNA 192 *1Ruminant as B = beef cows, D = dairy cows, G = goats, and S = sheep.*

**Table 1.**

*noncoding RNAs).*

miR33b miR192 *Important mediators associated with nutrient-gene interactions in ruminants via transcriptional regulatory factors (transcription factors) and epigenetic factors (DNA methyltransferases and* 

DNMT3b

*DNMT3B*

N/A

DNMT3a

*DNMT3A*

N/A

DNMT1

*DNMT1*

N/A

Maintenance of methylation patterns

Creates de novo methylation patterns. Present in

cytoplasm and nucleus

Creates de novo methylation patterns restricted to

nucleus

Regulates lipogenesis

Regulates myogenesis

D S

[139]

[30]

B and D B and D B and D

[137, 138]

[72], [136]

[72, 105]

RXRα

*RXRA*

9-cis-retinoic

acid

LXRα

*NR1H3*

Oxysterols/fatty

acids

**Protein** 

**Gene** 

**Agonist**

**Main function**

**Ruminant1**

**Reference**

**symbol**

**symbol**

The study of milk fat depression revealed the importance of SREBP1 in milk fat synthesis [25], which resulted in a deep understanding how *t*10,*c*12 CLA, and a milk fat-depressing diet consistently downregulate *SREBF1* in bovine mammary tissue [21]. Consequently, this effect downregulates the expression of genes associated with milk fat synthesis such as fatty acid synthase (*FASN*), lipoprotein lipase (*LPL*), and insulin-induced gene 1 (*INSIG1*) [21]. The importance of this TF for nutrigenomics in ruminants cannot be overstated since this was the first nutrigenomic effect documented. And the importance of this TF in the regulation of fat synthesis has also been observed in beef cattle and translated in marbling and meat quality [26]. A section on this commonality between dairy and beef cattle is dedicated at the end of this chapter.

Epigenetic mechanisms play a significant role as mediators of nutrient-gene interactions in ruminants and the ramifications of these effects in ruminant nutrition and physiology are only beginning to be uncovered, and they add another layer of complexity to our model (**Figure 1**). From a nutrigenomic standpoint, methyl donors present in common diets fed to ruminants such as folate, vitamin B (e.g., 2, 6, 12), choline, and methionine can regulate epigenetic modifications through the one-carbon metabolism where the intermediate s-adenosylmethionine (SAM) is produced and subsequently used as the universal methyl donor for DNA and histone methylations [27]. These effects are carried out through specialized enzymes such as DNA and histone methyltransferases. While the effect of dietary methyl donors on DNA methyltransferases has been evaluated in ruminants, the effect of methyl donors on histone methyltransferases in ruminants remains unknown. Similarly, other histone modifications such as acetylation and phosphorylation have not been investigated within the context of nutrigenomics in ruminants.

Among the various epigenetic mechanisms, noncoding RNA and specifically microRNAs have received a lot of notoriety in recent years [28], and, in contrast, long noncoding RNAs (LncRNAs) are only beginning to be evaluated in ruminants [29]. Examples of microRNAs with a potential application to improving milk and meat quality are the miR33b and miR192 (**Table 1**). The former has been previously associated with lipogenesis in the mammary gland of dairy cows as well as having the greatest upregulation from pregnancy into lactation [30]. In the case of miR192, it has been observed to influence muscle development through myogenesis in sheep. Since the interactions between microRNAs and coding mRNAs are one-to-many, meaning that a single microRNA can regulate the translation of several coding mRNAs, special caution should be applied when interpreting this type of data. The LncRNAs are relatively new in the context of ruminant nutrition and physiology and are commonly characterized by containing >200 nt that are not translated into proteins [31]. This work provided nuances on the role of LncRNAs in the mammary gland in terms of mastitis and milk quality and production.

The importance of understanding these multiple mediators of nutrient-gene interactions cannot be overstated. The authors envision that the continuous accumulation of this wealth of knowledge will lead to accurate and consistent manipulation of the ruminant genome to access or unlock the full genetic potential with the aim to produce ruminant products more efficiently, with a targeted effect on human health, and with a lesser cost for the environment.

## **2. Gene regulation in dairy cattle**

Milk is one of the most nutritious foods known to man, and milk from dairy cattle has been part of the human diet from approximately 9000 years ago [32]. And, currently, the consumption of milk and milk-derived products around the world is expected to

increase, primarily due to an increase in world population and increased consumption in countries where milk has not traditionally been considered popular [33]. Until now, the demand of milk worldwide has been supplied by a large increase in milk yield per cow, which has been a product primarily from the selection and enhancement of management practices, including improved nutrition. However, because of the ever-increasing demand for milk and milk-derived products as stated above, there is a need to continue increasing milk production efficiency.

Milk and milk products are an excellent source of macronutrients such as fat, protein, and carbohydrates, and contain a variety of bioactive molecules associated with health benefits, for instance, conjugated linolenic acid (CLA). The CLA has been associated with reductions in cancer development [25]. Because of its ability to contain bioactive molecules, milk has been considered a functional food. However, our ability to understand and yet manipulate the cow genome through nutrigenomic approaches to enrich specific bioactive molecules in milk is in its infancy. This calls for a continuous development of a wealth of knowledge around the various complex nutrient-gene interactions in dairy cows as well as development of nutritional models that can account for both traditional aspects of ruminant nutrition and more novel molecular regulation of nutrient metabolism.
