Impact of Unsaturated Fat Supplementation on Mammary Gland Growth and Development in Dairy Animals

Abstract

The dairy industry plays a pivotal role in promoting food security across human societies globally by providing high-quality protein sources, primarily raw milk, sourced from animal husbandry. A key factor contributing to the economy of the dairy industry is the enhancement of both the quality and quantity of milk produced in dairy farms. One of the strategies used to increase milk production is the use of fats in livestock feeding. Despite the long history of adding fats to animal diets, information on the effects of varying types and amounts of fat consumption at different stages of animal breeding remains scarce. Unsaturated fats, particularly polyunsaturated fatty acids, are commonly used in dairy farms. In addition to their nutritional value in providing energy for animals, they have been shown to have positive effects on growth and overall health. This has led to their categorization as functional foods. These compounds increase milk production by promoting the growth and development of mammary tissue through changing gene expression. This section aims to present a brief summary of the impact of consuming unsaturated fats on the growth and development of the mammary gland.

1. Introduction

The significance of milk as a nutrient is widely recognized in both academic and business settings. Ancient records found on stone tablets in the Great Sahara, located in North Africa, demonstrate that the importance of milk and dairy products has been understood since 4000 BC. Even today, the role of these substances in meeting the nutritional needs of human societies in terms of energy, high-quality protein, minerals, and key vitamins is well-defined [1].

The capacity for milk production is primarily determined by the number of milk-producing cells. Any manipulation of these cells can directly impact the amount of milk production [2]. Accordingly, the factors that influence the number of epithelial cells have the most significant effect on milk production. Mammary gland growth control is a complex process influenced by many hormones and external factors, such as photoperiods and diet [3].

Fat supplements have long been crucial for providing energy to dairy animals. However, the use of unsaturated oils instead of saturated fats not only avoids the adverse effects of the latter but also offers a range of benefits [4]. In recent years, unsaturated fatty acids have emerged as the primary regulators in biological tissues. Given their role as the key components of cell membranes, the composition of fatty acids greatly influences the function of cell membranes. Moreover, the presence of fatty acids in diverse biological systems and processes, such as the immune system, blood coagulation and vascular resistance, enzyme activities, cell proliferation and differentiation, and the expression of receptors, has been widely acknowledged. Omega-3 family fatty acids, owing to their unique biological properties like long chain length and the presence of double bonds on carbon number three, are considered potent regulators in these pathways [5].

Polyunsaturated fatty acids play a critical role in many physiological actions, particularly in stimulating cell growth. As such, feeding foods containing omega-3 and omega-6 fatty acids at the end of the first pregnancy is highly recommended. This period is crucial for mammary gland growth as it promotes an increase in parenchymal tissue relative to the surrounding stroma, thereby positively and stimulatingly affecting the growth, proliferation, and organization of mammary gland tissue. Notably, enhancing the growth and development of mammary gland tissue can lead to improved production performance in the animal. It is expected that the supplementation of dairy animal feed with polyunsaturated fatty acids during the physiologically critical periods for mammary gland growth will increase both the quantity and quality of milk produced. To achieve the optimal results, this feeding practice must be implemented during the appropriate developmental stage of the animal. Such an approach has the potential to be an effective strategy for enhancing milk production and quality in the dairy industry.

2. Mammary gland growth

The mammary gland is an epithelial skin appendage comprising different tissues and cell types that undergo significant morphological and functional changes throughout the reproductive cycle in association with growth, functional differentiation, and regression [6]. The growth and development of mammary tissue can be categorized into distinct stages, including: (1) mammogenesis, which encompasses the growth and differentiation of ductal and alveolar mammary tissue; (2) colostrogenesis, the transfer of immunoglobulins to mammary secretions before parturition; (3) lactogenesis, the initiation of the secretion of a suitable volume of prepartum milk; (4) lactation, which involves the continuous production and secretion of milk; and (5) regression, which entails the cessation of lactation and the return of the mammary gland to a less differentiated state [7].

The lactating mammary gland comprises parenchymal and stromal connective tissues. The former consists of epithelial structures, such as ducts and alveoli that are closely associated with the stromal connective tissue. The stroma is characterized by cellular and noncellular components, with the cellular constituents encompassing fibroblasts, endothelial cells of blood vessels, and leukocytes that accumulate in the tissue. The noncellular parts comprise collagen and other connective tissue proteins. Significant white fat tissue is formed in the mammary gland during the early stages of fetal development and remains until pregnancy in the animal, representing an extra-parenchymal tissue that forms part of the developing stroma of the gland [8].

2.1 Growth and development of the mammary gland during pregnancy

The mammary gland’s function is the primary determinant of milk production per animal. The differences in milk production between dairy and beef cattle are predominantly attributed not only to the increase in parenchymal mammary mass of dairy animals but also to the heightened activity of most secretory cells alone [3]. The structural development of the mammary gland can be classified into five (or six) stages: (1) development during the embryonic period, (2) development during the prepuberty period, (3) development during the postpuberty period, (4) development during pregnancy, (5) development during lactation [8], and (6) development during regression [9].

Mammary growth resumes during pregnancy after entering the isometric growth stage following puberty. Milk-secreting cells develop solely during pregnancy, and the amount of milk produced is directly dependent on the number of secretory cells and their functional differentiation. As such, factors that maintain or increase the number of existing alveoli during the lactation period, along with those that affect the functional differentiation of these cells, significantly affect milk production. Thus, this period is pivotal in determining the number of secretory cells and future milk production in the mammary gland. During the second half of pregnancy, the size of the alveoli continues to increase, and new alveoli are added until a vast area of the mammary gland is covered. Alveolar cells gradually undergo the necessary biochemical and structural changes to begin abundant milk secretion, lactogenesis, at the time of parturition. Although it was previously believed that terminally differentiated cells do not multiply, recent observations have shown that cells continue to proliferate during the initial phases of lactogenesis. At this time, epithelial cells exhibit a net appearance in the apical region of the cell, resulting from the abundance and accumulation of secretory vesicles. In fully differentiated cells, the basal-lateral part of the cytoplasm appears darker in staining and is devoted to absorbing precursors and synthesizing proteins and lipids. The cytoplasm of the apical region, which is full of Golgi apparatus, structures, processes and packages proteins and lactose for secretion from the cell [2, 8].

2.1.1 Metabolic changes during pregnancy

In the last 3 to 4 weeks of pregnancy, a rapid growth of the fetus occurs, along with mammary gland development and favorable metabolic adjustments for the mobilization of fat and other nutrients. However, during this period, nutrient intake is reduced, and the metabolism of glucose and fatty acids undergoes significant changes. Metabolic adaptations occur in other tissues of the mother’s body to ensure the supply of glucose required by the uterus and lactating mammary gland, even under conditions of maternal nutritional changes. Hormonal changes that occur before parturition are also well-defined. The adaptive changes start in the prenatal period and continue after parturition and lactation. These changes involve increased lipolysis, decreased lipogenesis, increased gluconeogenesis, increased glycogenolysis, increased consumption of fats, and decreased consumption of glucose as energy sources. They also involve increased mobilization of protein reserves, increased absorption of minerals and mobilization of mineral reserves, increased feed consumption, and increased absorption capacity for nutrients [10]. Extensive metabolic and hormonal adjustments occur during the transition period from late pregnancy to early lactation. During this period, the plasma concentration of insulin decreases, and the responsiveness of muscle and skeletal tissue to insulin decreases. These adaptations may be crucial factors in the initiation of catabolic activities of the transition period [11].

2.1.2 Metabolic changes during the transition period

The transition period in dairy breeds, which includes about 3 weeks before to 3 weeks after calving, is widely considered a sensitive and critical stage [12]. During this period, significant changes occur in the nutritional, behavioral, physiological, and anatomical patterns of livestock, particularly in the mammary, reproductive, immune, metabolic, and digestive systems. Appropriate management during this period is essential to ensure that the animal moves from the nonproductive to the productive stage with full health. The future performance and health of dairy cattle depends on successfully navigating this period [13].

2.2 Mammary gland growth and development during lactation

Mammary milk production is a two-step process called lactogenesis. The first stage commences during the final trimester of pregnancy, characterized by the limited structural and functional differentiation of the alveolar epithelium. The second stage follows immediately before parturition and is characterized by the completion of structural and biochemical differentiation, coinciding with the onset of milk production and secretion [3]. During lactation, mammary epithelial cells become highly specialized in the production of milk proteins, including casein and whey proteins [14]. The number and activity of these cells determine the shape of the lactation curve and, consequently, the volume of milk produced [15]. Notably, an increase in mammary cell growth can have severe and permanent effects on milk production in response to frequent milking in early-stage dairy cows [16]. The growth of the mammary gland is minimal when lactation is fully established. The relative contribution of hypertrophy and hyperplasia to this phenomenon remains unclear. Approximately 40% of the parenchymal tissue comprises closely packed alveoli, while the remainder comprises alveoli that are greatly expanded in the stromal matrix [2, 8]. The number of cells reflects the sum of the relative rates of cell proliferation and death. The mammary gland grows when the rate of cell proliferation exceeds the rate of cell death, and when the rate of cell death exceeds the rate of cell proliferation, it diminishes [15].

2.2.1 Metabolic changes during lactation

In the first 3 to 4 weeks after calving, there is a slight increase in dry matter intake, accompanied by a rapid drop in nutrients to support milk production. During this period, there is continued rapid growth of mammary tissue and hypertrophy of key digestive and metabolic organs. Metabolically, the cow mobilizes its nutrient reserves, primarily from fat tissue, labile proteins, and bones. A crucial adjustment during this period is the stabilization of the homeostatic control mechanisms of essential nutrients such as glucose and calcium to support lactation. This process is known as homeorhesis and is considered a long-term adaptation to a change in stage, such as a transition from non-lactating to lactating or non-ruminant to ruminant. It involves a coordinated set of changes in metabolism that enables the animal to cope with the challenge and adapt to the new stage. Any inefficiency in a metabolic process can undoubtedly affect the efficiency of other processes as well [10].

3. Genes involved in the growth and development of the mammary gland

Growth factors are central to the growth and development of many cells. These factors, which include EGF, IGF, FGF, and TNF-α, play a unique role in controlling growth [17]. The production of IGF-I is regulated by both growth hormone and nutritional status [18]. The concentration of IGF-I mRNA, which is essential for growth, is higher in the stromal part of the mammary gland compared to its epithelial parts, indicating that the stromal segment is responsible for the local synthesis of IGF-I. During pregnancy, as the population of IGF receptors increases, the role of IGFs in mammary gland growth becomes more pronounced. Moreover, the synergistic effects of IGFs, EGF, and TGF-α on cow udder growth have been observed in some studies. The growth factors EGF and TGF-α have a stimulating effect on mammary gland growth, and their activity increases significantly in the presence of IGF-I, indicating a mainly supportive role in intensifying growth stimulation. The regulation of mammary functions during pregnancy is also attributed to these growth factors [19].

IGFBP1 to IGFBP6 are six structurally related proteins that regulate the activity of IGFs. These IGF-binding proteins modulate the transport of IGFs to specific cells and tissues, enhancing their access to receptors, by prolonging the half-life of IGF. One of the vital functions of IGFs is to inhibit cell death in some cells, where programmed cell death is due to apoptosis, and growth factors play a crucial role in its regulation [20].

4. Nutritional solutions

Adequate consumption compounds providing net energy and metabolizable protein are crucial for the healthy functioning of cows [21]. However, during the transition period, animals face increased challenges due to the heightened demand for nutrients and the reduced capacity of their digestive system. Inadequate nutrition during the dry period can lead to postpartum problems such as metabolic diseases, susceptibility to infections, infertility, and reduced milk production [11]. Despite the high energy demand during late pregnancy and early lactation, cows cannot consume enough feed to satisfy their metabolic and production needs. To address this issue, it is advisable to incorporate energy-dense compounds, such as fat, into their diet during this period.

4.1 The importance of fats in animal nutrition

Fats are a rich source of energy for animals, owing to their high energy and storage properties [22]. Incorporating fat supplements into animal diets increases the energy density, resulting in improved lactation performance and metabolic efficiency in lactating cows. Other benefits of using fat supplements include improved reproductive efficiency and reduced incidence of metabolic abnormalities. Inadequate energy consumption during the prenatal period and early lactation is linked to increased risk of metabolic abnormalities and poor reproductive performance [23]. The essentiality of certain fats for optimal growth and development in animals and humans has been studied for almost 80 years, and the theory of their necessity is well-established. There is growing evidence that long-chain fatty acids with polyunsaturated bonds can modulate the expression of genes that regulate cell growth and differentiation [24]. When incorporating unsaturated fats into the diet of ruminants, it is important to be aware of the permissible limit of consumption of these fats in their unprotected form, which is up to 5%. If there is a need to consume more than this amount, it is recommended to use protected fats in order to avoid disturbing the rumen function and endangering the health of the animal.

4.1.1 The role of fatty acids in the growth and development of mammary tissue

Although the lipogenic activities of adipose tissue in non-lactating ruminants in response to long-chain fatty acids have been studied, few studies have examined the effects of these compounds on mammary gland and adipose tissue in lactating animals. The effect of fatty acids on the different tissue is summarized in Table 1.

Author	Subjects	Supplement type	Time of consumption	Effect
Bernard et al. [25]	Cow/Goat	Oilseeds	Middle of lactation	Suppress de novo lipid production/No effect.
Bagga et al. [26]	Women	Fish oil	Daily for 3 months	Increasing ω-3/ω-6 ratio approximately fourfold.
McFadden et al. [27]	Prepubertal ewe lamb	Formaldehyde-protected sunflower seed supplement	From 7 to 22 week of age	Increasing mammary parenchymal weight, parenchymal dry fat-free tissue, and DNA content.
Javaheri Barfourooshi et al. [28]	Cow	Fish oil	Dry period and early lactation	Increasing the mammary gland relative percentage of epithelial area.
Javaheri Barfourooshi et al. [29]	Primiparous Saanen goat	Roasted soybean and extruded flaxseed	Two months before kidding to 4 months after parturition	Improving the volume, environment, and size of mammary halves.
Mach et al. [30]	Cow	Rapeseed, soybean, linseed, or a mixture of three oils (unprotected)	Middle of lactation	Downregulation of gene sets related to cell development and remodeling, apoptosis, nutrient metabolic process, and immune system response.
Javaheri Barfourooshi et al. [31]	Cow	Fish oil	Dry period and early lactation	Decreasing of relative expression of IGF-I and TNF-α genes, increasing of relative expression of Bcl-2 gene, increasing the ratio of Bcl-2/Bax gene expression.
Seti et al. [32]	Human colorectal cancer cell line	Alpha-Linolenic acid (ALA) and Linoleic acid (LA)	24 hours (treatment time of cells)	A potential mitogenic effect on cells, reduction in apoptotic activity.
Manna et al. [33]	Rat (carcinogenic model)	Fish oil	7 weeks of age to 32 weeks of age	Increase in Bax immunolabeling and reduction of Bcl-2 immunopositivity and TUNEL-positive apoptotic cells.
Lennie et al. [34]	Human (Patients with heart failure)	Polyunsaturated fat/Omega-3	4 days	Reduction of sTNF-R1 and sTNF-R2.
Huang et al. [35]	Barrows	Linseed	Growing-finishing period	Increasing the expression of PPARγ in longissimus muscle and spleen, reduction in TNF-α gene expression level in longissimus muscle, spleen, and adipose tissue and serum concentration of TNF-α.

Table 1.

Effect of polyunsaturated fatty acids on the different tissue in human or animals.

Consumption of oilseeds in the middle of lactation by dairy cows has a suppressive effect on de novo production of lipids in the mammary gland, while no such effect is observed in goats [25].

Mammary epithelial cells grow and organize only when they come into contact with the fat pad. This interaction is influenced by specific fatty acids produced by the fat pad that can trigger changes in epithelial development. Unsaturated fatty acids, especially, stimulate the growth of mammary epithelial cells and can enhance the external effects of other growth factors such as IGF-I and EGF [8, 36]. A diet lacking essential fatty acids can stunt ductal growth and alveolar regression, while a diet rich in unsaturated fats can increase parenchymal growth and tumorigenesis. The growth of epithelial cells can be stimulated by unsaturated fatty acids and their derivatives. Furthermore, unsaturated fatty acids and their derivatives can enhance the proliferative effects of EGF. Fatty acids derived from adipocytes can regulate epithelial growth and potentially morphogenesis through direct and indirect mechanisms involving lipids and their derivatives [37].

Studies have shown that polyunsaturated fatty acids can increase prepubertal mammary growth in sheep. Additionally, studies using bovine and human mammary epithelial cells have suggested that mammary growth may be influenced by retinoids and conjugated linoleic acid (CLA) [2]. The fatty acid composition in adipose tissue reflects the long-term consumption of fatty acids through food. Altering the stored fatty acids in mammary gland fat is crucial for storing and releasing the fatty acids required for the differentiation, proliferation, and normal morphogenesis of mammary epithelial cells [26].

Omega-3 fatty acids have been shown to have a wide range of effects on cellular activity. These fatty acids are a source of energy production while also determining the physicochemical properties of cell membranes. Further, they act as substrates for the production of signaling molecules or acting mediators and moderate the regulation of gene expression. Therefore, omega-3 fatty acids have significant impacts on physiological activity and pathological processes through different mechanisms [38].

In most mammalian species, the histological appearance of the parenchymal tissue is similar, with wide areas of alveolar cavity and compressed stromal tissue between alveoli visible from late pregnancy until lactation. The relative area occupied by the epithelium and the alveolar cavity space is similar, with approximately 40% for each, and the remaining 20% is covered by stromal tissue. Alveolar cells undergo progressive biochemical and structural differentiations necessary for the initiation of abundant milk secretion (lactogenesis) at parturition [8, 39].

Studies conducted on laboratory animals have shown that polyunsaturated fatty acids stimulate the growth of mammary parenchymal tissue by promoting the proliferation of epithelial cells, while saturated fats inhibit growth. In ruminants, unsaturated fatty acids have a stimulating effect on the growth of mammary epithelial tissue in the prepubertal age, despite an increase in dietary energy. It has also been demonstrated that lambs receiving diets containing unsaturated fatty acids exhibit increased mammary growth before puberty and a larger amount of empty fat pad, which allows for further development of parenchymal parts [27]. In a recent study, Javaheri Barfourooshi et al. [28] showed that incorporating fish oil into the diet of Holstein cows during the dry period and the first 2 months of lactation led to an increase in the percentage of epithelial cells and a decrease in the stromal tissue of the mammary gland compared to cows consuming palm oil (Figure 1).

Figure 1.

Percentage of different parts of alveoli (LSMEANS±SEM) at the first and second biopsy (7 and 63 DIM, respectively) for two diets, palm oil (PO) and fish oil (FO). ٭ Referred to significance at p < 0.05 level.

It is important to note that histological assessments alone are insufficient to accurately measure the effects of treatments that may affect the total number of cells in the entire mammary gland. Akers and Capuco [3] demonstrated differences in the relative proportions of well-differentiated alveolar epithelial cells, which were related to higher milk production in Holstein cows compared to similar Hereford cows. Differences in the relative proportions of luminal versus stromal space have been shown to correspond with differences in milk production; that is, more luminal space per stromal tissue is associated with increased milk production.

Furthermore, the increase in the number of epithelial cells per alveolus in cows consuming fish oil suggests that the number of epithelial cells per alveolus was higher than in those receiving palm oil. Additionally, the number of alveoli with a diameter ranging between 50 and 90 micrometers in cows consuming fish oil increased significantly compared to those consuming palm oil, leading to uniformity in the size of the alveoli. This can increase the surface-to-volume ratio in the alveoli, which may be one of the possible reasons for higher milk production in this group [28].

Comparable results were obtained in Saanen goats that consumed extruded flaxseeds and roasted soybeans in their late pregnancy and early lactation diets, relative to goats that consumed palm oil or a diet without any fat source (Javaheri Barfourooshi, unpublished data). These changes were also visible in the context of mammary gland morphology, as the volume, circumference, and other dimensions of the mammary gland of goats that consumed extruded flaxseed and roasted soybeans were compared to those that received palm oil and those that did not receive any fat supplements in their diet [29]. These histological findings have indicated that the consumption of diets containing unsaturated fatty acids promotes the growth and development of the epithelial parts of the mammary gland. At the time of parturition, the mammary gland tissue had a fully mature structure with active epithelial cells, compared to the groups that consumed saturated fat or a fat-free diet (Figure 2).

Figure 2.

Mammary tissue sections for palm oil (PO) and fish oil (FO) groups at two different biopsy times. (A) FO mammary tissue section in the first biopsy, (B) PO mammary tissue section in the first biopsy, (C) FO mammary tissue section in the second biopsy, and (D) PO mammary tissue section in the second biopsy. The arrows show large and small alveoli.

It has been demonstrated that consuming a diet deficient in essential fatty acids leads to ductal growth damage and alveolar regression, while a diet rich in unsaturated acids increases the growth of epithelial cells. Research has shown that unsaturated fatty acids increase mammary gland growth during prepuberty in sheep [2].

In the context of dairy cow farming, it has been observed that incorporating unsaturated fatty acids into their diet induces significant alterations in the transcription of various genes in the mammary gland. Such findings are of notable interest as they provide insight into the complex mechanisms underlying milk production in dairy cows. Similar to the changes made in milk production and composition, the effects of unsaturated fatty acid supplementation on gene expression may also change with the stage of lactation and different amounts of protein and energy in the cow’s diet [30].

Nutritional status is known to regulate blood insulin-like growth factor-I (IGF-I) and insulin-like growth factor-binding proteins (IGFBPs) levels. During food deprivation, the somatotropin-IGF-I axis does not pair, leading to an increase in somatotropin levels and a decrease in IGF-I levels. The uncoupling of the ST-IGF-I axis causes somatotropin to increase the availability of nutrients by affecting the lipolysis of adipose tissue for non-mammary tissues while minimizing the consumption of nutrients by the mammary gland [19].

Several studies have reported the stimulatory effects of IGF-I on DNA synthesis or on increasing the number of cells in vitro in the mammary gland of ruminants. The growth-stimulating effect of IGF-I depends on the period of time when the tissue is affected by it, rather than the amount of IGF-I. In the mammary tissue of nonpregnant sheep or in early pregnancy, this effect is minimal, but in late pregnancy, its effect (tissue responsiveness to IGF-I) reaches its maximum value. While it is now known that growth factors affect the control of the cell cycle machinery both positively (IGF-I) and negatively (TGF-β), the mechanisms by which hormones capable of initiating mammary development control growth factors that may mediate their effects are still mostly unknown [40]. In some in vitro studies, IGF-I and IGF-II have been shown to be powerful mitogens for normal mammary epithelial cells, as well as mammary tumors in rodents, sheep, and cattle. Both IGF-I and IGF-II are expressed in the mammary fat plate of sheep, confirming the previous theory that IGFs produced in the mammary act as paracrine mitogens for the mammary epithelium. The interaction between the stromal and epithelial parts of the mammary gland has significant effects on cell growth and morphogenesis. Based on these results, it is suggested that the proliferating epithelium exerts a positive feedback on its surrounding stroma, likely through the local release of diffusible factors, to increase the expression of IGF-I and IGF-II [41].

The growth of mammary epithelial cells and the ex vivo effects of growth factors such as IGF-I and EGF have been the focus of multiple studies with regard to the impact of fatty acids, particularly unsaturated fatty acids. The fat pad of the mammary gland is believed to mediate the effects of synthetic hormones on the development of the mammary epithelium [42]. It is postulated that synthetic hormones alter the expression of growth factors, such as IGF-I, which then act through specific receptors on epithelial cells, resulting in either positive or negative effects on epithelial cell proliferation. The fat pad’s response to multiple signals might be altered by interacting with the epithelium, for example, by producing IGFBP or other growth factors. Proliferative responses are correlated with a higher concentration of mammary IGF-I protein and a lower concentration of mammary IGFBP-3 protein. As a result, proliferative processes are linked to a net increase in the biological supply of IGF-I in the mammary gland [43].

Few studies have delved into the effect of polyunsaturated omega-3 fatty acids on the proteins of the IGF pathway. These studies have shown that such fatty acids reduce proteins related to carcinogenesis and growth and increase the proteins linked to the negative regulatory effect on the IGF pathway, such as IGFBP-3 [44]. Mach et al. [30] have reported that the consumption of diets rich in unsaturated fatty acids affects the transcription of several genes related to cell growth, cell cycle, regeneration, apoptosis, and mTOR and JAK/STAT signaling pathways [40].

Adipocytes are primarily responsible for the expression of IGF-I in the mammary gland, and its expression is at its highest in the mammary tissue of heifers at the end of pregnancy, reaching its lowest level during lactogenesis and galactopoiesis [45]. With the onset of lactation, the expression of genes involved in milk component production is upregulated, while genes related to cell proliferation are inhibited. In goats, the growth and development of mammary tissue persist to a small extent during early lactation; in contrast, mammogenesis is completed at the time of parturition in sheep [46].

Javaheri Barfourooshi et al. [31] conducted a study on Holstein cows and found that fish oil, rich in polyunsaturated fatty acids, affects the expression and early production of IGF-I and, thereby, the proliferation and development of mammary epithelial cells. Fish oil significantly increased the number of epithelial cells and decreased the stroma in the mammary tissue of cows during the first week of lactation compared to palm saturated fat. However, while higher IGF-I was observed in the cows receiving palm oil, this may not necessarily indicate higher impact on mammary tissue, as IGF-I’s effects are influenced by various binding proteins (IGFBPs) [31].

Similarly, in a separate study conducted by Javaheri Barfourooshi et al. (unpublished data) on Saanen goats in their first pregnancy, it was discovered that the administration of fat supplements led to the maintenance of high expression of IGF-I throughout the entire period, with a greater effect observed during peak production. Sustaining high IGF-I expression levels may contribute to the modulation of apoptosis and stimulation of cell survival. The expression of the IGFBP-3 gene changed concurrently with the expression of the IGF-I gene, indicating that IGFBP-3 plays an intermediate role in controlling the growth, development, and activity of milk secretory cells. IGF-I and IGFBP-3 gene expression was not significantly different among goats at the time of parturition. However, during the first weeks of lactation until peak production, the activity of IGF-I in the goats consuming saturated fat, omega-3, and omega-6 stimulated cell development and preserved parenchymal tissue, resulting in better performance of the cells. In the absence of fat, a certain level of IGFBP-3 helped to maintain the existing situation to control the death of secretory cells and maintain the secretory activity of parenchymal tissue cells. The majority of IGFBPs in the mammary gland are in the form of IGFBP-3, and its concentration in blood and milk decreases during the lactation period compared to prepartum and postpartum stages [47].

Javaheri Barfourooshi et al. (unpublished data) found the impact of different fat sources on the expression of IGFBP-3 and IGFBP-5 genes during early and mid-lactation periods in Saanen goats. The study discovered that the expression of the IGFBP-3 gene decreased during early lactation and continued until mid-lactation, particularly for goats consuming omega-3 and omega-6 (Figure 3).

Figure 3.

Changes in IGFBP-3 and IGFBP-5 gene expression in Saanen goats during three sampling periods (0: time of parturition, 2: 2 months after kidding, 4: 4 months after kidding). C–: negative control group; C+: positive control group (palm oil); SB: omega-6 group (roasted soybean); FS: omega-3 group (extruded flaxseed). * Significance was considered at the level of 5% (p < 0.05).

Histological studies revealed that goats consuming omega-3 and omega-6 sources had a higher ratio of parenchymal tissue to stroma and more compressed secretory cells as well as more parenchymal cells compared to those consuming saturated fat and those not receiving any type of fat supplement. The increasing trend of IGFBP-3 gene expression at the end of the lactation period for goats consuming unsaturated fat and its decreasing trend in those consuming saturated fat may be due to the maintenance of the function of IGF-I in controlling apoptosis. The expression of the IGFBP-5 gene was higher in goats consuming omega-3 compared to other goats. Over time, the changes in the expression of the IGFBP-5 gene for goats receiving omega-3 and palm fat were completely opposite to the previous two genes, with its value increasing as it approached the production peak and decrease at the end of the period. In contrast, the trend of changes in this gene for goats consuming roasted soybeans and those not receiving fat supplements was the opposite of goats receiving omega-3 and palm oil.

IGFs play a critical role in inhibiting programmed cell death in some cells, with growth factors playing a vital role in regulating apoptosis [20]. Fatty acids affect both systemic physiological processes and intracellular events such as gene expression, apoptosis, signal transduction, and cell proliferation [32]. TNF-α inhibits the stimulation of IGF-I in DNA synthesis. Therefore, by removing IGFBP-3, the ability of IGF-I to increase DNA synthesis is weakened. The deletion of IGFBP-3 results in a decrease in base DNA synthesis, indicating that a certain level of IGFBP-3 is required for cell proliferation [20]. Additionally, the expression of the IGFBP-5 gene in mammary gland epithelial cells suggests that this protein is suitable for inhibiting IGF-I activity. Studies have also shown that IGFBP-5 regulates apoptosis by modulating IGFs [48].

Javaheri Barfourooshi (unpublished data) conducted a study on Saanen goats to investigate the expression of the Bcl-2 and Bax genes, which are anti- and pro-apoptotic factors, respectively. The study was conducted during peak lactation periods to examine the potential effects of dietary factors on gene expression. The results of the study indicated that the relative expression of the Bcl-2 gene increased and then decreased in goats receiving palm oil and omega-6. In contrast, the expression of the Bax gene increased significantly during peak lactation for goats receiving palm oil and omega-6, with lower expression levels in those consuming omega-3. The expression ratio of these two genes (Bax/Bcl-2) increased for goats receiving palm oil and omega-6 as the lactation peak approached, followed by a decrease in 4 months after parturition. However, this ratio was higher in goats receiving omega-6 than in those receiving palm oil. The increase in Bax gene expression during peak lactation in goats consuming omega-6 compared to other goats indicates an acceleration in the rate of the apoptosis process at this time. This leads to an earlier and more intense apoptosis process, resulting in the earlier cell death of epithelial cells and alveolar structures that produce milk. Consequently, this leads to a shorter duration of maximum milk production compared to the normal state. These findings suggest that the consumption of omega-6 in the diet may have an adverse effect on milk production in Saanen goats during peak lactation.

Previous studies have indicated that the expression of Bax protein in the mammary gland of goats is low during peak lactation and increases at the end of lactation and during the dry period. Additionally, it has been reported that docosahexaenoic acid (DHA), an omega-3 fatty acid, can stimulate the expression of Bcl-2 protein [49]. Another study revealed that feeding mice with fish oil resulted in morphological changes in the breast tissue, including changes in the size of the nucleus of the epithelial cells of the mammary gland, as well as distinct morphological changes in chromatin and nucleus [33]. Furthermore, omega-3 fatty acids have been reported to control the regression function and decrease apoptosis by upregulating the expression of Bcl-2 family genes [50].

Javaheri Barfourooshi et al. [31] conducted a study on the mammary gland of dairy cows that consumed fish oil. The study observed an increase in the expression of anti-apoptotic genes and a decrease in the expression of apoptosis-initiating genes. Therefore, they suggested that the consumption of fish oil and other omega-3 sources could delay the process of apoptosis that naturally occurs in the cells of the mammary epithelial tissue as the peak of lactation approaches. In their study, the expression of the Bcl-2 gene in the cows consuming fish oil tended to increase over time, and the expression of the Bcl-2/Bax ratio increased as the peak of production approached in the cows consuming fish oil. Conversely, the opposite trend was observed in the group receiving saturated palm oil. The expression of anti-apoptotic genes increased, while the expression of pro-apoptotic genes decreased in the cows consuming fish oil. Therefore, they concluded that by postponing the process of apoptosis that naturally occurs in the epithelial cells of the mammary gland during the peak of lactation, the alveolar structures that produce milk can be preserved, leading to higher milk production for a more extended period in cows consuming fish oil (Table 2) [31].

Gene Symbol	PO	FO	SEM	P value
				Treatment	Time	Treatment× Time
Bax	1.62	1.59	0.08	0.75	0.38	0.89
Bcl-2	1.67	1.70	0.02	0.29	0.05	0.67
Bcl-2/Bax	1.04	1.07	0.06	0.58	0.58	0.66
Cox-2	1.45	1.44	0.05	0.87	0.27	0.26
TNF-α	1.69	1.60	0.04	0.06	0.17	0.64

Table 2.

Mammary gland apoptotic gene expression 1 in cows during 8 weeks after parturition.

¹

Expression of genes is based on the logarithm in Section 10.

PO: Palm oil group; FO: Fish oil group.

In Saanen goats, the consumption of omega-6 has been found to result in a significant increase in TNF-α level compared to other groups, potentially leading to PGE2 production. Moreover, at the peak of production, the level of TNF-α was higher in goats consuming omega-6 than other goats, with goats receiving palm oil ranking second (Javaheri Barfourooshi et al., unpublished data). These conclusions were drawn based on the research conducted by Lennie et al. [34], that saturated fat has also been reported to elevate the level of inflammatory markers. A study conducted by Lennie et al. [34] found that the increase in the level of omega-6 compared to omega-3 and the imbalance between them is directly related to the increase in the level of inflammatory markers. Furthermore, their experiments compared the relationship between fatty acid consumption and the level of the inflammatory marker TNF-α in a group of heart patients. The excessive consumption of saturated fatty acid and trans fatty acid (unsaturated) was directly linked to increase TNF-α levels in cardiac patients. In contrast, Javaheri Barfourooshi et al. [31] reported that the expression of TNF-α in cows consuming fish oil was considerably lower than those consuming palm oil. Various animal species have demonstrated that omega-3 fatty acids with polyunsaturated bonds can reduce TNF-α production [47]. These fatty acids may regulate the expression of TNF-α through the activation of one or more transcription factors such as PPARγ [35].

Due to the limited number of studies investigating the effect of these fatty acids on the healthy mammary tissue of pregnant and lactating animals, it is challenging to provide a definitive opinion on this matter and identify the exact mechanisms involved. More research is needed in this area to gain a better understanding of the relationship between fatty acid consumption and the inflammatory response in lactating animals.

5. Conclusion

The following most important conclusions can be drawn from the latest research results discussed in this chapter.

1. Adding unsaturated fat sources, particularly sources containing omega-3 fatty acids such as fish oil or extruded flaxseed, to the diet during critical periods of mammary gland development (such as late first pregnancy or dry period) can stimulate the proliferation and development of alveolar epithelial cells.

2. By delaying the natural process of apoptosis in the mammary tissue, it preserves the milk-producing units for a longer period of time, leading to increased milk production and persistency of lactation.

3. Omega-3 fatty acids have been found to reduce the production of pro-inflammatory cytokine TNF-α in the mammary tissue. While maintaining the health of the mammary gland, they also significantly reduce the microbial load of milk.

4. The produced milk has a higher health index for human consumption due to having significant amounts of omega-3 fatty acids, offering countless benefits to the consumer.

Further research is needed to ascertain the optimal dosage and duration of unsaturated fat supplementation for maximum benefit.

Acknowledgments

University of Tehran financially supported the study [28] under the grant number 7108017/6/18.

Animal Science Research Institute of Iran (ASRI), Agricultural Research Education and Extension Organization (AREEO), and Ministry of Agriculture of Iran financially supported the study [33, 42, unpublished data], Project code: 01-13-13-022-95014.