LACTOGENESIS The term lactogenesis is used to describe the initiation of milk secretion after parturition (delivery of the fetus),

when the mammary glands have reached a suitable degree of development. The process of lactogenesis at parturition encompasses the hormones involved in its initiation, milk secretion by the epithelial cells of the alveolus, and removal of milk stored in the mammary gland. It should not be confused with pharmacological hormone-induced lactation in barren, nonlactating cows and heifers. Secretions by the Epithelial Alveolar Cells Alveolar cells of the lactating (postparturient) mammary gland synthesize and secrete milk proteins (casein, serum albumin, alpha-lactalbumin, beta-lactoglobulin, immu-noglobulins, and glycoproteins), fats, and lactose. Junctions between these cells enable them to carry on functions of adhesion (desmosomes), occlusion (tight junctions), and communication (gap junctions). At about midgestation, the epithelial alveolar cells begin to secrete appreciable quantities of specific milk products, which frequently accumulate in the mammary gland as precolostrum. This lactogenesis stage I is a prerequisite for lactogenesis stage II, which is the onset of copious colostrum and milk secretions at parturition. Lactogenesis I The onset of lactogenesis I, that is, secretory activity in the mammary alveoli, is marked by production of fire-colostrum. The appearance of lactose in the precolostrum is of physiological importance, since it indicates that the intra-cellular apparatus for milk synthesis is functionally differen-tiated. It occurs at midgestation in goats, in the last third ot pregnancy in rabbits, a month before parturition in ewes, 15 days before foaling in mares, at 10 days before calving in cows, and a day before delivery in mice and rats. During this period of late pregnancy, when the mammary gland remains unmilked, a local raccor maintains "leaky" tight junctions to keep the epithelial paracellular pathway open, and the ducts permeable. Transfers between extracellular fluid and precolostrum ar then possible. The precolostrum contains some large molecules such as immunoglobulins, and its aqueous phase shows fluctuating levels of chloride, sodium, po-tassium, and lactose. Eat globules, protein granules, desqua-mated epithelial cells, and leukocyces also accumulate in the lumen of the alveoli. Disappearance of this factor, with removal of precolostrum (sucking or milking), results in closure of the paracellular pathways in the alveolar epithelium and duct system. Lactogenesis II Lactogenesis II usually begins shortly before parturition, when the mammary gland first releases colostrum, then normal milk, which are not identical in all animal species. For instance, the colostrum of some animals (cows, bitches, ewes, goats, mares, and sows) contains globulin antibodies, which are essential for transmitting passive immunity to their offspring. The milk of carnivores, with high-protein and low-sugar contents, is noticeably different from the milk of equidae (protein-poor and sugar-rich). Colostrum Colostrum, the transitional fluid between precolostmm and normal milk, is the first food of the neonate. In addition to its rich nutritive value, it transfers passive immunity, and it has a slight laxative effect to aid in clearing the intestine of meconium (greenish mucilaginous material in the intestine of the full-term fetus). Secreted and stored in the mammary gland during the last 2 co 7 days of gestation and the first 2 or 3 days poscpartum, colostrum contains more proteins (including immunoglobulins), lipids, and ash

(sodium, and chloride) and less lactose than milk (except in mares). It also contributes fat-soluble and water-soluble vitamins and all the essential amino acids to the rapidly growing neonate (Table 59- 2). In the colostrum of sows, a trypsin inhibitor offers protection to the immunoglobulins in the gut of piglets. Colostrum is essential for supplying immunoglobulins to the neonates from females of species (cows, ewes, goats, mares, and sows) in which antibodies are not transferred from maternal to fetal blood (Fig. 59 1). During their first 2 days of life, kittens and puppies need colostrum to supplement the immunoprotection conferred through the placenta during gestation. A calf should ingest about 6 percent of its body weight in colostrum within 6 hours after delivery. A dairy cow produces about 50 Kg of colostrum during the first six milkings after parturition (the first milking is the richest in immunoglobulins). After fermentation (souring), refrigeration, or freezing, colostrum can be fed to calves until the age of 3 weeks, as a source of energy. Colostrum can be frozen in cartons and kept for several months. In the newborn lamb, bovine colostrum is a satisfactory substitute for natural ovine colostrum. A synthetic ovine colostrum, adequate for its nutritive value but without protective antibodies, can be made with one beaten egg, 1 teaspoon (10 ml) of cod liver oil, and 2 teaspoons (20 g) of sucrose added to 750 ml of cow's milk. Commercial preparations containing concentrated immunoglobulins are available for calves, lambs, and piglets. Equine colostrum (300 to 500 ml) must be administered within 6 hours to foals born to mares with anticipated lactogenesis II (running milk), that is, mares producing only milk, without any colostrum, at parturition. During the first 3 days of lactogenesis II, the composition of colostrum, far from being static, changes, with the gradual modification of colostrum into normal milk (Table 59-3). The immunological experiences of the mother determine the mix of antibodies available to the fetus and the neonate. Domestic carnivores and primates deliver anti- bodies to their fetuses across the placenta. Important additional immunoglobulins (Igs), mostly IgA, and lesser amounts ofIgG and IgM, particularly significant for protection against viral diseases, are also provided in colostrum. Domestic herbivores and swine have placentas that allow very little transfer of maternal antibodies to the fetus. These species are very dependent upon the immunoglobulins in colostrum to provide passive immunity to protect the newborn for the first few weeks of life. Their colostrum is particularly rich in IgG (Fig. 592). Substantial amounts of IgA and IgM are also present. The neonatal animal has little proteolytic activity in the gut, and colostrum contains trypsin inhibitors. Consequently, the Igs pass intact to the ileum, where they are absorbed via pinocytosis for a limited time only. Permeability to and absorption of the Igs in the digesta is at a peak during the first few hours after birth and starts to decline after about 6 hours. Often, by 24 hours, because of a phenomenon known as "closure," little further absorption of proteins occurs. In species that have nearly proteinimpermeable placentas, it is imperative that the newborn receive an adequate supply of colostrum within the first 24 hours after birth. For example, calves are born with very low plasma levels of Igs. These levels rise rapidly after a colostrum meal, peaking during the first day. This rise is accompanied by a transient proteinuria, resulting from renal filtration of smaller milk proteins, such as beta-lactoglobulin, that are also absorbed. The plasma IgG levels persist for several weeks, providing passive immunity until the young animal's immune system becomes activated. Some IgA is protected from digestion by a protein called secretory component, which also reduces its absorption. Lymphocytes, which may also be absorbed from colostrum, convey cell-mediated immunity, but also some infectious agents such as viruses may be absorbed. Normal Milk Normal milk contains proteins derived from amino acids taken up by, and synthesized in, the alveolar epithelium of the mammary gland. In the lactating mammary gland, the entire duct system

is impermeable and acts as a storage region. The epithelium of the alveolus is also impermeable, blocking all paracellular (between cells) transfers and all transport is via the transcellular route across epithelial cells. This holds even for the aqueous phase of milk, which constitutes 88 percent of cow's milk and contains the main ions (potassium, sodium, and chloride) and other substances (citrate, phosphate, calcium, urea) that make milk isoosmolar to blood plasma (Fig. 59-3). After synthesis in the endoplasmic reticulum, milk proteins for secretion pass to the Golgi vesicles, where caseins undergo phosphorylacion and associate with calcium to form micelles. The Golgi vesicles move to the apical surface, and their protein contents are released by reverse pinocytosis (exocytosis, exo-pinocytosis, merocrine secretion). Lactose, the predominant milk sugar, is synthesized from glucose, enters the Golgi apparatus as a complex (since the Golgi is impermeable to lactose), and is released into the alveolar lumen by exocytosis, together with milk proteins. In humans, intolerance to lactose, a deficiency in lactase (enzyme) within the small intestine mucosa, causes diarrhea and upsets the gastrointestinal tract in certain populations (e.g., Eskimos, American Indians, blacks, Asians, and New Guinea and Australian aborigines) after weaning. Triglycerides constitute over 95 percent of milk lipids. Fatty acids are either derived from the diet or synthesized in the mammary gland. Lipid droplets migrate to the apical membrane, are enveloped, and then extruded from the cell into the lumen of the alveolus. The composition of milk varies with the animal species, and within a given species the composition of secreted milk is not static but changes with the stage of lactogenesis and the level of nutrition. Usually, toward the end of lactogenesis, when milk production is declining, the concentrations of proteins, milk fat, and sodium (600 mg per liter) steadily increase, whereas lactose and potassium (1450 mg per liter) decrease. In mare's milk, except for sodium and potassium, the concentrations of calcium, phosphorus, magnesium, copper, and zinc decrease during the course of lactation. In 16 weeks, the content of total solids in mare's milk can pass from 12 to 10 percent, and that of ash can be halved from 0.6 to 0.3 percent. Milk is isoosmotic to plasma and has a pH near 6.8 (Table 59-4). Epithelial cells, macrophages, mononuclear leukocytes, and cell fragments are normal elements in milk, but not polynuclear neutrophils associated with a diseased or infected mammary gland. The number of cells considered as normal in average bovine milk (0.2 X 106 cells per milliliter) varies with the time of day, the stage of lactogenesis, and the quarter of the udder. Equine milk usually contains very few leukocyric cells (8,000 cells per milliliter). A high-quality bovine milk must meet the major standards of low counts of somatic cells and of viable bacteria. Certain plant alkaloids (colchicine, vincrisrine, vinblasrine) and some plant lecrins (concanavalin A) interfere with the transport and discharge phases of the secretory process, and bring a retention of the synthesized milk within the alveolar cells. Ergot derivatives (e.g., bromocriprine) can depress or stop lactogenesis II in sows by inhibiting release of prolacrin (PRL) from the adenohypophysis. Removal of Milk Stored in the Mammary Gland Milk secreted into the lumen of the alveoli dilates and fills the alveoli, the duct system of the mammary gland, and, depending on the species, the enlarged terminal portion of the ducts (the sinus, or cistern). In ruminants, only the milk present in the larger ducts and sinus, or cistern, can be removed by cannulation of the teat (Fig. 59-4). The mammary glands of the sow do not have a sinus or cistern. The bulk of the milk is retained in the alveoli and small ducts until the neuroendocrine milk-ejection reflex occurs. The neurohumoral reaction is activated by sensory receptors in the teat, at suckling or milking, and by conditioned responses to sound and sight at milking or suckling. The nervous signals finally reach the neurohypophysis through still-unknown paths and result in oxytocin release. Oxytocin causes myoepithelial cells surrounding the alveoli to contract and to increase milk pressure in the duct system and also causes scored milk to be forced down the galactophore (Fig. 59 5). Oxytocin, released in a single spurt, has a half-life of 2 to 3 minutes. In lambs and kids, the tongue and mouth of the sucking ruminant acts on the teat like the hand of a milker with suction added. The milk is trapped in the teat cistern by compressing the base of the teat

and is then expressed through the teat canal at the tip of the teat. By cineradiography, with milk rendered radiopaque in the doe's udder, it has been possible to get the following description of the sucking sequence in the kid's mouth. The kid compresses the base of the teat between its upper gum and the tip of the tongue resting on the lower gum. Then, it raises its tongue to indent the teat from the base toward (he tip, closing the lumen of the teat sinus and expressing the milk to be swallowed. At the same time, it lowers its jaw and tongue to allow the teat sinus to fill again with milk, and the cycle begins again. Emotional stress, pain, fright, and epinephrine inter-fere with milk removal through inhibition of oxytocin release from the neurohypophysis. The inhibitory effects of eo nephrine on milk ejection are also mediated by vasoconstric tion of the mammary blood vessels, reducing access ofoxvtocin to the myoepithelial cells, and by competition for receptor sites on the myoepirhelium. Some ergot derivatives (alpha-ergocryptine, ergoramine, ergocornine) are also potent inhibitors of the milkejection reflex. In postparturient sows, oxytocin administration may correct some forms of a condition of agalactia, which is considered part of a syndrome also involving mastitis and metritis. Hormonal Control The initiation of lactogenesis is controlled mainly by lactogenic hormones from the adenohypophysis (PRL and growth hormone [GH]) and the placenta (placental mammogenic hormones or placental lactogens [PL] in cactle sheep, and goats). PRL, GH, and PL have similar structures, but within a given species each hormone is believed to have a distinct biological role, not yet precisely characterized. Adenohypophysial lactogenic hormones can partly replace their placental counterparts in studies on initiation of lactogenesis, but they cannot do so completely. A growing body of experimental evidence gives a role to PLs in the normal growth of the udder. The metabolic hormones, such as adrenal corticoids (corticosteroids), insulin, and glucagon from the pancreas, and thyroid hormones, also directly affect the mammary gland; these same hormones exert an indirect effect on the metabolic precursors needed for milk synthesis. In most periparturient females, the levels of progesterone and of PL decrease. These declines are followed by a rise in levels of PRL, estrogens, PGF2a, oxytocin, and adrenal corticoids. Two basic hormonal concepts are invoked to explain the triggering of lactogenesis stage II at parturition: 1. The release of the mammary gland from the inhibitory effect of progesterone, and 2. A rise in the levels of some lactogenic hormones (PRL and GH) to overcome the progesterone inhibition. In beagle bitches, PRL levels rise steadily from mid-pregnancy to early lactation, decline slowly as lactation progresses, and fall abruptly upon weaning. PRL levels are not significantly lower in pseudopregnant (diestrous) bitches. During the last week of pregnancy, the mare shows a major increase in plasma PRL concentration, which fluctuates but stays high during early lactation, then declines to basal levels in 1 to 2 months. If the foal dies shortly after birth, plasma PRL falls rapidly, and estrus occurs as early as 4 days postparrum, rather than a week after parturition, as the usual "foal heat." PRL appears to be important for the completion of mammary development and the initiation or milk secretion. Suckling triggers increases in postpartum PRL secretion. In ruminants, after lactation is established, PRL secretion can be completely suppressed without arresting milk secretion. The release of PRL and GH, episodic in ruminants, is mostly spontaneous and due only in part to stimuli such as suckling or stress. In cows and goats, PRL plays a decisive role only for establishment of milk secretion. Al-though PRL release is not related to the short ruminant sleep, and to circadian activity, morning and afternoon milkings cause an immediate release of PRL and initiate a gradually decreasing chronic release of the hormone. The concentrations of PRL in blood, dependent on season (day length), are highest during summer and lowest during winter in cows, goats, and sheep. This PRL periodicity is maintained from birch to maturity in heifers and bulls, in rams, in pregnant cows, and in response to milking in cows and goats and to suckling in ewes. Goats and ewes, pregnant during the winter months, have an increase in PRL

levels only a few days before parturition. Similarly, PRL levels rise sharply in the last 2 days of bovine gestation and, because of seasonal variations, are higher in autumn calving than in spring calving cows. PRL release is inhibited by PRL-inhibiting factor (PIF, or dopamine) and is stimulated by a PRL-releasing factor (PRF), which may well be thyrotropin-releasing hormone (TRF). Growth hormone is more important than PRL for galactopoiesis in ruminants. Levels of GH are low in pregnant ewes and cows but are somewhat higher and more fluctuating in ewes carrying twins or triplets. Lactating or not, goats do not liberate GH in association with sleep, air temperature, time of day or night, or husbandry routines, or in response to plasma levels of PRL, insulin, glucose, or free fatty acids. The final release ofGH is thought to be the result of a balance between the opposing effects of two hypothalamic peptides: GHRP (growth hormone-releasing factor, or somatoliberin) and somatoscatin (growth hormone release-inhibiting hormone). Bovine GH produced by biotechnology, that is, by fermentation in bacteria from recombinant DNA, is known as recombinant somatotropin (rbST). In lactating cows receiving supplemental bovine GH (bovine somatotropin, or bST), milk production is increased by 10 to 25 percent, and the feed efficiency also becomes 5 to 15 percent higher. Sustained-release formulations have been developed that are effective for over 2 weeks. Industrial production of bovine somatotropin made by recombinant DNA technology in bacteria (rbST) has made possible the manipulation of bovine lactation physiology. It seems that bST produces an increase in insulin-like growth factor I (IGF-I) and that this agent increases milk synthesis. A large increase in milk production, on the order of 5 to 25 percent, depending on the standard of herd management, occurs after the first 2 to 3 months of lactation. Given daily injections of rbST, the increased yield persists throughout the remainder of the lactation. New sustained-release preparations, lasting over 14 days, avoid the undesirable feature of daily injections. The only negative effect ofrbST treatment is an increase in the average number of services (from about 2 to 2.5) required to achieve conception, which causes the cows to remain open (unbred) for about 21 days longer. In one trial, the milk yield (x: 20 % = 3.7 kg per day) increased promptly (2 to 3 days) after initiation of bST injection, at 9 weeks after calving (Fig. 59-6). After a lag of about 8 weeks, dry matter intake increased by 1.7 kg per day. Feed efficiency improved by 6 percent, because of the reduced contribution of the maintenance requirements per gallon (about 4 L) of milk. In the treated cows, an initial negative energy balance was then compensated for, through the delayed increase in food intake. Plasma PL levels rise to a plateau of highest values during the last third of gestation in goats and cows but only 10 days before full term in ewes. Concentrations of ovine PL in peripheral plasma begin to fall 5 days before parturition and disappear rapidly after delivery. GALACTOPOIESIS The term galactopoiesis refers to the maintenance or stimulation of an already established milk production (lactogenesis II). Galactopoiesis is related to the removal of milk from the mammary gland by either milking or suckling. It is a commonly known fact that lactogenesis II slows or ceases altogether if the frequency and the extent of milk removal is reduced. The secretory activity of the mammary gland declines slowly and eventually ceases, even though milk is removed regularly. The natural decline in lactogenesis often coincides with weaning of the young. A negative-feedback mechanism operating locally within the gland may participate in this slow, direct, and gradual involution of the alveolar epithelium during the later stages of lactogenesis. In marsupials, suckling is continuous during the first part of pouch life, which is equivalent to the fetal period of placental animals (the "fetus" is firmly anchored to a nipple). In some animals, milk is produced during a period far exceeding the weaning period of the offspring. These main milk-producing species are the goat, water buffalo, sheep, reindeer, and

cattle. As a result of genetic selection for milk-producing ability, the typical modern cow produces more than four times the milk required to raise her calf, and top producers yield milk that would suffice for more than 20 times the physiological needs of the normal calf. Posrpartum anestrus occurs in cows and ewes. In ewes, the duration of the postpartum anestrus is related to the degree of mammary stimulation during suckling. In cows, which are usually pregnant for most of the period of lactation, the degree of udder stimulation does not influence the duration of postpartum anestrus. In the absence of a new pregnancy, and with continual suckling or milking, lactation may be prolonged far beyond the normal weaning period (e.g., as long as several years in cows). Hormones Prolactin (PRL), growth hormone (GH), thyroxine (T4), insulin, corricosteroids, and oxytocin are the hormones recognized as also associated with maintenance of lactogenesis (galactopoiesis). However, adenohypophysial release of PRL at milking or suckling is essential for galactopoiesis in most animal species, except the goat and cattle. In these two ruminants, PRL has a decisive role only in establishing lactogenesis II. The conditioned stimuli of events preceding milking, and the tactile sensations at the udder and teat during milking or suckling, normally increase maternal plasma prolactin and oxytocin levels in all species. In most dams, except the cow and the nanny goat, failure to empty the milk from a producing mammary gland leads to lower levels of PRL and consequent hypogalacna whereas inhibition of circulating PRL with an ergot alkaloid derivative (bromocriptine) induces agalacria. Besides its classic role in the milk-ejection reflex, oxytocin also influences lactogenesis and galactopoiesis. Oxytocin mjecred into lactating animals without immediate milking generally inhibits lactogenesis, whereas lactogenesis is stimulated it th(. ejected milk is removed immediately. Oxytocin would contribute to galactopoiesis by loosening the right junctions between epithelial alveolar cells and by accelerating the transfer of milk constituents into the alveoli. Suckling or milking causes the release of growth hormone in nanny goats and rats but not in cows and bitches. In nanny goats, the release of GH is unpredictable and may not be entirely related to the tactile stimulation of the mammary eland. In contrast with the situation in nonruminants, in which PRL is more important for lactogenesis, GH assumes a more prominent galactopoietic role in ruminants (cows and goats). GH is required for maintenance of galactopoiesis in cows and goats and increases milk yield in cattle without requiring a proportionate increase in food intake or a proportionate reduction in body tissue reserves in the short term. A normally functioning thyroid gland is required for maintenance of lactogenesis. Lowered milk yield and reduced milk fat percentage resulting from thyroid deficiency are reversed by prolonged iodinated protein feeding. The role of thyroxine in maintaining ruminant lactogenesis is not clear, since there is less hormone in lacrating cows than in nonlactating cows, and the levels are negatively correlated with milk yield. Insulin is recognized as essential for the maintenance of normal lactogenesis, but its exact mammary gland action is difficult to distinguish from its general anabolic role. Insulin treatment decreases milk yield and milk lactose in lactating cows and goats and increases milk protein and milk fat. In goats, exogenous insulin favours synthesis of milk fat containing more Ciz to Cig fatty acids. Intact adrenal glands are essential for the maintenance of lactogenesis in both ruminant and nonruminant species, and replacement therapy is best effected by a combination of mineralocorticoids and glucocorticoids. In cattle, plasma cwticwteroids are higher in lactating animals than in nonlactaring ones, and high-yielding cows have more corticosteroids than lowyielding ones. A disagreement remains to be resolved on the relationship between plasma level of corticosteroids and the stage of lactogenesis in cattle. Some claim that the basal postpartum level increases as lactation progresses, whereas others support the opposite view, that is, a high postpartum level that decreases with progressing lactogenesis. Cessation of Calactopoiesis by Involution of the Mammary Gland

In animals, involution of the mammary gland may be "initiated" or "gradual." Initiated involution refers to the regression of the gland as a result of sudden cessation of milk removal during galactopoiesis. Gradual involution is the regression of the mammary gland during the normal course of galactopoiesis. Initiated involution is of practical value in sows, since the production objectives aim at increasing the annual number of piglets per dam, rather than the annual milk yield. Regression of the mammary gland by early and sudden weaning of piglets (i.e., at 3 weeks instead of at 6 weeks) is recommended in order to decrease the length of lactogenesis and the interval between farrowing. In this sow-breeding system, sudden stoppage of milk removal ends lactogenesis II at 3 weeks postpartum, when it is actually reaching its peak. Withholding feed for the first 2 or 3 days after withdrawal of the piglets reduces the milk flow, and gonadotropins (pregnant mare serum gonadotropin and human chorionic gonadotropin) are then injected to speed up and synchronize the onset ofestrus after early weaning. Complete involution of the gland occurs after 8 days in sows. Sudden weaning of animals in full lactogenesis usually results in gross distencion of the mammary gland for 3 or 4 days. The interstitial fluid is laden with milk constituents (milk edema). During initiated involution, milk secretion is unaffected for 16 hours in cows, and for 12 hours in ewes. Distention is concomitant with neurtophilic infiltration, which is followed by the appearance of macrophages (foamy cells, bodies of Donne) before lymphocytic dominance sets in, usually from the fourth day in ewes. Gradual involution does not proceed uniformly throughout the gland. Areas adjacent to the abdominal walls are the last to involute. Even at the height of lactogenesis II, as much as 20 percent of the mammary gland of the cow shows areas of alveoli without secretory activity. These areas resemble those of a nonlaccaring gland, with prominent interalveolar and inrerlobular connective tissue. Srilbesrrol (unauthorized drug) fed to cows, and injections of GH or of small doses ofescradiol to nonpregnant cows, prolong galactopoiesis or decrease the gradual involution of the mammary gland. Gestation stops galactopoiesis in goats and reduces milk production in cows. In mares and bitches, large doses of estrogens are used to put a sudden end to lactogenesis. In the involuting gland, lactoferrin production by mammary epithelial cells increases (as in acute mastitis) to exert a bacterioscaric effect. Lactoferrin, the major ironbinding protein of milk, is a natural protective factor. By binding to iron, lactoferrin withholds an essential nutrient from pathogenic bacteria.

(Ruckebusch, 1991)