LactationResource Library

Overview

Overview

Recall from The Beginning Independent Study Module that lactation is the combined processes of milk secretion and milk removal. This current Module on Lactation will focus on the regulation of milk secretion and milk removal. Other lessons also are relevent to this section. For example, the Module on Mammary Structure: Macro & Micro includes sections on the structure and cell biology of the alveolar epithelial cells, the cells that synthesize and secrete milk. Many of the underlying principles of regulation of lactogenesis, dicsussed in the Mother & Neonate Module, hold true for the maintenance of lactation once it is established.

Galactopoeisis is the maintenance of lactation once lactation has been established. Two key interrelated components contribute to the maintenance of lactation, galactopoietic hormones and removal of accumulated milk. Because of the importance of galactopoietic hormones in milk production, sometimes the word galactopoiesis also is used to indicate enhancement of lactation, especially in dairy animals. Inhibition of secretion of key galactopoietic hormones will depress milk production to varying degrees depending on the species, stage of lactation, and the particular hormone being suppressed. Much of the fundamental knowledge that we have on galactopoietic hormones comes from classic studies demonstrating that inhibition of hormone secretion will inhibit milk production. Conversely, administration of additional amounts of galactopoietic hormones during lactation can enhance milk protuction, again depending on the species, stage of lactation, and particular hormone. The most widely known example of this has led to the common practice of administration of bovine somatotropin (bST, or bovine growth hormone) to lactating dairy cattle resulting in relatively dependable increases in milk yield. Any substance that is administered to a lactating animal and that results in increased milk production would be considered a galactagogue.

The role of galactopoietic hormones such as prolactin in maintenance of lactation is well established (reviewed by Tucker 1994), although specific cellular mechanisms of action continue to be investigated. Prolactin is considered the major galactopoietic hormone in nonruminants. Prolactin is released at the time of milk removal in ruminants and nonruminants, and it remains a key systemic modulator of milk secretion during lactation. Conversely, growth hormone is generally considered to be the predominant galactopoietic hormone in ruminants (Bauman 1992; Tucker 1994). Inhibition of prolactin secretion or administration of prolactin to lactating cows has little effect on milk yields (Karg and Schams 1974; Plaut et al. 1987). However, these apparently clear-cut roles of prolactin vs. growth hormone in maintenance of lactation in nonruminants vs. ruminants are probably an oversimplification (Wilde and Hurley 1996). For example, in lactating sheep both prolactin and growth hormone seem to be important for galactopoiesis (Hooley et al. 1978; Tucker 1994). Even in the rat, recent studies have demonstrated an important role for growth

hormone, independent of the role of prolactin (Flint et al. 1992; Flint and Gardner 1994).

Regardless of the hormones involved, all attempts to evaluate milk secretion must account for continued removal of milk. This is a reminder of the critical role of local mammary factors in maintenance of milk secretion. One such factor that plays a major role in regulating milk secretion in many species is a feedback inhibitor of lactation (FIL) found in milk (Wilde et al. 1995). FIL is thought to be produced by the mammary cells as they synthesize and secrete milk. Accumulation of FIL in the milk-producing alveoli results in feedback inhibition of milk synthesis and secretion. Frequent removal of milk from the gland minimizes local inhibitory effects of FIL and increases milk secretion (Wilde et al. 1987; Wilde and Knight 1989; Wilde and Peaker 1990).

Milk removal involves several mechanisms that impact milk production, including removal of local inhibitory components, regulation of local blood flow, and even physical factors in the alveolus. The effects of frequency of milk removal are tied closely with the local regulation of milk secretion. The mechanism by which the alveoli physically express milk from the lumen during milk removal is called milk ejection. Stimulation of the mammary gland, particularly the teats or nipples, results in secretion of the hormone oxytocin from the posterior pituitary. Oxytocin travels via the blood to the mammary gland and causes contraction of the myoepithelial cells surrounding the alveolus (for a reminder of myoepithelial cells, see the lesson on Mammary Structure: Macro & Micro). This results in expulsion of the luminal milk from the alveolus into the ducts and out of the gland, resulting in the physical removal of milk from the alveoli.

The role of milk removal complicates interpretation of the hormonal requirements for milk synthesis and secretion. Without frequent emptying of the mammary gland (milk removal), milk synthesis will not persist in spite of adequate hormonal status. Conversely, maintenance of intense suckling or milking stimulus will not maintain lactation indefinitely. Nevertheless, suckling or actual removal of milk from the gland is required to maintain lactation.

This module will begin with a discussion of the major hormones involved in galactopoiesis, followed by discussion of milk removal and its implications, and finally by the mechanisms of milk ejection.

Role of Hormones in Galactopoiesis

Role ofHormones

In most species, the mammary gland is not static during most of the lactation period. Although it is thought that any given cell can not divide and lactate at the same time (this is still controversial), it should be remembered that the gland is made up of many epithelial cells and alveoli, and different

alveoli may be in different functional states at any given time.

The lactating goat offers an example of the dynamic changes which occur in the mammary gland during a lactation (see Table below). The increase in milk yield in early lactation is caused by increasing mammary cell numbers (mammary growth during lactation) and some increase in milk secreted per cell. Nutrient availability, metabolic state of the animal, and environmental factors will affect all stages of lactation, but particularly the period of peak and declining lactation. The nanny is often pregnant in very late lactation and concurrent pregnancy with lactation will affect milk yield. In very late pregnancy, there is cell loss (via programmed cell death, called apoptosis), as well as a decline in the milk secreted per cell. A similar scenario can be assumed for the dairy cow.

Qualitative changes in milk yield, mammary secretory cell number, and milk yield per cell occurduring lactation in goats. For example, mammary DNA in lactating goats increases by over 25% during the initial three weeks of lactation, but milk production does not peak until about the eight week (Knight and Peaker, 1984). Activity of lactogenic enzymes continue to increase beyond the third week of lactation, indicating an increase in differentiation of secretory cells. The table below is a summary of work by Knight, C . and M. Peaker (1984, Quarterly J. Exper. Physiol. 69:331).

Weeks of Lactation

Phase of the Lactation Curve

Milk Yield Cell Numbers

Yield per Cell

1 Ascending Increasing Increasing

2 Ascending Increasing Increasing

3 Ascending Increasing Increasing Increasing

4 Ascending Increasing Static Increasing

5 Ascending Increasing Static Increasing

6 Ascending Static

7 Peak Static

8 Peak Static Declining

8 - 11 Peak Declining Declining

11 - 23 Declining Declining Declining

23 - 36 Late lactation, pregnant

Declining Static Declining

In dairy cattle, mammary DNA also increases in early lactation (Akers et al., 1981), but the precise timing and extent of this postpartum mammary growth has not been defined. Few other estimates of mammary gland changes in DNA content in cattle are available, but postpartum mammary growth may be expected for only a short period in early lactation.

The changes in mammary cell numbers (by growth or by cell death) and in milk yield per cell are regulated in part by galactopoietic hormones and in part by local mammary factors. The other sections of this Module discuss specific hormones and their role in regulating established lactation, includinhg Prolactin, Growth Hormone, Placental Lactogen, Adrenal Corticoids, Thyroid Hormone, and Ovarian Steroids.

Role of Prolactin

Prolactin

Prolactin is a primary component of the galactopoietic complex of hormones.

Considerable species variability exists in the importance of PRL in maintenance of lactation. For example, in hypophysectomized rabbits, exogenous PRL administration alone restores lactation to normal (Cowie et al., 1969, J. Endocrinol. 43:651) and PRL is galactopoietic in the intact lactating rabbit (Cowie, 1969, J. Endocrinol. 44:437).

In the hypox. rat, PRL alone does not quantitatively restore lactation to prehypox. levels (see Cowie, 1966, Anterior pituitary function in lactation. In The Pituitary Gland, Vol. 2, pp. 412-443, Eds. Harris and Donovan, Butterworths, London). Minimum requirement in hypox. rats to restore lactation to levels adequate to rear pups is administration of PRL plus glucocorticoid. Administration of GH plus PRL and glucocort.) improves lactational performance.

In the hypox. goat, administration of PRL, GH, glucocort., plus thyroid hormone are required to restore lactation to prehypox. levels (Cowie et al. 1961 J. Endocrinol. 23:79; Cowie et al. 1964 J. Endocrinol. 28:267)

Exogenous PRL is galactopoietic in intact lactating rabbits, while in intact lactating rats, administration of exogenous PRL stimulates milk secretion in early lactation (and reduces the time required for refilling of the mammary gland following suckling). Continuous infusion of PRL into lactating rats may prevent the expected decline in milk production in late lactation.

In many nonruminants, suppression of blood PRL concentrations by ergot alkaloids (such as bromocriptine) does reduce lactational performance. Administration of L-DOPA (which also inhibits PRL secretion from the pituitary) reduces milk yeild, although L-DOPA also inhibits milk ejection. Injection of antisera to PRL receptors can inhibit PRL binding in the rat mammary gland. This inhibits PRL-induced casein synthesis and milk yield is decreased.

So, PRL secretion rates from the pituitary may be limiting in rats and rabbits. The importance of PRL in maintaining lactation in nonruminants is well established, as the above examples illustrate. However, in most nonruminants and ruminants PRL is only one component of a complex of hormones which regulate lactation. Furthermore, the role of PRL in maintaining lactation is less clear in some other species, especially ruminants.

The role of PRL in galactopoietic in cattle is ambiguous. Suppression of PRL in cows and goats

(and in guinea pigs) by ergot alkaloids (bromocriptine) has minimal effects on milk yield, especially compared with decreases of 50% or more when comparable experiments in rodents or near complete failure of lactation in rabbits. An exception to this seems to be the lactating ewe, where bromocriptine administration markedly reduces milk yield (Hooley, R.D., J.J. Campbell, and J.K. Findlay. 1978. The importance of prolactin for lactation in the ewe. J. Endocrinol. 79:301-310), and drugs which stimulate PRL release enhance milk yield (Bass, E., J. Shani, Y. Givant, R. Yagil, and F.G. Sulman. 1974. The effect of psychopharmacological prolactin releasers on lactation in sheep. Arch. Int. Pharmacodynami. Ther. 211:188-192).

So, blood concentrations of PRL in ruminants generally do not seem to be limiting for maximal milk yield (except perhaps in the ewe). However, some evidence does indicate that a 2 hr infusion of PRL into lactating goats starting 30 min after milking may result in a slight (2.6%) but statistically significant increase in milk yield (see Jacquemet and Prigge, 1991, J. Dairy Sci. 74:109).

For more on experimental models used to study lactogenesis and galactopoiesis see Wilde, C.J. and W.L. Hurley (1996. Animal models for the study of milk secretion. J. Mammary Gland Biol Neoplasia 1:123-134).

Mammary PRL receptors:

In the rat mammary gland, PRL receptors increase 4 fold within 2 days of parturition, then decline gradually over lactation. PRL receptors can also be affected by presence or absence of galactopoietic hormones, as indicated in the table below.

Treatment

decline in PRL receptors in rat mammary tissue

ovariectomy 59%

adrenalectomy 45%

hypophysectomy 35%Suckling or milking induces a Prolactin surge in the blood

(see Tucker HA, 1994 above; also see Jakubowski and Terkel, 1986, Endocrinology 118:8)

There is a milking-induced or nursing-induced release of PRL (see graph below; adapted from Tucker 1994). This surge of PRL (green line in the graph) is small compared with the peripartum surge of PRL associated with lactogenesis; about a 3-fold increase over non-stimulated PRL concentrations (blue hatched line). However, the milking-induced PRL surge is a direct link between the act of nursing or milk removal and the galactopoietic hormones involved in maintaining lactation. The surge occurs over a period of about a half of an hour after milking or nursing. This compares with the oxytocin surge which only lasts about 5 to 10 minutes (red stippled box in the figure). Part of the galactopoietic response to nursing intensity (litter size) in pigs or rodents may involve the amount of PRL released at nursing.

Suckling or milking probably works by decreasing prolactin inhibiting factor (PIF) from the hypothalamus, and therefore increasing PRL levels. It may also act to increase the response of the pituitary to prolactin releasing factors (Samson et al., 1989, Endocrinology 124:812). The effect of suckling on PRL declines with advancing lactation, even if nursing stimulus is kept equivalent throughout lactation.

Relationship of milk yield to blood prolactin

(see Koprowski and Tucker, 1973, Endocrinology 92:1480)

In dairy cattle, there is no correlation between milk yield and PRL levels 2-4 hr before milking or 1 hr after milking. However, there is a correlation between milk yield and PRL levels 5 min. post-milking. This may reflect the post-milking PRL response which only lasts for about an hour.

Photoperiod on Growth and Milk Production in Cattle

Photoperiod

For a review of this topic see Dahl et al. (2000) in the reference list.

Effects of light on growth in heifers: Giving heifers 16 hr of light daily increased rate of body growth 10-15% during fall and winter months (this work was done in Michigan, where natural lighting was 9-12 hr light/day). This growth occurred without a proportional increase in feed intake. Increased body wt. was due to protein gain, not body fat. This effect did not occur in growing steers. The increased length of day light did increase PRL, but did not increase GH concentrations in the blood. The response also may involve the effect of light on melatonin (see Stanisiewski et al., 1988, J. Anim. Sci. 66:464). [See also: Tucker and Ringer, 1982, Science 216:1381; Petitclerc et al., 1983, J. Anim. Sci. 57:892; Tucker et al., 1984, J. Anim. Sci. 59:1610; Zinn et al., J. Anim. Sci.

62:1273 , 1986; and Zinn et al., 1989, J. Anim. Sci.67:1249.]

Effects of light on mammary growth in heifers: Mammary growth was stimulated by 16 : 8 (hr light : dark cycle) compared to 8 : 16, in prepubertal and postpubertal heifers. [See Petitclerc et al., 1985, J. Dairy Sci. 68:86.]

Effects of light on milk yield: Cows given 16 hr of light daily increased milk yields by about 10% during the fall and winter. Intake was increased sufficiently to account for increased milk yield. [See Peters et al., 1978, Science 199:911; Peters et al., 1981, J. Dairy Sci. 64:1671; Marcek and Swanson, 1984, J. Dairy Sci. 67:2380; and Stanisiewski et al., J. Dairy Sci. 68:1134, 1985.]

Many stimuli can alter blood PRL levels, especially TEMPERATURE and LIGHT:

Generally, as temperature increases, blood PRL concentrations increase, and as day-length increases, blood PRL concentrations increase, although this response lags a few days behind the change in day-length. Blood PRL concentrations also respond to other stimuli such as stress. Great care must be taken when collecting blood samples from animals not to stress the animal.

Growth Hormone - bST

GrowthHormone

Acknowledgments: Paola Piantoni (Masters of Science, University of Illinois) contributed to the content of this page.

Somatotropin is another name for growth hormone (GH). Bovine somatotropin is abreviated bST.

Nursing causes increased release of GH from the pituitary in the rat and goat, but not in the lactating cow or the lactating woman.

Blood GH decreases with advancing lactation. Thyroid Releasing Hormone (TRH) is the hypothalamic hormone which stimulates release of Thyroid Stimulating Hormone (TSH) from the pituitary. TRH can also release GH from the pituitary. The release of GH from the pituitary in response to TRH administration also decreases with advancing lactation.

In rats - GH administration has little effect on an established lactation.

In ruminants - GH is clearly galactopoietic!

In hypophysectomized lactating goats, GH is necessary to maintain lactation. (see Cowie et al., 1964, J. Endocrinol. 28:253 and 28:267).

In cattle, growth hormone stimulates galactopoiesis, so it is considered a galactogogue. Bovine Somatotropin (bST, the same thing as bovine Growth Hormone) can be mass-produced by genetically engineering bacteria. This has had a significant impact on the dairy industry when its use was approved by the US Food and Drug Administration (FDA). The name for this product of biotechnology is rbST (recombinant bovine Somatotropin hormone) and it is a protein with essentially the same structure and biological activity than GH.

Some important things to consider with rbST treatments in cows:

Effects:

Increase milk production (it is increased within a day and maximized within a week; 10-15% increased in milk yield when administered in early to mid-lactation and up to 40% when administered during late-lactation). The treatment results in coordinated changes in tissues and physiological processes that support the increases in synthesis of lactose, fat, and protein in the mammary gland (i.e. increase in blood flow to the gland which will provide more milk precursors).

Increase persistency of lactation (which means higher levels of milk production for more time).

Increase in DMI within several weeks after start using rbST (6 to 8% per day). For a normal cow, intake response is a predictable function of increased milk production; overall metabolic rate does not increase, so the cow will only eat more to support the increased milk production, at least in the case of long term administration of bST.

The effects are maintained as long as the treatment is continued. Overall, it will improve dairy productive efficiency and reduce negative effects on

the environment if compared to conventional dairying.

Slight effects, or no effects on:

Milk composition (fat, protein, calcium).

No effects on:

Sensory characteristics (flavor, color). Incidence of Mastitis or other health problems: increase of mastitis insignificant

compared to an increase for other reasons, such as seasonal variation (9 times more influence than rbST treatment). Problems normally associated with high milk production, but not directly caused by bST administration, could be seen.

Concerns about consuming milk or meat of bST-treated cows:

bST concentration: within normal variation. It was shown that: 1) milk concentration of rbST treated cows is similar to milk concentration of non treated cows (~10ppb); 2) 90% is destroyed by pasteurization and the rest is digested in the GI tract as

another protein; and 3) GH shows species-specificity, so even if it survived all these obstacles and got to the blood stream intact to exert its effects in the body it would not be active in humans.

IGF-1 level in milk: modest rise, but within normal variation.

Protocol for bST administration:

1st administration after the 9th week (>60d) post-partum (related with peak in milk production (6-8wk) and peak in DMI (10-12wk), which will determine a 60 days post-partum cow at or near positive energy balance).

500 mg/cow every 2 weeks. Body Condition Score (BCS): >= 2.5.

For further information about bST in dairy cattle, see the link to Reference List on the ANSC 438 Home page.

Placental Lactogen

Cattle often are lactating and pregnant at the same time, unlike many species. Consequently, placental hormones may affect mammary function. Although relatively little placental lactogen is released into the mother's blood in the cow, bovine placental lactogen administration during lactation will increase milk yield in dairy cattle. In addition, the mechanism of this galactopoietic effect seems to be different from that of BST. [See Byatt et al., 1992, J. Dairy Sci. 75:1216.]

The goat generally is not lactating and pregnant at the same time (except sometimes in very late lactation). In contrast to the cow, very high levels of caprine placental lactogen are found in the pregnant goat's blood (see section on Mammary Growth and Development for more on Placental Lactogen), and these high levels of PL have a dramatic effect on mammary development in the goat.

Adrenal and Thyroid Hormones

Adrenal andThyroid Hormones

Adrenal Corticoids

*** are essential for maintenance of lactation.

Adrenal glucocorticoids can inhibit lactation at high doses. However, at lower doses (more physiological) exogenous glucocorticoid stimulates milk yield in rats in early lactation and prevents the expected decline in milk yield in later lactation. In ruminants, the effects of high doses of glucocorticoid is inhibitory, but the effects of lower doses are ambiguous, depending on the study evaluated.

In the adrenalectomized rat, milk secretion is impaired and casein mRNA levels are decreased by 85-92%. Cortisol administration will reverse this. Adrenalectomy-ovariectomy stops lactation completely.

In the rat, adrenal secretions are limiting. ACTH in the pituitary declines 68% during extended lactation. Adrenal corticosterone content also declines, but blood concentrations are unchanged. Adrenal corticoid content is highly correlated with mammary nucleic acid content during lactation. Corticoid binding globulin (CBG) declines in blood during early lactation, then increases during later lactation. So, even though blood corticoids stay the same during lactation, the effective concentration declines.

In cattle, there is no change in CBG during lactation. Corticoids are not limiting to milk yield in cows, but are nevertheless essential.

Thyroid Hormones

*** are essential for maximal secretion of milk.

Several observations on the role of thyroid hormone in maintenance of lactation:

Thyroidectomy can be achieved by surgical procedures or by irradiating the thyroid by ingesting radioactive iodine which is sequestered into the thyroid and essentially destroys the thyroid function. Thyroidectomy in cattle by either method results in decreased milk yield.

Injection of thyroid hormone into cattle for 7 weeks at 25% above the normal thyroid secretion rate results in increase milk yield by 27%. However, this is not a permanent effect because continued thyroid hormone administration at 150% for 7 more weeks. had little or no effect.

Feeding thyroprotein (iodinated casein) to cows increases milk yield by 10 % in early lactation and by 15-20% in late lactation. But, the effect only lasts 2-4 mos. and subsequent yields are below normal. There generally is no net benefit in feeding thyroprotein over the entire lactation. The transient increase in milk yield in cows fed thyroprotein feeding in cows only occurs when feed intake is increased.

Dairy cattle are under very high metabolic demands in early lactation. In addition, there is an inverse relationship between a cow's milk yield and blood levels of thyroid hormones in early lactation. Curiously, this apparent hypothyroid state of the cow is in contrast with the increased milk yield attained when lactating cows are administered thyroid hormone. This apparent inconsistency becomes clearer if the metabolism of thyroid hormones by non-thyroid tissues, including the

mammary gland, is taken into account.

Mechanisms of thyroid hormone action:

Thyroid stimulating hormone is secreted by the anterior pituitary and stimulates secretion of thyroid hormones from the thyroid gland. The primary thyroid hormone produced in the thyroid gland is thyroxine (3,5,3',5'-tetraiodothyronine; or T4). The T4 is an alanine molecule with a tyrosyl ring and a phenolic ring, each of which is iodinated at the 3 and 5 and 3' and 5' carbon positions, respectively. However, T4 has a relatively low affinity for thyroid hormone receptors in target tissues and is considered a prohormone requiring further modification to achieve full activity. The major biologically active form of thyroid hormone is T3 (3,5,3'-tri-iodothyronine). Deiodination of T4 can occur in the thyroid gland or it can occur in the various target tissues. Several specific deiodinase enzymes have been identified, each belonging to a family of selenocysteine-containing enzymes. Type I deiodinase is found predominantly in the liver, kidney and thyroid, but is also present in skeletal muscle, heart, lung, intestine and mammary gland, at least in the rat. Type I deiodinase can catalyze either 5'- or 5-monodeiodination. The 5'-deiodination removes an iodine from the outer phenolic ring, resulting in formation of the active T3 (3,5,3'-tri-iodothyronine), while 5-deiodination removes an iodine from the inner tyrosyl ring, resulting in the inactive reverse T3 (3,3',5'-tri-iodothyronine). Type II deiodinase is found in the central nervous system (cerebral cortex, cerebellum, hypothalamus), pituitary, and brown adipose tissue. Type II deiodinase catalyzes only removal of the 5'-iodine. Therefore, type II deiodinase will convert T4 to T3, while rT3 is converted to the inactive T2 (3,3'-di-iodothyronine). Type III deiodinase has 5-deiodinase activity and therefore converts active T4 or T3 to inactive rT3 or T2, respectively. Type III deiodinase is found in cerebral cortex and skin, but also in several tisues of the fetus. It may be involved in metabolizing active thyroid hormone in the fetus to protect the fetal brain and other tissues from exposure to high levels of thyroid hormones.

As indicated above, the mammary gland of the rat has Type I deiodinase activity. However, there seems to be considerable species diversity in which type of deiodinase is present in the mammary gland. For example, the mammary deiodinase activity is Type II is the only activity present in the cow and mouse mammary glands, while both Type I and II are found in the sow's mammary gland. Deiodination of thyroid hormones by peripheral tissues, including the mammary gland, plays a primary role in regulating thyroid homeostasis, and therefore is indicative of the metabolic state of the animal and the particular tissue. With locally high activity of deiodiinase in the mammary gland results in local production of T3 which in turn stimulates metabolism in the gland.

So, in early lactation in the cow the excessive local conversion of T4 to T3 in the mammary gland may be reducing blood levels of T4. In addition, administration of T3 to the cow bypasses the T4-T3 conversion at the mammary level and provides greater T3 to the gland resulting in enhanced milk yield.

For more on deiodinases in general and in mammary tissue, see the reference list.

Ovarian Steriod Hormones

OvarianSteriods

Ovarian Steroids

Ovariectomy has no effect on postpartum mammary growth or lactation, suggesting that ovarian steriods are not necessary for maintenance of lactation. On the other hand, increased blood concentrations of estrogen may affect milk production.

Although relatively few species are concurrently lactating and pregnant, concurrent pregnancy does influence persistency of milk yield in the declining phase of lactation. This is particularly evident in dairy cattle. Inhibitory effects of pregnancy on lactating cows do not become apparent until about mid pregnancy (Wilcox et al. 1959; Bachman et al. 1988). An inhibitory effect of pregnancy on lactation has been noted in a number of other species, as well (Wilde and Knight 1989; Tucker 1994). The mechanism of this effect is not fully understood. Progesterone seems to have no effect on milk yield in the lactating cow because (1) progesterone has a higher affinity for milk fat than for glucocorticoid receptors, and (2) there are no progesterone receptors in the mammary gland during lactation. However, the timing of inhibition of milk yield in cattle coincides approximately with the period of increasing placentally-derived plasma estrogen (Robertson and King 1979).

Administration of pharmacological doses of estrogen will decrease milk yield. In fact, administration of estrogen to cows in late lactation can enhance the rate at which the mammary gland undergoes involution at drying off. A role for estrogen in mammary gland involution in dairy cattle has been indicated (Athie et al. 1996). Estogen was administered to late lactation dairy cattle for 4 days prior to dry-off, or milk stasis, resulting in a more rapid rate of involution in the estrogen treated cows. However, cows used for that study were already producing low yields of milk (means of ~11 kg daily with 3X/day milking) and already had elevated lactoferrin levels in milk (an indicator of mammary gland involution) prior to drying off. Lactoferrin is usually relatively low in concentration in lactating cattle that do not have mastitis. The administration of estrogen significantly reduced milk yield even further prior to drying off (halting of any further milk removal). In effect, this treatment gave the estrogen treated cows a 4 day start on the involution process, accounting for some of the 6 day difference noted in involution rate. Nevertheless, that study and other studies on the effects of estrogen on milk composition during lactation (Bachman 1982) strongly suggest that estrogen, and perhaps pregnancy, may have an effect on mammary gland involution in cattle.

Role of Milk Removal in Galactopoiesis

MilkRemoval

Milk removal is required for maintenance of lactation!!!

Milk removal from the lactating mammary gland is the major factor in maintaining milk secretion. Removal of milk from mammary alveoli results in a range of effects on mammary gland function manifested as immediate, short and long term effects (Wilde et al., 1987). Immediate effects of milk removal occur within seconds to minutes of stimulation of the gland and removal of milk. As milk is secreted from the mammary epithelial cells into the alveolar lumen milk components accumulate in the lumen. In addition, an autocrine factor, termed feedback inhibitor of lactation (FIL; Wilde et al., 1995; Knight et al., 1998), is secreted from the alveolar epithelial cells and accumulates in the alveolar lumen. The FIL then feedsback and inhibits further milk secretion from the alveolar epithelial cells. Removal of milk at milking or suckling removes FIL and allows for continued milk secretion. Milk accumulation in the gland also causes an increase in intramammary pressure, which reduces blood flow to the tissue. Furthermore, the stimulus during suckling or massage of the gland by the piglets causes increased prolactin concentrations in the sow's blood (Bevers et al., 1978; Kendall et al., 1983; Algers et al., 1991; Spinka et al., 1999). Short term effects of milk removal are thought to occur through the enhancement of epithelial cell differentiation (Wilde et al., 1987; Wilde and Knight, 1989). This work has been done primarily in ruminants and is based on increasing frequency of milk removal. Higher frequency of milking in ruminants results in greater activities of milk synthesizing enzymes per cell in lactating tissue. Long term effects of more frequent milking in ruminants are thought to occur over a period of weeks or longer and occur in response to increased cell proliferation (Henderson et al., 1985).

Stimulation Intensity :

In the case of a dairy cow that is milked by a milking machine, the intensity of milking is controlled by the machine and stays mostly constant throughout a lactation. However, in nature there may be variable numbers of offspring suckling at any given time, the nursing offspring may be of different sizes and have different levels of aggressiveness in nursing. The greater the nursing intensity the more mammary growth and the more milk produced. Nursing intensity means the number of nursing young (litter size), especially in litter-bearing species such as the pig. Although it is less well documented, this effect of stimulation intensity also probably means the vigor with which the young nurse, perhaps involving the degree of gland emptying which occurs at each nursing or the intensity of stimulation of the nipple. (What is the effect of litter size on mammary growth during lactation? If you do not remember, then see the Mammary Development section?).

The pig will be used as the example in this section. Factors which contribute to suckling intensity in pigs include litter size, suckling interval (frequency of milk removal), piglet size, limitations of milk letdown, piglet behavior, and a number of maternal characteristics (Hartmann et a., 1997; Brooks and Burke, 1998; King, 2000). All of these factors will contribute to the overall process of milk removal. It is reasonable to expect that the cell proliferative effects of very frequent milk removal occurring in the sow may provide a primary stimulus for mammary growth observed during lactation. While the relationship between milk removal from the sow's mammary glands and cell proliferation in those glands has not been demonstrated directly, there is considerable indirect

evidence for such a relationship.

Litter size is related to total milk production by a sow and extent of postpartum mammary gland growth, as discussed above. The more piglets suckling a sow, the more total milk is removed. Size of the suckling piglet is positively correlated with sow milk production and average daily gain of the piglet (Pluske and Dong, 1998; King, 2000). More effective milk removal is expected when a larger piglet is suckling a gland. Several studies have demonstrated that shorter suckling intervals result in greater total milk removal (Sauber et al., 1994; Auldist et al., 1995; Brooks and Burke, 1998). Maximal refilling of the gland occurs within no more than 35 min after a suckling bout (Spinka et al., 1997). Unlike the ruminant, which has large cisterns and non-alveolar milk storage areas, the sow has little non-alveolar milk storage volume. Milk accumulation in the sow's mammary gland after a suckling bout should rapidly diminish the rate of further milk secretion. Nonnutritive suckling bouts occur frequently in domestic sows and are part of the natural behaviour of these animals (Brooks and Burke, 1998). While not resulting in milk removal, nonnutritive suckling bouts probably stimulate further prolactin secretion and contribute to maintenance of lactation. The total number of nutritive sucklings per 24 h period is an important determinant of total milk removal from the mammary glands. Small differences in suckling intervals could result in significant differences in total milk removed from the gland within a day.

Health and aggressiveness of the suckling piglet, sow mothering behaviour, and perhaps teat size and shape are among a range of other factors which could directly or indirectly affect milk removal (Fraser, 1990; Brooks and Burke, 1998). Age, parity and sow body condition all affect milk production in sows (King, 2000) and would be expected to affect milk availability and ultimately milk removal.

Some other observations related to milk removal and galactopoiesis:

There is no evidence for nerves directly controlling secretory activity. Mammary glands which have been transplanted to the neck of goats can still secrete milk.

Nursing stimuli which occurs without milk removal (ie. when the nipples are ligated) in lactating rats results in a slower rate of involution, but does not quantitatively prevent losses of mammary cells or metabolic activity.

In rats, repeated replacement of litters with new foster litters can prolong lactation, for up to a year.

In cows, if you do not milk 2 quarters for 2 weeks (but continue milking the other 2 quarters) then start milking the unmilked quarters again, they will recover milk secretion, but the yield is typically lower than in control quarters (the quaters milked throughout the period). However, throughout the next lactation, the experimental quarters produce more than the control quarters. These types of experiments indicate that local, intra-mammary factors are responsible for the observed effects. Each gland is exposed to the same blood or the same systemic stimuli.

Autocrine Control of Lactation

AutocrineControl

Conrtol of lactation is clearly regulated by hormones, however local factors are also important. More frequent milk removal in both cows and goats increases milk yield. Consider studies where cows or goats have had one side of the udder milked more frequently than the other side (for example 3X/day milking or hourly milking vs. 2X/day). Rate of milk secretion increases only in the gland which is milked more frequently.

Furthermore, if one udder side is milked 2X/day and the other side milked only once per day, or if milking is incomplete from one side, milk yield is decreased only in the less frequently emptied gland.

These unilateral effects cannot be attributed to systemic (hormonal) control because both sides of the udder are exposed to the same concentrations of galactopoietic hormones.

The response requires actual removal of milk from the gland. For example, hourly massage of the gland without milk removal does not have the same effect (Linzell and Peaker, 1971).

In addition, the response is not the result of increased pressure of the stored milk. Studies using goats where one gland (remember that goats only have two glands) was milked 2X/day and the other gland milked 3X/day have been used for this type of experiment. In glands milked 3X/day the volume of milk from the extra milking was replaced back into the gland by an equal volume of an inert sucrose solution (so, intramammary pressure was the same in both sides of the gland even though the milk was removed once more often on the one side). The result was that secretory rate was still increased by 3X/day milking, even though intra-mammary pressure was maintained the same as in the 2X/day milked gland.

These types of observations gave rise to the hypothesis that a milk constituent acts as an inhibitor of milk secretion and that removal of this inhibitor at milking regulates the rate of milk secretion.

A milk whey protein (~7 kDa molecular weight) has been identified as a Feedback Inhibitor of Lactation (FIL; see Wilde CJ, Addey CVP, Boddy LM, Peaker M (1995) Autocrine regulation of milk secretion by a protein in milk. Biochem J 305:51-58). This inhibitor is thought to be secreted by the mammary epithelial cells and in turn inhibits further milk secretion as its own concentration increases in the alveolar lumen. The exact mechanism of how this feedback inhibitor works is unknown. In vitro, the feedback inhibitor seems to reduce secretory rate and key enzymes in mammary cells, stimulates intracellular degradation of newly synthesized casein, reduces prolactin receptor numbers on the cells, and can inhibit differentiation of mammary cell function.

The balance between systemic (hormonal) and local (FIL) control of milk secretion is illustrated in the following discussion.

Each time milk is removed:

Prolactin release is stimulatedIntra-mammary pressure is relievedFIL is removed from the alveoli

If milk is not removed:

There is no stimulation of PRL release

There is an acute accumulation of milk in the gland, resulting in:

Increased intra-mammary pressureActivation of sympathetic nervesDecreased mammary blood flowDecreased availability of hormones and nutrients to the gland

Rate of milk secretion declines

This interplay between systemic and local factors in control of galactopoiesis can be thought of as a seesaw.

The gland is under the influence of the systemic factors shortly after milking and maximal secretion rate is achieved. This gradually slows as the role of the local factors becomes dominant. If milk is not removed, then the secretion rate will eventually drop to zero (see below), however, under normal nursing or milking intervals the secretion rate does not go to zero. Once milk is removed, the cycle begins again.

(see Wilde and Peaker 1990 J. Agric. Sci. 114:235)

Milk Secretion Rate

SecretionRate

Milk yield is dependent on (1) the amount of secretory tissue and (2) the rate of milk secretion (per unit of time). Secretion rate is affected by the accumulation of milk in the alveolar lumen. Accumulation of milk in the lumen increases the intra-mammary pressure (see figure below). Once the intra-mammary pressure reaches a certain level (probably about 8 to 10 hours after the last milking int he dairy cow), secretion rate declines. If the pressure increases enough (in the cow at about 70 mm Hg), then secretion stops and milk starts to be resorbed. In the dairy cow, secretion rate stops (reaches zero) at about 35 hours after the last milking. Instruments have not been available to measure intra-alveolar pressure. Generally the intra-mammary pressure has been measured in the teat cistern using a teat cannula. So these intra-mammary pressure estimates reflect total gland pressure from accumulation of milk and not directly the intra-alveolar pressure. These types of studies are done with great care to assure that milk ejection is not stimulated, which would also increase intra-mammary pressure.

[Adapted from Schmidt, G.H., 1971, Biology of Lactation, W.H. Freeman and Co., p. 150.]

The inhibition of milk secretion that accompanies increasing intra-mammary pressure is probably caused by a chemical inhibitor (FIL) rather than the increased pressure of the fluid itself (see above).

Oxytocin and Milk Ejection

Oxytocin

Oxytocin is a 9 amino acid long peptide. The amino acid structure of oxytocin is:

Cys-Tyr-Ile-Gln-Asn-Cys-Pro-Leu-Gly

It has a molecular mass of 1007 daltons. Oxytocin has a disulfide bond between the two cysteines. Reduction of the disulfide bond inactivates oxytocin. One IU (international Unit) is approximately 2

micrograms of pure peptide.

Hypothalamic Nuclei and Oxytocin Synthesis

Oxytocin is syntheized in the hypothalamus in specific nuclei, the paraventricular nucleus and the supraoptic nucleus in the hypothalamus. [A cluster of nerve cells in the brain is often called a nucleus. This is different from the nucleus of a single cell.] Neurons in these hypothalamic nuclei synthesize the oxytocin precursor and package it into vesicles. Oxytocin is initially synthesized as a large molecular weight precursor which also consists of the oxytocin-carrier peptide neurophysin. The precursor is proteolytically cleaved in the neuron in the oxytocin-containing vesicle to yield oxytocin bound to neurophysin. The oxytocin-neurophysin complex is the intracellular storage form of oxytocin.

The oxytocin-containing vesicles are transported from the cell body (which is in the hypothalamus), down the axons to the neuron endings in the posterior pituitary. This is called the hypothalamo-neurohypophysial tract. The oxytocin-neurophysin complex is stored in neurosecretory granules called herring bodies in the axon ending.

The synthesis of oxytocin in the cell bodies and its transport to the axon endings occur separately from the milk ejection reflex.

Oxytocin Surge

The oxytocin concentration in the blood normally is increased within 1 to 2 min. after udder stimulation, but the amount released is declining during milking.

Why is pre-stimulation of the cow needed before milking?

hygiene - for prevention of mastitis and for maximizing milk quality. milk ejection - [see J. Dairy Sci. 1980 63:800] In this study they compared milking after

manual stimulation (washing the teats) vs. nonstimulated (milkers were put straight on the gland with no pre-washing or other stimulation). The manual stimulation resulted in shorter machine-on times (higher milking efficiency) and higher peak and average milk flow rates. Mean peak oxytocin was not different, but the pre-stimulated cows' oxytocin peaked at 2 min. after stimulation, compared with 5 min. after machine-on time for the unstimulated cows.

milk flow rate - [see J. Dairy Sci. 1985 68:1813] In this study, average milk flow rate increased with increasing duration of udder stimulation (30, 60, 120 sec.) and milking machine-on time decreased. However, oxytocin concentration was not different, and when premilking stimulation time was added to machine-on time the difference in average milk flow rate was not significant.

*** The timing of oxytocin release relative to milk removal is an important factor affecting milk ejection.

For example, the image below illustrates the milk flow rate of a cow that A) had teats stimulated for 1 min prior to attaching the milking machine, and B) had the milking machine put on immediately without any prior manual stimulation. Note that the machine-on-time is shorter for the pre-stimulated cow and the peak flow rate is higher for the pre-stimulated cow. Also note the initial rise and fall of

flow rate during the first min of milking in B. In this case, the milking machine is initially removing the milk present in the cisterns (does not require milk ejection) and is providing the tactile stimulation necessary to elicit the normal release of oxytocin, which causes the second increase in milk flow rate. In both A and B, the final small increase in milk yield probably was caused by machine stripping by the person milking the cow.

The sensitivity of the neuroendocrine reflex seems to decline as lactation progresses. Peak oxytocin seems to come later after mammary stimulation as lactation progresses. Peak oxytocin occurs at 1 min. at 1-2 weeks of lactation , 2 min. at 5-6 weeks, and 15-16 weeks. Maximum oxytocin concentration during milking also declines as lactation progresses. Extra-tactile stimuli often can release oxytocin before milking, but the occurrence of this declines as lactation progresses.

The dry or nonlactating period may serve to restore the sensitivity of the neuroendocrine reflex. Nonlactating cows will release oxytocin in response to udder stimulation. But, virgin heifers do not respond substantially to udder stimulation. Apparently maximum oxytocin release in response to udder stimulation occurs only if the mammary gland is lactating or has lactated.

Maximal prolactin release from the pituitary in response to tactile stimulation of the udder depends on the presence of a fully developed mammary gland.

It is estimated that the bovine pituitary has about 800 micrograms of oxytocin. This is about 40X what is in the blood under resting conditions. Only about 1/3 of pituitary oxytocin is released at a milking.

How much oxytocin is needed to elicit milk ejection? Peak oxytocin is about 11to 65 microunits/ml serum; 40 liters of blood in a cow = about 0.4 to 2.6 IU. Normally you inject 10 IU to cause milk letdown, but as little as 0.02 IU into the jugular can result in milk ejection (see Sagi et al. J. Dairy Sci. 1980 63:2006).

*** Oxytocin receptors on myoepithelial cells can respond to very low levels of oxytocin.

*** Oxytocin has a short half-life in the blood = 0.55 to 3.6 min. This means that the removal of milk by machine or by nursing must be closely timed with stimulation of the teats.

Other Roles of Oxytocin

Injection of oxytocin into the ventricles of the cerebellum in rats induces maternal behavior. Oxytocin has insulin-like activity and it may be lipogenic. The mother is rapidly losing a

great deal of lipid when milk is removed. Both oxytocin and prolactin are implicated in osmoregulation. The mother is rapidly losing a

great deal of water when milk is removed. Oxytocin-containing neurons from the paraventricular and supraoptic nuclei go to other

brain regions which are involved in autonomic regulation (such as cardiovascular effects). In these cases, oxytocin is acting as a neurotransmitter.

Oxytocin may be directly or indirectly involved in prolactin release during proestrus in the rat, but this may not be the case in prolactin release caused by suckling or milking (Johnston and Negro-Vilar, 1988, Endocrinology 122:341). The interaction between oxytocin (or oxytocenergic neurons in the hypothalamus and brain) and prolactin release

from the pituitary remains an area of investigation (see also Mori et al., 1990, Endocrinology 125:1009).

Involvement of Autonomic Nervous System and Stress

Mammary Nerves& Stress

The autonomic nervous system is part of the central nervous system. It mainly controls viseral function. The autonomic nervous system is made up of two types of nerves, the parasympathetic nerves and the sympathetic nerves.

Parasympathetic nerves: The neurotransmitter of parasympathetic nerves is acetylcholine.

There is no parasympathetic innervation in the mammary gland.

Sympathetic nerves: The neuroendocrine components of sympathetic nerves are epinephrine and norepinephrine. Epinephrine (adrenaline) is primarily from adrenal medulla. Norepinephrine is a neurotransmitter from peripheral nerves and nerves in the brain. Norepinephrine also can from the adrenal medulla.

The effect of sympathetic nerves on milk ejection depends upon the type of neurotransmitter receptor: Generally,

- alpha-receptors are vasoconstrictive. - norepinephrine can stimulate milk ejection via brain alpha-receptors. - norepinephrine can inhibit milk ejection via brain beta-receptors. - it is the location of these receptors that is the most important thing.

Most sensory receptors (neurons) are located in the teat. There are pressure-sensitive neurons around the cisterns and the large ducts.

*** There is no direct innervation of alveoli or myoepithelial cells!

If you electrically stimulate the cut end of the mammary nerves, you do not get milk ejection.

Norepinephrine and epinephrine can inhibit oxytocin-induced contraction of myoepithelial cells.

Stressful stimuli will inhibit milk ejection. This occurs via epinephrine or norepinephrine derived from

the adrenal gland or the sympathetic nerves by the following mechanisms :

Norepinephrine reduces myoepithelial cell contractial response to oxytocin; this is a direct inhibition at the myoepithelial cell level.

Norepinephrine decreases mammary blood flow (amount of oxytocin to the gland); this is an inhibition at the mammary tissue level.

Norepinephrine reduces oxytocin release from the pituitary; this is an indirect effect mediated by inhibition of oxytocin release at the hypothalamic level.

In the bovine species norepinephrine is the primary catecholamine. Injections of norepinephrine into cows which increase blood levels to 2 to 5X above normal will cause a decrease of milk yield by 10%. Oxytocin is not altered.

Emotional disturbances can cause inhibition of the CNS part of the milk ejection reflex. This may especially occur after calving in the first-calf heifer. Inject of oxytocin may be needed to remove milk because failure to remove the milk will result in reduced yield through lactation. This may need to be continued for a time.

Other Mechanisms of Milk Ejection

Myoepithelial cells will also contract in response to vasopressin (ADH or antidiuretic hormone). Vasopressin has about 1/5 the oxytocic activity of oxytocin. It probably is not of physiological significance in milk ejection.

Visual or auditory stimuli can cause milk ejection. Milk ejection is a condition response.

Stimulation of the genital tract such as vaginal distention causes release of large amounts of oxytocin.

The Mechanical Tap Stimulus does not involve oxytocin. It will occur under anesthesia or denervation of the udder. It is not inhibited by epinephrine. Kneading or butting of the udder by the young may elicit this response. This may involve distortion of the alveolar structure or the myoepithelial cell structure, resulting in milk ejection.

Residual Milk

Residual Milk

About 15-25% of the total amount of milk in the udder at the start of milking is not removed during milking. This milk is referred to as residual milk. Residual milk is also called complementary milk. The only way to get residual milk out is by injection of oxytocin. Usually this is done by intravenous injection of 10 IU of oxytocin or injection of 20 IU oxytocin either subcutaniously or intramuscularly. However, 0.5 IU (intravenous) can be as effective as 16 IU. The response dependsup on the cow and other circumstances. In some cases as little as 0.02 IU may work. Oxytocin injection (given 3 min. after start of udder preparation, but prior to attachment of milking machine) for an entire 305-day lactation resulted in over 800 Kg more milk during the lactation (see J. Dairy Sci. 1991 74:2119)

Twice daily injection of oxytocin immediately after normal milking followed by remilking resulted in substantially greater milk yields, but, the percentage of milk collected at the normal milking declines over time, while the percentage of residual milk increases. Repeated injections of large doses of oxytocin interferes with normal secretory activity of mammary epithelia and inhibits the normal milk ejection reflex.

Continuous oxytocin infusion (at 100 or 200 IU/day) decreases milk yield (see J. Dairy Sci. 1973 56:181).

After injection of oxytocin and removal of residual milk, concentrations of Na, Cl and whey proteins (probably serum-derived) are increased and lactose concentrations decreased in the next milking . This change in milk composition persisted for several milkings (see Wheelock et al., 1965, J. Dairy Res. 32:255).

Lactating heifers have less residual milk than older cows. The percentage of residual milk is greater for lower producing cows than for higher producing cows. Cows with a higher percentage of residual milk usually have a lower persistency of lactation

Residual milk decreases in proportion to milk yield as lactation progresses; that is, the percentage of residual milk remains the same throughout a lactation. The genetic heritability of residual milk is ~.34, and the repeatability between lactations for a cow is ~.77

Percentage of milk present in the udder at the beginning of milking that remains as residual milk after milking.

Residual Milk

Milking intervals ranging from 4 -24 hr. 9.6 - 17.8 %

Comparison of lactation number:

First 10.0 %

Second and third 15.2 %

Fourth and later 17.9 %

Comparison of effect of machine stripping:

Machine-stripped cows 12.8 %

Nonmachine-stripped cows 14.0 %

Cows receiving oxytocin subcutaneously 16.8 %

Cows measured for one year 12.2 %See Schmidt GH 1971, Biology of Lactation, p. 151.

Mammary Gland Involution

Mammary GlandInvolution

The lactation function of the mammary gland is maintained by a delicate balance of systemic blood-borne factors and local mammary-derived factors, many of which are directly affected by the process of milk removal. Systemic factors include galactopoietic hormones such as growth hormone and suckling-induced prolactin secretion which generally stimulate milk secretion, but also include factors arising from competing physiological states such as pregnancy which may inhibit lactation function. Additionally, local control of milk secretion is directly linked to physical removal of milk. The impact of these factors on mammary function in dairy cattle is evident from the known effects of frequency of milk removal on milk yield, the effects of galactopoietic hormones on milk secretion, and the effects of milk stasis-induced mammary involution on mammary function. For a review of galactopoietic factors see the sections in the Lactation Resouorces on Hormones and on Milk Removal.

Maintenance of Lactation Function

The role of galactopoietic hormones such as prolactin in maintenance of lactation is well established (reviewed by Tucker 1994), although specific cellular mechanisms of action continue to be investigated. Prolactin is considered the major galactopoietic hormone in nonruminants. Prolactin is released at the time of milk removal in ruminants and nonruminants, and it remains a key systemic modulator of milk secretion during lactation. Conversely, growth hormone is generally considered to be the predominant galactopoietic hormone in ruminants (Bauman 1992; Tucker 1994). Inhibition of prolactin secretion or administration of prolactin to lactating cows has little effect on milk yields (Karg and Schams 1974; Plaut et al. 1987). However, these apparently clear-cut roles of prolactin vs. growth hormone in maintenance of lactation in nonruminants vs. ruminants are probably an oversimplification (Wilde and Hurley 1996). For example, in lactating sheep both prolactin and growth hormone seem to be important for galactopoiesis (Hooley et al. 1978; Tucker 1994). Even in the rat, recent studies have demonstrated an important role for growth hormone, independent of the role of prolactin (Flint et al. 1992; Flint and Gardner 1994).

Regardless of the hormones involved, all attempts to evaluate milk secretion must account for continued removal of milk. This is a reminder of the critical role of local mammary factors in maintenance of milk secretion. One such factor that plays a major role in regulating milk secretion in many species is a feedback inhibitor of lactation (FIL) found in milk (Wilde et al. 1995). FIL is thought to be produced by the mammary cells as they synthesize and secrete milk. Accumulation of FIL in the milk-producing alveoli results in feedback inhibition of milk synthesis and secretion. Frequent removal of milk from the gland minimizes local inhibitory effects of FIL and increases milk secretion (Wilde et al. 1987; Wilde and Knight 1989; Wilde and Peaker 1990).

Lactation in Decline

In spite of continued milk removal (with associated removal of FIL and stimulation of a post milking prolactin surge), milk yield in dairy cattle declines as lactation progresses. This decline occurs even with routine administration of growth hormone (bovine somatotropin). Lactation persistency is of particular concern in the production of milk. Mammary tissue function declines after peak lactation and this is at least in part due to a decrease in mammary cell number (Knight and Peaker 1984; Wilde and Knight 1989). The cell loss during the declining phase of lactation in the goat and cow apparently is the result of programmed cell death, also called apoptosis (Quarrie et al., 1994; Wilde et al., 1997). The mechanisms that control lactation decline remain important areas of investigation. Mammary involution is a greatly enhanced extension of these processes leading to a complete cessation of lactation function.

Concurrent pregnancy also influences persistency of milk yield in the declining phase of lactation. Inhibitory effects of pregnancy on lactating cows do not become apparent until about mid pregnancy (Wilcox et al. 1959; Bachman et al. 1988). An inhibitory effect of pregnancy on lactation has been noted in a number of other species, as well (Wilde and Knight 1989; Tucker 1994). The mechanism of this effect is not fully understood. However, the timing of inhibition of milk yield in cattle coincides approximately with the period of increasing placentally-derived plasma estrogen (Robertson and King 1979). Estrogen may have an effect on the transition of mammary function from a lactating state to an involuting state (Athie et al. 1996; Bachman 1982).

Bovine Mammary Gland Involution

Cessation of milk removal leads to rapid changes in the mammary tissue and initiation of the process of mammary involution (Hurley 1989). Changes in composition of mammary secretions during the early phases of involution indicate rapid changes in the normal mechanisms involved in milk synthesis and secretion (see discussion below; also Hurley and Rejman 1986; Hurley 1987; Hurley et al. 1987; Rejman et al. 1989; Hurley 1989). These changes in mammary gland secretion composition include a rapid decline in lactose concentration in the mammary secretions, indicating that lactose synthesis, and the associated water transport mechanism, decline soon after cessation of milk removal. However, total protein concentrations increase in early involution, partially because of water resorption from the secretion and partly due to increased concentrations of lactoferrin, serum albumin and immunoglobulins. Lactoferrin is a major protein found in mammary secretions during involution (Rejman et al. 1989). Its synthesis is increased during involution in contrast to milk-specific proteins such as casein whose synthesis is decreased (Hurley and Rejman 1993; Hurley et al. 1994a). Lactoferrin has a number of potential functions in the mammary gland, particularly as a nonspecific disease resistance factor (reviewed by Sanchez et al. 1992).

Involution-associated ultrastructural changes in bovine mammary cells begin within 48 hours after cessation of milk removal (Holst et al. 1987; Hurley 1989). The most apparent change is the formation of large stasis vacuoles in the epithelial cells (Holst et al. 1987), formed largely as a result of intracellular accumulation of milk fat droplets and secretory vesicles (Hurley 1989). These vacuoles persist to at least 14 days of involution and are usually gone by day 28 (Holst et al. 1987). Alveolar lumenal area declines during this period, while interalveolar stromal area increases. A substantial reduction in fluid volume in the gland occurs between day 3 and 7 of involution (Hurley 1989), probably accounting for the reduction in lumenal volume. By day 28 the collapsed alveolar structures remaining are considerably smaller than during lactation, with a very small lumen. General alveolar structure is maintained thoughout involution in the cow.

Histological and ultrastructural work on the bovine mammary gland during involution (Holst et al. 1987; Hurley 1989) provides no evidence for the extensive tissue degeneration observed in other species, such as rodents and others (Helminen and Ericsson 1968a,b; Helminen and Ericsson

1971). Limited autophagocytic processes occur only transiently during the initial two days after cessation of milking. Formation of autophagocytic structures in rodent mammary tissue is characteristic of involution (Helminen and Ericsson 1968a,b; Helminen and Ericsson 1971). A detachment of epithelial cells from the basement membrane and their loss from the tissue has been reported in rodents and other species (Wellings and DeOme 1963; Verley and Hollman 1967; Helminen and Ericsson 1968b; Richards and Benson 1971). This leaves characteristic bare spaces on the basement membrane and myoepithelial cells are thought to fill the space. No such situations are observed in the involuting bovine gland (Holst 1987; Holst et al. 1987). More recently, the involution process in the mouse has been characterized by examining the role of apoptosis.

Apoptosis and Mammary Gland Involution

Mammary involution in the mouse is characterized by a rapid loss of tissue function and degeneration of the alveolar structure and massive loss of epithelial cells. This cell loss is due to programmed cell death or apoptosis (Strange et al. 1992, Walker et al. 1989). Apoptosis is a both a natural and systematic method of cell suicide which takes place during normal morphogenesis, tissue remodelling and in response to infection or irreparable cell damage (Wyllie et al. 1984, Schwartzman & Cidlowski, 1993). There are two distinct types of cell death, apoptosis and necrosis, which may be distinguished by morphological, biochemical and molecular changes in dying cells. The process of apoptosis was originally distinguished from necrosis on the basis of its ultrastructure (Kerr 1971, Kerr et al. 1972). Apoptosis may be identified by a characteristic pattern of morphological changes: nuclear and cytoplasmic condensation, nuclear fragmentation and formation of apoptotic bodies (Walker et al. 1989, Strange et al. 1992). These changes are associated with cleavage of chromatin into discrete sized oligonucleosome fragments by a calcium dependent endonuclease (Arends et al. 1990), resulting in the appearance of oligonucleosomal DNA laddering in ethidium bromide stained gels (Wyllie et al. 1980).

A morphology consistent with apoptotic cell death can be observed in the murine mammary gland within two days of milk stasis. The nucleus and cytoplasm condense, the chromatin becomes fragmented and marginated, and apoptotic bodies are formed (Walker et al., 1989; Strange et al., 1992). This cell loss results in extensive disintegration of alveolar structure during the early period of involution in the mouse. DNA laddering characteristic of apoptosis also has been detected in goat mammary tissue during early and late lactation (Quarrie et al., 1994) and during late lactation in the cow (Wilde et al., 1997). This would suggest that removal of secretory epithelial cells by apoptosis is a normal physiological event in the ruminant mammary gland, even during lactation. In addition, milk stasis has been demonstrated to stimulate DNA laddering in both goat and cow mammary tissue (Quarrie et al., 1994; Wilde et al., 1997). These observations suggest that mammary epithelial cells are indeed lost during involution in the bovine mammary gland. However, this process of cell loss does not seem to be as dramatic as that observed in the mouse. In spite of the loss of cells, bovine mammary alveoli retain general structural integrity throughout involution (Holst et al. 1987). While the role of cell loss in the mouse mammary gland during involution is clear, the impact of mammary apoptosis in the bovine is not fully characterized.

Dry Period in Dairy Cattle

DryPeriod

Dry Period and Subsequent Lactation

*** The mammary gland of the dairy cow requires a nonlactating (dry) period prior to an impending parturition to optimize milk production in the subsequent lactation.

This period is called the dry period, and it includes the time between halting of milk removal (milk stasis) and the subsequent calving. Generally, 45 to 50 days is recommended. If less than 40 days, then milk yield in the next lactation will be decreased. [see Swanson 1965; Coppock et al. 1974; Dias and Allaire, 1982.]

The normal procedure to dry off a cow is to withdraw all grain and reduce the water supply several days before the start of the dry period. This drastically reduces the milk production during that time. Then milking is halted about 45 to 50 days before expected date of parturition. Infusion of the udder with antibiotics can help prevent infections that may occur in early involution. After milking is stopped intramammary pressure increases, milk products accumulate in the gland, and further milk secretion is inhibited. Sometimes if the udder becomes extremely congested, it may need to be re-milked. However, this practice stimulates further milk synthesis because intramammary pressure is reduced and pituitary hormones (oxytocin and prolactin) are released. Perhaps more importantly re-milking removes the leukocytes from the udder at a time when many are needed to prevent infection. It usually is unnecessary to re-milk if production is reduced below about 50 lbs per day before milking is stopped.

In studies with identical twins, milked 2X/day the twin with no dry period gave only 62-75% as much milk as the twin with 60 day dry period.

If you milk 1/2 of the udder throughout the dry period while the other 1/2 is dry, then the milked side gives less milk in the subsequent lactation. (DNA concentrations are the same in both halves)

Cows with a dry period of 10-40 days produce 450-680 kg less milk than those with 40 or more days dry.

Conclusions :

There is an optimum length of dry period. A dry period shorter than 40 days will decrease subsequent production (also long dry

periods over 70 or 80 days will result in lowered production in the next lactation). Changes occur in the mammary gland during the dry period which influence mammary cell

proliferation and mammary function in the subsequent lactation.

A dry period may not be required for goats (see Fowler et al. 1991). The requirement for a dry period between lactations may be peculiar to the dairy cow. Consider that most species are not concurrently pregnant and lactating (they exhibit some level of lactational anestrus or other inhibitory effect of lactation on reproductive function). Therefore, they only start reproductive cycling after weaning (the end of lactation), so there indeed will be a nonlactating period prior to the next parturition.

Summary of Changes in Composition of Mammary Secretions During the Dry Period

Milk component Active Involution

Steady State Involution

Redevelopment and Colostrogenesis

Lactose decreasing low increasing (late)

Milk Proteins decreasing low increasing

Milk Fat decreasing low increasing

Udder fluid volume decreasing low increasing

Concentrations of:

Milk components decreasing low increasing

Leukocytes increasing high low

Lactoferrin increasing high low

Immunoglobulins increasing high increasing

Other Factors Affecting Milk Yield and Composition

OtherFactors

This section has information on the role of various factors in milk yield and composition, including Genetics Stage of Lactation and Persistency, Milking practices, Age and Size, Estrous cycles and pregnancy, Environment, and

metabolic diseases.

Genetics

A. Milk composition:

Heritabilities : Correlations between: % Fat = 0.58 % Fat and % Protein r= .45 to .55 % Protein = 0.49 % Fat and % SNF r= .40 % Lactose = 0.55 % SNF and % Protein r= .81 Milk Yield = 0.27 Milk Yield and % Fat r= -.15 to -.30

Milk Yield and % SNF r= -.10 Milk Yield and % Protein r= -.10 to -.30 (SNF is Solids-Not-Fat)

B. Breed differences - Fat is the most variable constituent of milk, minerals (ash) and lactose are the least variable. However, differences among individuals within a breed are often greater than differences among breeds. See Introduction and Milk Composition Lesson for more on differences between breeds in milk composition.

C. Diameter of the milk fat droplet - varies from 1 to 10 microns. Guernseys have the largest and Holsteins and Ayrshires the smallest fat particles. In general, the higher the fat percentage in milk the larger is the diameter of the fat particle. Also, its size usually decreases as the stage of lactation advances.

D. Carotene - is a precursor of vitamin A, and is a yellow pigment. Guernsey and Jersey cows convert much less carotene to vitamin A than other breeds of dairy cattle. Thus, milk from Guernsey and Jersey cows is yellow. Humans can convert the carotene to vitamin A; thus, milk from these cows provides as many vitamin A equivalents am milk from other breeds.

Stage of Lactation and Persistency

Colostrum vs. Milk

See also the Colostrum sections in the Mother & Neonate Lesson.

Colostrum is produced by the udder immediately after parturition. The composition of colostrum is considerably different from the composition of normal milk. Three to 5 days immediately postpartum is needed for the secretions to change to the composition of milk. During this period the total solids, especially the immunoglobulins, are elevated. Newborn calves are practically devoid of immunoglobulins, the antibodies against various disease organisms. Calves must ingest the immunoglobulins from colostrum to acquire a passive immunity against common calfhood diseases. Feeding colostrum after birth is especially critical during the first 24 hours of a calf's life. After this time, enzymes in the digestive tract degrade the antibodies and the permeability of the gut to antibodies decreases.

Lactose content is depressed in colostrum, whereas fat and casein percentage is rather variable. High lactose in the intestine can cause scours in calves, and presumably the reduced lactose content of colostrum helps to prevent this disease.

Calcium, magnesium, phosphorus, and chloride are high in colostrum, potassium is low. Iron is 10 to 17 times greater in colostrum than in normal milk. This high level of iron is needed for the rapid increase in hemoglobin in the red blood cells of the newborn calf.

Colostrum contains 10 times as much vitamin A and 3 times as much vitamin D as that found in normal milk. The newborn calf is practically devoid of vitamin A, and since it provides a degree of protection against various diseases every calf should be fed colostrum.

B. Stage of Lactation and Persistency

At parturition milk production commences at a relatively high rate, and the amount secreted continues to increase for about 3 to 6 weeks. Higher-producing cows usually take longer than low producing cows to achieve peak production. After the peak is attained, milk production gradually declines. The rate of decline is referred to as persistency.

After peak lactation, on average the decline in milk yield will be ~6% per month for first lactation heifers, and ~9% per month for mature cows.

Nonpregnant cows will decrease in production about 94 to 96% of the preceding month's yield after the peak of production is attained. Nonpregnant cows can continue to secrete milk indefinitely, but at a reduced rate.

Milk composition changes during lactation:

Fat percentage in milk decreases slightly during the early lactation and then increases as total production decreases with advancing lactation. Milk protein content gradually increases with advancing lactation. Lactose and mineral concentrations increase slightly during advancing lactation. Most of the increase in SNF components of milk is associated with advancing stages of concurrent pregnancy rather than stage of lactation.

Milking Practices

A. Milking Interval and Duration - for more on this see the sections in the Lactation Lesson on Frequency of Milk removal.

B. During a Milking - see also the Introduction and Milk Composition Lesson.

Milk first removed from the udder contains much less fat (as low as 1 to 2%) than the milk removed at the end of the milking process (as high as 7 to 9%). The reason for this distribution of the fat globules is not known. Fat globules may aggregate in the alveoli which may retard their passage toward the teat, whereas the fluid portion more readily passes around the fat globules out of the alveolus. Immediately preceding milking, the milk in the larger ducts has less fat than milk in the

alveoli.

C. Milking Duration -

Having a set milking time: Comparing 4 min. vs. 8 min. duration with machine on, the cows milked for 4 minutes for an entire lactation produced less milk, especially during early lactation, and were incompletely milked. The 8 minute group was somewhat over-milked.

Optimal milking time for most cows is just over 5 minutes to achieve maximal milk removal.

Leaving 4 lbs of milk in the udder after milking for 10 consecutive days permanently reduces milk yield for the entire lactation.

Age and Size of Cow

Milk yield increases (at a decreasing rate) until about the 8th year of age and then decrease at an increasing rate.

Mature cows produce about 25% more milk than 2-year-old heifers. Increased body weight accounts for about 1/5 of this increase. The remaining 4/5 results from increased udder development during recurring pregnancies.

Heifers should be bred to calve at 24 months of age or earlier if they are of sufficient size to permit delivery of the calf. Although heifers will produce more milk during the first lactation if breeding is delayed to the point where she calves after 30 months of age, total lifetime production will be reduced.

Large cows generally produce more milk than small cows, but milk yield does not vary in direct proportion to body weight. Rather, it varies by the 0.7 power of body weight, which is an approximation of the surface area of the cow (metabolic body size). A cow which is twice as large as another usually produces only about 70% instead of 100% more milk.

Estrous Cycle and Pregnancy

A. Estrus - may temporarily depress milk yield, but this is not occur in all cases. This effect on milk yield is primarily from the increased physical activity of the cow in heat and lowered food intake, rather than an effect of elevated estrogen at estrus.

B. Follicular cysts - Cows with follicular cysts on the ovary produce significantly more milk (adjusted for days not pregnant) than normal herd mates. These same cows produced equivalent amounts of milk before the cystic condition was present. This suggests that circumstances associated with the cystic ovary condition caused the increased milk production and that high milk production does not cause follicular cysts. The longer that cows are cystic, the greater the production in comparison with normal herd mates. Anestrous cystic cows produce more milk than nymphomaniac cystic cows.

[Image kindly provided by D Kelser]

C. Pregnancy - reduces milk production during concurrent lactation. For example, a cow bred at 90 days of lactation will produce 750 - 800 lbs less milk in a 365-day period than if bred at 240 days. By the 8th month of gestation, milk production may be reduced 20% for that month in comparison with nonpregnant cows lactating the same length of time.

D. Twinning - in dairy cattle can result in increased dystocia, and decreased milk and milk fat yields.

Environment

A. Environmental Temperature -

Environmental temperature will increase the respiratory rate which is the primary mechanism whereby European-evolved breeds of dairy cattle dissipate heat.

Respiratory rate increases ~5 fold when temperatures rise from 50 to 105 F. Heat produced by lactating animals is about double that of nonlactating cows. Milk production and feed consumption are reduced automatically in an effort to curtail body

heat production when temperatures become elevated. Depressed appetite is the primary cause of reduced milk yields. Heat stress is especially harmful at the peak of lactation.

Optimal temperature for European breeds of dairy cattle is about 50 F. A rising temperature above 50 F is more detrimental than a similar fall below 50 F.

High humidity adversely affects production only when temperatures exceed 75 F. Provision

of shade, use of fans, showers, or refrigerated air alleviate thermal stress.

B. Season - Milk yields for the entire lactation are usually greater when the cow calves in the fall. Yields decrease progressively in freshening occurs in winter, spring, and summer. The greater production in cows which calve in the fall is probably due to optimal temperatures, absence of flies, and more digestible feeds that are available in the fall, winter, and early spring in comparison with summer conditions. Photoperiod plays a major role in the effects of season. See the section on Photoperiod in the Lactation Lesson for a review.

C. Exercise - Moderate exercise is conducive to high milk production. Too much or too little is detrimental. Cows in stanchion barns should be turned out at least once per day for exercise and heat detection.

Metabolic Diseases Related to Lactation

Milk production starts suddenly and increases daily in early lactation. Each day greater amounts of nutrients are needed for lactation. Large quantities of nutrients are leaving the body, including: amino acids, fat, glucose, calcium, phosphate and water. The body has stores of amino acids and fat to draw upon, but not much glucose is stored in the body, and Ca stores are difficult to mobilize rapidly. Several metabolic diseases can arise under these conditions.

This section contains discussions on Ketosis and Milk Fever (Parturient Paresis)

Ketosis

Results from a rapid drain of blood glucose at exactly the same time that there is an underlying negative energy balance. Occurs usually 10 days to 6 wks postpartum. All cows are at least borderline ketotic, but only 4-12% develop clinical symptoms. Animals rarely die from ketosis, rather the symptoms persist until a new equilibrium is reached, of course this is at a reduced level of production.

Symptoms include: depressed appetite, decreased milk yield, loss of weight, listless behavior, increased milk fat %, acetone breath, and constipation, ketoneurea, and sometimes fatty liver.

How does ketosis arise? For each kg of milk produced, the cow must produce 50 g of glucose to make lactose, and production is increasing daily. There is an underlying negative energy balance during early lactation. The cow may not obtain sufficient feed to yield enough propionate to maintain blood glucose levels. This is sensed as an energy deficit and -

fat metabolism is increased (catabolism) (they can't make glucose from fat) fatty acids are transported to the liver in greater quantities than can be metabolized protein breakdown can yield glucogenic amino acids, but also yields more ketogenic aa's.

This results in over production of ketone bodies (and development of fatty liver). Other tissues can metabolize ketone bodies, but this is too much for them. Excessive prepartum conditioning can also accentuate the problem because the excess fat will tend to depress appetite, and will also lead to greater fat mobilization in early lactation.

Cause: unknown. A number of factors have been implicated, including: Insulin/glucagon imbalance,

abnormal liver function, adrenal corticoids, thyroxine, and mineral and/or vitamin deficiencies. None of these have been proven to be the cause.

Prevention: Overfeeding during the dry period leads to excessive weight gain, and reduced ability to mobilize nutrients in early lactation. Therefore, moderate feeding during the dry period until 2-3 wk prepartum is recommended. Increase feeding rapidly during the postpartum period, but the cow has depressed intake and appetite.

Treatments: All are aimed at increasing blood glucose

IV glucose - 500 ml of 40% dextrose. This is not particularly effective, relapses are common. Only amounts to enough glucose for about 8 liters of milk (at 5% lactose). Some glucose is lost via the kidney.

Hormone treatments - There may be insufficient corticoid release. Cortisone or ACTH is administered. Not good for the cow for the long term.

Feed sugar or molasses - Not particularly useful. Results in production of VFAs in the rumen.

Polypropylene glycol - Most effective. It is converted to propionate. This treatment is often used in combination with IV glucose.

Nicotinic acid - Nicotinic acid is a precursor of the vitamin niacin. In pharmacological doses it may have benefit as a treatment.

Milk Fever (Parturient Paresis)

Concentrations of blood calcium are very tightly regulated, therefore difficult to change rapidly.

General control of calcium metabolism -

parathyroid hormone (PTH) is secreted in response to lowered blood Ca, causes Ca resorption from the bone,

also causes conversion of vitamin D to its active form - in the kidney (1,25 dihydroxy vitamin D3)

1,25-D3 causes increased release of bone Ca and increased intestinal absorption of Ca elevated blood Ca releases calcitonin from the thyroid, resulting in increased Ca deposition

in bone

Milk has 120 mg Ca / 100 ml vs. blood ~10 mg/ 100 ml. The avg. Holstein cow secretes 50 g Ca / day There is a rapid removal of Ca from the animal after parturition. In general, immediately postpartum the cow is not sufficiently capable of mobilizing bone Ca, therefore, she is dependent on intestinal absorption. The problem of obtaining sufficient Ca is accentuated by the postpartum depressed appetite.