Nutrient Uptake and Endocrine Regulation of Milk Synthesis by Mammary Tissue of Lactating Sows'

Nutrient Uptake and Endocrine Regulation of Milk Synthesis by Mammary Tissue of Lactating Sows'

R. Dean Boyd*$, Ronald S. Kensingert, Robert J. HarrelF, and Dale E. Bauman*

*Pig Improvement Company, Franklin, KY 42135; +Department of Dairy and Animal Science, Pennsylvania State University, University Park 16802; and

$Department of Animal Science, Cornel1 University, Ithaca, NY 14853

ABSTRACT The neonatal pig has greater capacity for growth than is currently realized. Artificial rearing data demonstrate that the biological potential for growth is a t least 400 g/d (average to 21 d ) vs approximately 230 g/d for sow-reared pigs (+70%). Milk secretion becomes inadequate for maximum neonatal growth after 8 t o 10 d of lactation, yet preliminary information suggests that the sow has greater capacity for synthesis than is being expressed under normal conditions. Nutrient output is a function of nutrient uptake by the mammary gland and biosynthetic capacity of mammary epithelial cells. Some quantitative estimates of nutrient uptake have been derived using the [arterial] - [venous] technique. This and radiotracer technology confirms that glucose is the major precursor for milk synthesis. Dietary amino acid input is integral but secondary. The ideal amino acid profile for lactating sows is different when

assessed by anteriovenous difference vs milk profile because the former accounts for potentially important roles in mammary metabolism. We propose that the capacity for milk synthesis may be partially limited by our understanding of nutrient needs, but we antici- pate that an endocrine constraint largely accounts for differences between maternal supply and that required for maximum neonatal growth. Somatotropin and prolactin are involved in coordinating the partition of nutrients and stimulation of milk synthesis, but their exact roles remain unclear. Critical studies on mammary gland metabolism, regulation of its synthetic capacity, and tissue adaptations will be required to enhance milk nutrient secretion commensurate with the growth potential of neonatal pigs. Quantitative information on milk precursor-product relationships is important for predicting changes in nutrient needs with changes in milk production.

Key Words: Swine, Lactation, Neonatal Growth, Mammary Gland, Milk Synthesis, Endocrine Regulation

Introduction

The growth rate of neonatal pigs is determined largely by milk nutrient output (Noblet and Etienne, 1989). In turn, the weight achieved by the time of weaning determines adaptability to the nursery and ultimately how rapidly market weight is achieved (Harrell et al., 1993). However, the nursing pig has much greater growth potential than is being realized because milk secretion is inadequate. We estimate that nutrient output limits preweaning progeny

'The authors express their appreciation to P. Bandy and M. Boyd for typing the manuscript and to S. Shoulders for preparing figures. The critical comments of N. Trottier on anatomy of the sow mammary gland and on the arteriovenous metabolite difference section are recognized and appreciated.

2Formerly at Cornell Univ. Present address and reprint re- quests: Pig Improvement Co., Box 348, Franklin, KY 42135.

J. h i m . Sci. 1995. 73(Suppl. 2):36-56

growth by at least 40%. In pigs, average growth rates of approximately 400 g/d to 21 d of age (vs 230 gld) are achievable with unlimited nutrient supply (Har- re11 et al., 1993).

The biological basis for this apparent limitation in milk secretion is unclear. It could be the result of inadequate precursor supply to mammary tissue, a deficit in endocrine stimulation of milk synthesis, and(or) coordination of metabolism in extra-mammary tissues (e.g., adipose, muscle) to provide nutrients for milk synthesis. The relationship between maternal dietary intake and milk secretion has been studied, but surprisingly little information is available on specific nutrient needs and endocrine regulation of porcine mammary tissue. We believe that this is due to a failure to appreciate the benefits of optimized milk production combined with the more obvious benefits of increased lean deposition and prolificacy. More detailed studies on nutrient and endocrine needs

36

PIG GROWTH POTENTIAL AND LIMITS TO MILK SYNTHESIS 37

of mammary tissue are necessary if we are to realize the biological potential for neonatal pig growth and overall growth potential.

The objectives of this paper are 1) to illustrate that milk secretion is a major constraint to preweaning pig growth, 2) t o review nutrient uptake and metabolism by porcine mammary tissue during lactation, and 3) to summarize efforts to enhance milk output through exogenous homeorhetic hormones. A metabolic approach to defining nutrient needs of mammary glands is warranted because they dominate the total nutrient needs of the sow.

Milk Nutrient Secretion and Neonate Growth

Milk Nutrient Output and Neonate Growth. The relationship between preweaning pig gain and milk energy and nitrogen output by the sow is high ( r2 = -87 to .90; Noblet and Etienne, 1989). This estimate is substantially higher than previous estimates, but methodological weaknesses of past studies were ad- dressed (see discussion by Noblet and Etienne, 1989). Thus, nutrient secretion by the mammary gland is the main determinant of progeny growth rate. The next logical question is whether milk nutrient output by high-producing sows is adequate to allow for maximum neonatal growth.

Artificial rearing studies suggest that the nutrient requirements for maximum growth of nursing pigs exceed the milk nutrient supply of modern sows. For example, 21-d weights of artificially reared pigs fed less than ad libitum amounts of a milk replacer were at least 7.5 to 8.6 kg/pig (Jeppeson, 1981; J. Corley, personal communication, 1985) compared with that normally observed in sow-reared pigs (5 .5 to 6.5 kg/ pig). Thus, the lactating sow seems to be a limit- feeding system, but we are not aware of artificial rearing studies in which sufficient energy and nutrient intake was ensured to probe the biological potential for preweaning growth.

Biological Potential for Growth. A series of studies are being conducted by our group a t Cornel1 to study the role of somatotropin and insulin-like growth factors in the control of early postnatal growth. Artificial rearing was used as a technique to ensure ad libitum energy and nutrient intake during the nursing phase so endocrine regulation could be studied under conditions in which nutrient supply was not limited. Data from these studies allowed us to explore the biological potential for preweaning growth and the associated milk energy requirement during a conventional preweaning period. Two groups of eight male pigs each were removed from sows at 2 d of age and acclimated to a semi-automated liquid milk replacement feeding system. The milk replacement mixture approximated sow’s milk with respect to amino acid levels but was lower in fat content. It was calculated to contain 4,015 kcal of ME, 293 g of protein, and 225

g of fat per kilogram of DM and was mixed to provide 15% solids. Daily intake of milk replacer was determined through 21 d of age, but growth rates were monitored through 23 d. The potential for shifting the postweaning growth curve by allowing for more rapid preweaning growth was studied with the second group of eight pigs. They were compared to eight littermate male pigs that were reared by four sows in litters of 8 to 10 pigs each through 21 d of age. Litters that farrowed within 24 h were selected as a source of pigs to compare the impact of maximized preweaning growth on growth rate in the nursery and to 110 kg BW (see Harrell et al., 1993).

The artificially reared pigs grew an average of 70% faster (395 vs 232 f 24 g/d; mean k SE) and were 53% heavier at 21 d of age than sow-reared pigs (9.8 vs 6.4 f .5 kg/pig). The former maintained a faster growth rate from weaning to 47 d of age (533 vs 317 f 37 g/d) but exhibited no incremental advantage thereafter. Thus, a 3.4-kg difference at weaning translated into a 9.0-kg difference by 4 wk after weaning, and this difference was maintained to the 110-kg end point. The net effect was to shiR the growth curve and reduce the time required to attain market weight by a t least 10 d. No difference in body composition was evident from measures of backfat thickness, loin eye area, or length at slaughter (Harrell et al., 1993). The advantage of improved early nursery growth ( 2 3 to 47 d ) is consistent with results of a previous study in which growth rates of sow-reared pigs were enhanced but not optimized by a liquid milk supplement (Boyd et al., 1985). In this experiment, a 1.4-kg weight advantage at 22 d of age resulted in a 2.8-kg difference 21 d after weaning.

The artificial rearing studies by Harrell and co- workers (1993) suggest that the biological potential for neonatal pig growth is a t least 450 g/d (average from birth to 21 d ) based on performance of the fastest-growing pigs. This resulted in BW of 10.8 to 11.3 kg at 21 d of age. However, average growth rate to 21 d fails to illustrate both the potential and the progressive nature of growth during the neonatal period. Consequently, the average rate of growth achieved over 4-d intervals is shown in Figure la. Artificially reared pigs were already gaining 432 g/d between 11 to 14 d of age, but averaged 610 g/d from 21 to 23 d of age. The latter represents a daily increase of approximately 6.1% of BW. It is noteworthy that the two fastest-growing pigs achieved growth rates of 810 and 820 g/d between 21 and 23 d of age (data not shown). Furthermore, growth rate increased in parallel with a progressive increase in milk energy intake (Figure lb) . These data illustrate the minimum quantitative need for milk energy in order to achieve the growth potential.

The point at which milk production of the sow begins to limit progeny growth was estimated by comparing energy intake curves for our artificially- reared pigs and sow-reared pigs from the literature

38 BOYD ET AL.

2500

600 7 0 0 1 A u l

358

432 455

61 0 m

5-8 8-11 11-14 14-17 17-21 21-23

Interval of Growth. d

Artificial w

- m 2 1000

500

0 -- 0 3 6 9 12 15 18 21 24

Day of Lactation

Figure 1. Potential for growth and energy requirement of neonatal pigs using artificial rearing (unpublished data by Harrell, Thomas, and Boyd). Growth rates were determined in 16 pigs over 4-d intervals (Panel A; CV = 14%). Baby pigs were removed from the sow at 2 d of age and fed a milk replacement mixture for ad libitum intake through 23 d of age. Daily intake of ME was measured through 21 d of age assuming a computed value of 4,015 kcal ME/kg DM (Panel B: Harrell et al., 1993). This was compared to published data for pigs reared by eight primiparous females with ME estimated (95% x gross energy) from gross energy output (6,335 kcal of ME/kg of DM; Noblet and Etienne, 1989).

(Figure lb). These data suggest that milk energy output by high-producing, primiparous females failed to satisfy the energy needs of offspring (9 to 10 pigs/ litter) after d 8 of lactation. The disparity progressively increases so that maternal milk output is

approximately 50% of neonatal need at 21 d of age. We estimate that 18 to 20 kg of milWd would be required to meet the energy needs of a 10-pig litter at 21 d (assuming 1.14 Mcal of MEkg of milk; Noblet and Etienne, 1986). This research clearly shows the relevance of lactation research in the pig that is directed toward improving maternal nutrient secretion.

Maternal Biosyn thetzc Capacity. The next question is whether the capacity for milk production is greater than that expressed in lactating pigs. Sauber and co- workers ( 1994a) recently investigated the lactation capacity of primiparous females ( n = 8 ) . Each day, the females alternatively nursed two groups of pigs at 40-min intervals for 18 h/d and then one group continuously for the remaining 6 h over a 22-d lactation period. This nursing intensity was applied from 12 h after birth, and the number of pigs in each group was equivalent to the number of functional glands (14 minimum). Recognizing that milk production could be constrained by energy and amino acid intake, nutrient density of the lactation diet (5.5 kg/d) was significantly greater than the NRC (1988). The energy level (Mcal/d) offered exceeded their expectations for ad libitum consumption. Milk energy secretion was predicted indirectly in 4-d periods from 2 t o 22 d of lactation by calculating the ME needed for maintenance and weight gain of the litter. An estimated 14.1 kg/d of milk was secreted, assuming 1.14 Mcal of MEkg of milk. The data suggest that the extraordinary nursing stimulus increased average milk output by approximately 1.5 times that reported for high-producing females (van Kempen et al., 1985; Noblet and Etienne, 1989). This response is consistent with that of Harkins et al. (1989), who stimulated milk secretion of sows by elevating plasma somatotropin ( ST) levels in lactating sows with daily injections of highly purified porcine ST and thus increased progeny weight gain. Thus, lactational capacity seems to be greater than exhibited under normal conditions. These two different approaches provide evidence that a significant constraint to milk output exists.

Determinants of Milk Nutrient Secretion. Milk production is a function of nutrient uptake by mammary tissue and biosynthetic capacity. There are at least four determinants of milk nutrient secretion: 1) number and activity of functional mammary epithelial cells (synthetic machinery); 2 ) endocrine stimulation of cell differentiation, mammary metabolism, and coordinated partitioning of nutrients to the mammae by extra-mammary tissues (adipose, muscle); 3 ) precursor availability to and uptake by the mammary gland (availability = [blood concentration] [flow ratel 1; and 4) degree of glandular stimulation and milk removal by progeny (prevents premature involution). Milk output is a function of the most limiting factor. The endocrine system con- tributes directly or indirectly to each of these deter-


minants, but milk yield is ultimately constrained by nutrient availability.

A comprehensive review of each component is beyond the scope of this paper. We will focus on our present knowledge of 1 ) precursor uptake and metabolism by porcine mammary tissue and 2 ) possible endocrine constraints and efforts to enhance milk synthesis through use of exogenous hormones during lactation. Our ability to enhance milk output will depend on our understanding of both nutritional needs and endocrine coordination of the sow’s mammary glands and supportive tissues.

Nutrient Uptake and Metabolism by Mammary Tissue of Lactating Sows

Substantial quantities of nutrients are required by mammary tissue for milk synthesis. They are made available to mammary epithelial cells through diet and body reserves (adipose, protein, bone; Bauman and Currie, 1980; Bauman and Elliot, 1983). Surpris- ingly little is known about mammary uptake and metabolism of specific nutrients in lactating sows. This is remarkable because mammary glands are the primary users of absorbed nutrients in lactating sows and virtually dictate the dietary nutritional needs. This is illustrated by the fact that 65 to 70% of the total energy requirement of a lactating sow is needed to support milk production (Aherne and Williams, 1992). This is largely a reflection of the glucose requirement. However, this is also true for other nutrients (e.g., amino acids, calcium, phosphorus etc.), and we estimate that even a higher proportion of amino acids is needed for milk synthesis (shown later). Thus, the sow is similar to the lactating dairy cow, in which the needs of mammary tissue for milk precursors is so dominant that the cow is seemingly an appendage to the udder rather than vice versa (Bauman and Elliot, 1983).

Model of Precursors and Products in Sow Mammary Gland Metabolism. A schematic of circulating nutrients used in the synthesis of milk components in sows appears in Figure 2 (Pettigrew et al., 1993). Glucose, triglyceride fatty acids, and amino acids represent approximately 95% of the total mass (or carbon) utilized by mammary tissue (Spincer et al., 1969). The availability of glucose, however, is widely regarded as a primary limit to milk synthesis. An understanding of precursor-product relationships and precursor interrelationships will allow us to predict how the amount and pattern of nutrient uptake must vary with level of milk output.

Studies on mammary uptake of plasma nutrients and their quantitative contribution to the synthesis of milk nutrients have been undertaken almost exclusively in lactating ruminants. There are similarities but also some important differences that limit the application of ruminant data to the pig (Kensinger,

1993). For example, pregastric fermentation in ruminants results in differences in the products of digestion. Glucose is absorbed to a lesser extent and is more highly conserved for lactose synthesis in ruminants by differences in whole-body and mammary gland metabolism (Baldwin and Smith, 1983). The pattern of milk constituents also differs. Although the sow and ruminant species tend to have similar concentrations of the osmoregulator lactose (approximately 4.8%; Baldwin and Smith, 1983), the sow produces milk that is approximately 70% higher in protein (5 .5 vs 3.2%) and 60 to 70% higher in gross energy than milk from dairy cows. The latter is primarily due to differences in fat content (6.8 vs 3.7% respectively), which is the most variable component in milk.

Techniques for Quantitative Estimates of Mammary Nutrient Uptake. The methodology required to quantitatively estimate nutrient uptake and to establish precursor-product relationships in lactating mammary glands has been reviewed by pioneers of the techniques (Linzell, 1974; Linzell and Annison, 1975; Annison, 1983). A minimal statement is warranted to appreciate contributions and weaknesses of available data for lactating sows. In studies with ruminants, quantitative estimates of nutrient uptake have been made by 1 ) simultaneously measuring the difference in metabolite concentration between arterial blood entering and venous blood leaving the mammary glands ( A - V) and 2) measurement of blood flow (BF) rate to the glands. Uptake (U) by mammary tissue is then computed by the equation U = (A - V ) BF. If coupled with isotope techniques, the amount of specific milk nutrients formed from each blood metabolite can be estimated. Extraction ( E) by mammary glands (E = (A - VVA) is useful as a qualitative measure of transfer, but it is not a quantitative measure of metabolite uptake.

Obviously, this approach requires an accurate measure of mammary BF rate and sampling of blood that is representative of that entering and leaving the mammary glands. A valid arterial sample can be acquired from any artery because systemic blood is well mixed and constant with respect to metabolite concentration. However, great care must be taken to ensure that placement of the venous cannula allows for sampling of blood that is exclusively from the mammary gland, because the composition of venous blood varies with the tissue being drained. Blood flow must be assessed in conscious animals and under steady-state conditions because position and move- ment can introduce considerable variation in BF rate.

Blood flow rate can be estimated by dilution of a nonmetabolizable indicator (e.g., para-amino hippuric acid), thermal dilution, ultrasonics, electromagnetic probes, or by measuring mammary uptake (A - VI and total milk output of a substance for which uptake equals output. Examples of nutrients for which uptake seems quantitatively to equal output include calcium

40 BOYD ET AL.

Absorbed Nutrients Triacylglycerol _ _ _ _ _ _ _ _ _ _ _ - - _ - - - - - - - +

Amino Acids Lysine Glucose Acetate Fatty Acids

1 + v I I

I in Milk in Milk in Milk 1

I I I I CO,, NADPH, production I

Figure 2. Schematic of absorbed energy components and amino acids and their metabolic fate in lactating sows (adapted from Pettigrew et al., 1993).

and certain amino acids (tyrosine, phenylalanine, isoleucine) (Clark et al., 1978; Annison, 1983). The virtually complete transfer from blood into milk without metabolism to other products allows one to estimate mammary gland BF by knowing the mean A - V difference of the precursor and output in milk (by using the Fick principle): BF (mL/h) = output (mg/h) / A - V (mg/mL). This method has been successfully applied in cattle (Clark et al., 1978) and ewes (Davis and Bickerstaffe, 1978) and might be beneficial in studies with lactating pigs because accurate BF estimation seems to be a greater challenge.

A timely and thorough anatomical description of the mammary vascular system of the lactating sow was made recently by Trottier et al. (1995; see also Trottier, 1995). The vessels supplying the mammary gland of the sow, however, are anatomically more diffuse (and compartmentalized) than those in ruminants, so conventional techniques for measuring BF may not be completely applicable. At the present time, it is not clear how to measure venous BF in the lactating sow accurately. Validation is needed to establish that venous blood flow is truly confined to the compartment being studied.

Precursors and their Role in Milk Synthesis. Pub- lished studies on porcine mammary gland uptake and metabolism of nutrients are confined to three classic papers (Linzell et al., 1969; Spincer et al., 1969; Spincer and Rook, 1971). Their pioneering work led to identification of the major blood precursors of milk nutrients and to semiquantitative estimates of precursor-product relationships. Their studies are limited by the number of animals used and inability to fully apply techniques used with ruminants. With those

limitations in mind, we have taken the liberty to propose potential relationships as a basis for scientific challenge by future research.

Spincer and co-workers (1969) used the A - V difference technique to identify milk precursors in five lactating sows (two anesthetized, three conscious) between their 5th and 6th wk of lactation. Nutrients that had a high percentage of extraction by mammary glands included glucose (3 1%) , indispensable amino acids (22 to 38%) and the triglyceride fatty acids (TGFA) oleate (23%) and palmitate (19%) (conscious sows). Percentage of acetate extraction was high (46%), but its net contribution is small because of a typically low plasma concentration. Percentage extraction of NEFA, ( -6%), P-hydroxybutyrate (BHBA, 11%) and citrate (-1%) also suggest a minor role for these metabolites in milk synthesis. The contribution of NEFA and PHBA is conceivably more important in early lactation when negative energy balance and body fat mobilization may result. Fatty acid uptake occurs largely from the TGFA fraction, and fatty acids are liberated from circulating triglyceride by lipoprotein lipase action in mammary capillaries (Barry et al., 1963). They mix with the capillary NEFA pool, which complicates estimation of A - V differences of NEFA. Nevertheless, we expect a vigorous transfer of milk fatty acids, as is observed in goats (Annison et al., 1967). Of the total uptake of plasma metabolites by mammary tissue (sum of the A - V mass difference), glucose accounted for approximately 61%, amino acids 24%, triglyceride fatty acids 12%, and acetate 1% (Table 1). These are in good agreement with data computed from an abstract by Linzell et al. (1967; see Spincer et al., 1969 for

PIG GROWTH POTENTIAL AND LIMITS TO MILK SYNTHESIS

Table 1. Relative uptake of plasma constituents by the mammary gland of lactating sows”

41

Constituent

A - V Relative

mg/dLb extraction % totalC synthesis, g/dL milkd difference, Percentage uptake, Uptake for milk

~ ~ ~~ ~

Glucose 37.5 31 61 14.1 Acetate .6 46 1 0.2 Wacylfatty acids 7.2 16-23 12 2.7 Amino acids 15.1 23-4 1 24 6.2 &Hydroxybutyrate .2 11 Lactate 1.4 12 2 0.5 Citrate 0 0

- -

- - *Summarized from Spincer et al. (1969). bArterial minus venous difference. ‘Percentage of total mass of constituents extracted. dAssumes 375 mL of plasma flow through the udder per 1 mL of milk secreted.

assumptions). Incorporation of plasma-derived TGFA into milk fat seems to be quantitatively less important for the sow than for the goat (uptake vs content). This suggests that de novo synthesis of milk fat is greater in pigs, and from glucose as opposed to acetate and PHBA for ruminants (Spincer et al., 1969). If the positive relationship between arterial TGFA concentration and A - V difference exists for pigs as observed for lactating dairy cows, raising TGFA levels by dietary means would result in increased TGFA uptake by the mammary gland (Baldwin and Smith, 1983). Consequently, more glucose could be available for use elsewhere in mammary metabolism. This may explain why Boyd and co-workers (198 1) observed an increase in milk production (and milk fat) with dietary fat addition (+8%) t o lactating sow diets. Therefore, it is important to understand the relationship between plasma metabolite concentration and uptake (A - V) and the resultant metabolic effect on other precursors. We cannot accurately predict how dietary changes would affect milk production and composition without this information.

Although Spincer and co-workers (1969) did not measure BF rate, they derived estimates of mammary uptake of major plasma precursors by assuming 375 mL of plasma flow through the mammary gland per

milliliter of milk secreted (Table 1). This was based on the relationship established with goats at peak milk production and is used because of observed similarities between the sow and goat in milk output, oxygen uptake, and C02 output per 100 g of mammary tissue (Linzell et al., 1969). The estimate of plasma flow allowed estimation of the grams of metabolite uptake per 100 mL of milk secreted. Albeit tentative, this information represents the first approximation of the precursor requirement for milk synthesis when computing precursor needs for different milk production levels (Table 1).

Estimates of the energetic efficiency of milk synthesis for lactating rats (87%) and cows (86%) suggest that it is a very efficient process (Baldwin and Yang, 1974; Plucinski, 1976; computed by Baldwin and Miller, 1991). Comparable estimates have not been made for the lactating sow. Therefore, we calculated the theoretical efficiency of milk nutrient synthesis to be 89% using information from Table 1 and the average composition of sow milk described previously in this paper (Table 2). Deviation of the theoretical estimate from actual is expected to be due largely to variation in fat output, the most variable component of milk nutrients.

Linzell and co-workers (1969) observed that arterial extraction of plasma glucose was 26% for the

Table 2. Estimation of the energetic efficiency of milk synthesis in the sow mammary glandajb

UptakddL milk OutpuVdL milk

Precursor mmol kcal Product mmol kcal

Glucose 78.2 52.5 Lactose 13.3 18.0 Triacylglycerol 2.9 20.9 Fat 7.25 47.4 Amino acids 42.8 24.2 Protein 37.9 21.7 Total - 97.6 - _. 87.1

milk assumed to be: lactose, 4.8%; fat, 6.8%; proteins, 5.5%. ‘Precursors account for 97% of total uptake as computed fmm Spincer et al. (1969). Composition of

bEffciency = (output + uptake) x 100.

42 BOYD ET AL.

Table 3. Arterio-venous (A - V) difference of amino acids across the mammary gland and comparison of amino acid pattern in

sow milk to the pattern of uptake (A - V)

A - V difference, pmol/L Proportion relative to lysine

Amino acida Linzellb Spincerb Trottierb Linzell A - VC Sow milk‘

Leucine Valine Lysine Arginine Isoleucine Threonine Phenylalanine Histidine Methionine Tryptophan Glutamate

69.4 66.6 54.0 47.1 43.5 31.9 26.6 26.4 12.7

126.4 -

145.6 133.2 73.9 66.6 90.0 61.3 46.0 39.3 23.5

194.4 -

53.8 42.6 31.0 26.8 28.0 24.6 18.2 12.8 9.2 7.4

82.6

115 99

100 104 72 48 56 52 23

( 2 3 ) d 236

114

100 66 59 59 56 40 28 19

262

78

~ ___ ~ ~

aNutritionally indispensable amino acids except for glutamate. bA - V differences obtained from Linzell et al. (19691, Spincer et al. (1969), and Trottier et al. (1994). CExpressed as a mass ratio (not molar). The A - V difference for each amino acid was converted to

milligramsfliter of uptake and expressed as a percent of lysine. The amino acid to lysine ratio was also computed for milk composition data (d 28 of lactation) from King et al. (1993b). Lysine was set equal to 100.

dEstimated from Trottier et al. (1994): 7.4 kmol tryptophadL + 31.0 pmol lys inf i .

sow’s mammary glands, which compares well with estimates by Spincer et al. (1969; 31%) and recent work by Trottier et al. (21%; 1994). Linzell estimated glucose kinetics by continuous infusion of [U- 14C]-glucose into a single lactating sow (d 43). Glucose uptake by the mammary glands accounted for 54% of the glucose turnover for the whole body (4.8 mg.min-l.kg B W - l ) . This estimate compares with 50 to 80% for the lactating cow (Annison, 1983) but is less than would be expected because measurement occurred late in lactation with only six functional mammary glands (vs 9 to 11 glands presently). Approximately 53% of the glucose taken up by mammary tissue was partitioned to lactose and 34% was oxidized. The remaining 13% was used for triglyceride-glycerol synthesis (glucose supplied 40% of total glycerol) and synthesis of milk fatty acids and amino acids. Linzell et al. (1969) estimated that 54% of the mammary C02 was accounted for by glucose oxidation so that considerable C02 may be derived from amino acids. These findings illustrate the pre- eminence of glucose as a precursor and the need to establish a glucose requirement in relation to potential milk secretion.

Linzell et al. ( 1969) noted that the net contribution of glucose to amino acid carbon was minor. However, perfusion of two isolated mammary glands with labeled glucose showed that de novo synthesis of milk fatty acids was extensive. It was not possible, however, to derive quantitative estimates of the contribution of glucose to fatty acids as compared to blood-derived fatty acids in milk triglycerides. Exten- sive use of glucose for generation of NADPH (via pentose monophosphate pathway to support fatty acid synthesis) is anticipated, but not quantitatively esti-

mated to our knowledge. The pathway by which the reducing equivalents are produced in sows was shown to be the same as that identified for rat mammary tissue but different from that in the dairy cow (Bauman et al., 1970). The extent to which hepatic gluconeogenesis operates to support the glucose requirement for metabolism is unknown.

Mammary tissue of the high-producing sow must be one of the most active tissues with respect to transport and metabolism of amino acids. For example, mammary glands of the sow yielding 10 kg of milk per day will secrete approximately 560 g/d of amino acids as milk protein. Substantial A - V differences across the mammary gland were observed for all indispensable amino acids (IDAA) in studies by Linzell et al. (1969) and Spincer et al. (1969; Table 3). Arterial extraction was calculated to be in the order of 18 to 36% and 23 to 39%, respectively (data not shown). The highest extraction percentage was observed for the nutritionally dispensable amino acid ( DAA) glutamate (37 and 41% for Linzell et al., 1969 and Spincer et al., 1969, respectively). This corresponds with it being the amino acid of highest concentration in sow milk (262% relative to lysine, King et al., 199313). Little or no net absorption was evident for other DAA (e.g., alanine, glycine, asparagine). The low A - V difference observed for most DAA suggests that they are synthesized within the gland.

We used the data by Linzell et al. (1969) to calculate A - V differences for each amino acid as a ratio to lysine because the latter is considered to be the first-limiting amino acid in diets of both lactating and growing pigs (Table 3). This allowed ranking of each amino acid on the basis of relative uptake by mammary tissue. The amino acid:lysine ratio was also


Dispensable

$ 1 0 0 Q, x (II CI

50

n

Amino Acid

uptake mg/h 145 79 105 55 65 32 48 44 40 84 20217 58 11 8 output mg/h 61 59 105 62 89 32 49 46 53 37 233 44 69 96 154

Figure 3. An estimate of the uptake of plasma amino acids by the mammary gland and output as milk protein in a single lactating sow. Estimates for nutritionally indispensable amino acids were derived from Linzell et al. (1969), but data for dispensable amino acids were computed by the authors using arterial minus venous data of Linzell et al. (1969) and the milk pattern of dispensable amino acids relative to lysine (King et al., 1993b; see text for details). Uptake by the mammary gland (mg/h) was expressed as the percentage of output as milk protein (mg/h). Actual milligram/hour values are provided on the x-axis for amino acid uptake and output, respectively.

computed for amino acids in milk protein so that the relative abundance of each in milk could be compared to the A - V difference ranking. The order of amino acids seems to be almost identical when comparing mammary uptakes and milk composition (true also for A - V difference data). However, the relative uptakes of valine, isoleucine, and arginine were higher than expected based on the milk amino acid composition. The ratios of arginine and valine to lysine in milk protein are 66 and 78%, but significantly more of each is apparently taken up by the mammary in comparison to lysine (104 and 99%, respectively). If genuine, this suggests that the involvement of branched-chain amino acids and arginine in mammary metabolism may be greater than suggested by the milk amino acid pattern.

A quantitative estimate of plasma IDAA uptake vs output was made for a single gland of a single sow (d 43 of lactation) by measuring BF rate, A - V difference, and composition of the milk protein secreted (Linzell et al., 1969; Figure 3). Although

data are limited, their results and our estimates for DAA balance are consistent with ruminant data (Clark et al., 1978). More trustworthy prototypes of precursor-product balance sheets have been published for ruminants. They illustrate the power of this tool for amino acids and other precursors (Clark et al., 1978; Annison, 1983; Baldwin and Kim, 1993). The relatively close balance between uptake and output for most IDAA suggest that their use is almost exclusively for milk protein synthesis. Valine, and possibly arginine, is in excess of milk output; almost twice as much valine is absorbed as is secreted. Branched- chain amino acids (valine, leucine, isoleucine) are consistently taken up by the ruminant mammary gland in excess of their output in milk protein (Annison, 1983). They represent potential sources of carbon and cr-amino nitrogen for synthesis of DAA (e.g., glutamate and aspartate; Wohlt et al., 1977; Mepham, 1982). The limited sow data suggest that valine is the amino acid that is used most extensively in alternative uses for milk protein and mammary

44 BOYD ET AL.

metabolism. It remains to be determined whether the metabolic role is entirely obligatory and whether its potential contribution to DAA needs are modifiable. Recent work involving more extensive sampling, plasma flow estimates, and input/output balance demonstrated significant net uptake (vs output) of branched-chain amino acids, especially leucine, by mammary glands of lactating sows (Trottier, 1995).

Linzell and co-workers (1969) also determined A - V differences for some DAA but did not include them in their calculations of the balance between mammary uptake and milk output. We computed the balance assuming a plasma flow of 127 d L h (calculated from IDAA information and Fick equation) to estimate whether extensive DAA synthesis may be occurring in the gland. Milk DAA output was estimated by multiplying the ratio of each to lysine in milk (King et al., 199313) by 89 mg of outputh for lysine (Linzell et al., 1969). In general, most DAA seem to be taken up in insufficient quantities compared with milk output (Figure 3) . This suggests that a significant proportion of DAA are synthesized in the gland from IDAA. The source of carbon and nitrogen may at least involve valine and arginine (no evidence of ornithine uptake). This is consistent with data from lactating cows: leucine and isoleucine, and possibly ornithine, also serve as precursors (Clark et al., 1978). The apparent glycine imbalance in lactating pigs does not have a ruminant equivalent.

Limitations of this Pioneering Work and Recent Contributions. Notwithstanding the excellent contributions made by this pioneering work, there are significant limitations that confront researchers with both challenge and opportunity. Foremost is the fact that vascular architecture of the porcine mammary system is not adequately documented (Turner, 1952) and is more diffuse than the ruminant system. The porcine mammary system extends over the entire abdominal surface and can be divided into three regions (thoracic, abdominal, inguinal) based on the vascular arrangement for arterial supply and venous drainage of glands (Trottier, 1995). In contrast, the circulatory system of ruminant mammae is localized in the inguinal region. Second, data for pigs were limited almost exclusively to A - V differences, with the exception of limited use of transfer quotients. Blood flow was either estimated or determined in only one sow because conventional dilution techniques may result in undue error (Linzell et al., 1969). Third, authors had difficulty in maintaining catheters, so relatively few samples were acquired and were confined to a short time frame. Fourth, only one to three sows were sampled at one point, late (by present standards) in lactation. Information on dietary and feeding protocols were virtually absent. Evidence that the surgical procedure did not alter milk production (reflected by litter weight gain) is important but was not provided. Finally, there are pools in the blood that

may serve as a source of amino acids besides free amino acids of blood plasma. These include blood cells (may serve to transport amino acids) and di- and tripeptides.

A technique for cannulation of the main anterior vein of pigs has also been proposed that could be an important innovation for porcine mammary physiology studies (Trottier, 1995; Trottier et al., 1995). The procedure was less invasive with less risk of reducing BF than earlier efforts. Venous drainage was confined to mammary tissue. Cannulas remained patent for a minimum of 15 d with validation that litter performance was not adversely affected.

Is Biosynthetic Capacity Limited by Nutrient Uptake? We propose that estimates of biosynthetic capacity for milk nutrients is at least partially constrained by our present understanding of nutritional needs. Milk production is both energy- and amino-acid dependent. However, recent work by Tokach et al. (1992) has shown that milk production is ultimately energy- dependent, so amino acid needs for milk synthesis depend on the amount of available energy consumed. Glucose is the major precursor given its extensive involvement in lactose formation and as an energy source (Table 1). Glucose utilization rate in ruminants is positively correlated with lactose output and milk secretion (Annison, 1983). However, glucose input is a linear function of digestible energy intake and therefore potentially limited by feeding management. Conservation of glucose by TGFA may also be important. We expect the latter to become more important with increased capacity for milk synthesis.

Dietary amino acid level is secondary but integral to lactation because both milk yield and muscle loss are affected (Sauber et al., 1994a). Recent empirical studies confirm factorial calculations that daily lysine intake in excess of 50 g (vs 32 g/d, NRC 1988) is required for the higher milk yield of modern sows (Johnston et al., 1991; Sauber et al., 1994b). The pioneering work on mammary uptake of amino acids and that of Trottier et al. (1994) suggests that the relative importance of valine may be greater than that of lysine and of increasing importance with increased milk synthesis. The A - V data (Table 3) give credence to the higher relative valine requirement published by the NRC (1988), which places it in a 1:l relationship with lysine. This ratio was determined empirically and includes the maintenance component, but in sows with a relatively low milk production (Rousselow and Speer, 1980). The A - V data suggest that the ARC pattern ( 198 11, which reflects the milk amino acid pattern (.70 valine:1.00 lysine), is low and would constrain milk protein output and(or) lead to greater body protein mobilization. Preliminary work suggests that the valine requirement of the high- producing, lactating sow is at least 117% (gram percent, not molar percent) of dietary lysine based on litter weight gain alone (Richert et al., 1994). Valine


may be co-limiting with lysine (depending on ingre- dients in the diet) for high-producing, lactating sows ( 2 9.5 kg milWd) when both mammary need for milk protein synthesis and body protein conservation is considered. Fundamental studies are needed to determine whether the metabolic needs for valine by mammary tissue are obligatory.

The ideal amino acid profile has been used in growing pigs to estimate the need for other amino acids aRer the requirement for one amino acid has been established empirically. Proposal of an ideal amino acid profile for lactating sows would be more accurate if the production component were based on A - V difference of amino acids across the mammary gland rather than the milk amino acid profile ( a s done by the ARC, 1981). The latter ignores the possibility of tissue amino acid metabolism. Development of an ideal amino acid pattern also requires that we establish the ideal ratio between dietary IDAA and DAA. We are unclear about the minimum supply of DAA needed for milk production and the extent to which mammary synthesis occurs. Mepham ( 1976) concluded that the supply of DAA was unlikely to limit the rate of milk protein synthesis in ruminants. This assumes that rates of intra-mammary synthesis of DAA are adequate even at high milk production levels. The significance of DAA for lactation in pigs needs to be established through mammary gland uptake-milk output balance studies for IDAA and DAA in which amino acid nutrition is not limiting. Sows secreting high levels of milk ( 9 to 10 kg/d) are the most relevant model.

Endocrine Regulation of Milk Nutrient Synthesis: Requirements and Enhancement

of Synthetic Capacity

The biological capacity for milk synthesis depends on the number of mammary epithelial cells, optimum endocrine stimulation for both milk synthesis and removal, and proper coordination of extra-mammary tissues and feed intake to support lactation (homeorhesis). It is conceivable that constraints might be imposed in pregnancy because the number of secretory cells is established prepartum. We believe, however, that the major progress to be made in capturing the biological potential for neonatal growth depends on addressing endocrine regulation of milk synthesis during lactation. If cells are not stimulated maximally, differences in cell number might not be that important. The remainder of this review concen- trates on possible endocrine constraints to milk synthesis, with particular emphasis on attempts to stimulate milk secretion in an established lactation.

Mammary Growfh and Difmmtiafion. The sow’s capacity for milk synthesis is dependent on the formation of mammary epithelial cells (mammogene-

sis). They are dependent on a complex of hormones and growth factors for their development, maintenance, and function (Akers, 1990). The critical time points in mammary development are not clear. Work with dairy cattle has shown that puberal development is a critical phase in which improper nutrition (excess energy) results in adverse effects on mammary development and lowers milk production in each successive lactation (Sejrsen, 1978). In pregnant sows, there is evidence that excess energy intake, but not protein, is detrimental to cell number if fed during d 75 to 90 of gestation (Head et al., 1991; Weldon et al., 1991). We presume cell number to be integral to maximizing milk secretion; however, the relationship between cell number and milk nutrient output in pigs needs to be established. Knowledge of timing and factors affecting cell formation is potentially important and in need of investigation so that the potential for enhancement or detriment are clear.

Mammary growth is initiated by the conceptus and is a function of litter size (Hacker and Hill, 1972; Kensinger et al., 1986a,b). It was hypothesized that estrogens were the signal from the gravid uterus to the mammae (Kensinger et al., 1986a). Increases in mammary DNA concentration begin by d 30 of pregnancy and accelerate markedly between d 75 and 90. Although the mammary gland continues to enlarge during the last 3 wk of pregnancy, there are only minor increases in DNA concentration (Kensinger et al., 1982). The most significant changes in mammary structure occur between d 90 and parturition, which suggests that differentiation has occurred and the secretory cells have acquired the biochemical machinery required for milk synthesis (Kensinger et al., 1982). The degree to which mammary cell growth continues into early lactation is unclear, but additional accumulation of mammary DNA seems to be minimal (Hacker, 1970; Kensinger et al., 1986~) . Therefore, the interval between d 75 and 90 is a critical interval in development of milk secretory cells and a logical point to investigate the effects of nutrition and(or) endocrine management.

The specific hormone requirements for differentiation of mammary epithelial cells in pigs are not known, but the complex of prolactin ( PRL), cortisol, and insulin plays a primary role in lactogenesis of rodents and ruminants (Cowie et al., 1980). Biochem- ical differentiation of mammary tissue in pigs is dependent on these hormones in vitro (Jerry et al., 1989; Dodd et al., 1994) and seems to be suppressed by progesterone (de Passille et al., 1993). This also occurs in ruminants, in which the decline in progesterone and the rise in PRL are key components of the lactogenic “trigger” in the onset of mammary synthesis of milk components and milk secretion. The immedi- ate prepartum decline of progesterone in cows removes the inhibitory effect on the mammary gland (Bauman and Elliot, 1983). Furthermore, inhibition of PRL

46 BOYD

secretion around parturition in pigs completely in- hibits lactogenesis (Whitacre and Threlfall, 198 1).

Possible Endocrine Constraints to Milk Synthesis During Lactation. Two lines of evidence demonstrate that the lactating sow has greater potential for milk synthesis than is expressed under normal conditions. The report by Harkins et al. (1989) showed that administration of a highly purified preparation of ST during lactation could increase milk production by 22%. This suggests that hormone signals are not optimum for eliciting the inherent capacity for milk synthesis, and that expression of regulatory elements downstream from ST have not been maximized in the sow. The second line of evidence was presented earlier and also suggests that hormone signals may not be optimum because an extraordinary nursing stimulus (two litters alternatively nursing one sow) increased milk energy output (Sauber et al., 1994a). This is supported by Australian work in which a linear increase in milk production was observed with increases in litter size up to 14 pigs (R. King, personal communication). The effect of nursing intensity might be due to more complete milk removal in early lactation and(or) the result of altered endocrine signals associated with nursing (Ellendorf et al., 1982). Complete milk removal is an important determinant of milk output because milk stasis in alveolar spaces is the primary stimulus for involution of alveoli (secretory units). Nursing also stimulates an increase in circulating hormones such as PRL and oxytocin (van Landeghem and van de Wiel, 1978) and these, or other hormone(s), may coordinate greater synthesis/ secretion in response to nursing stimulus.

A simplified model depicting the regulation of nutrient flow between body pools to support milk synthesis is presented in Figure 4. In this model, nutrients in the plasma pool are removed for milk synthesis by mammary tissue under the influence of a stimulatory hormoneh). At the same time, this hormone coordinates the net flux of nutrients from adipose tissue to support milk synthesis. Adipose tissue, muscle, and gastrointestinal and other tissues undergo metabolic adaptations in support of lactation (Bauman and Elliot, 1983; Vernon, 1986). The uniqueness of homeorhetic control originates with the dynamic nature of tissue response to different physiological states and the multiple loci of action for the hormone(s1.

Somatotropin and PRL are candidates for homeorhetic control that may play a pivotal role in determining the level of milk nutrient synthesis (see reviews by Bauman and Currie, 1980; Bauman and Elliot, 1983). The possibility of eliciting greater milk production in the lactating sow via use of exogenous ST ( o r growth hormone-releasing factor, GRF) is suggested by the extensive body of literature in lactating dairy cows ( + l o to 40%; see review by Bauman and Vernon, 1993). In some species, PRL

ET AL.

Milk

. 1 Nutrient > Hormone Food 111) Intake Pool / / ,

Figure 4. A simplified model for homeorhetic regulation. A homeorhetic hormone indirectly or directly stimulates mammary tissue for milk synthesis with nutrients being derived from the blood pool. This hormone simultaneously causes adipose tissue (and perhaps muscle and bone) to buffer the blood pool by mobilization of substrates in support of lactation. Adipose tissue is dynamic in that uptake and mobilization of substrate occurs simultaneously but the homeorhetic hormone ensures that net mobilization of NEFA occurs, as required, to support mammary needs. Level of food intake may be modulated by the ability to maintain the blood nutrient pool and overall body energy balance from body stores. Prolactin and somatotropin are two examples of homeorhetic controls that have been shown to orchestrate physiological processes. Insulin-like growth factor I is a potential indirect mediator of homeorhetic control (adapted from Bauman and Elliot, 1983 and Bauman and Vernon, 1993).

also seems to be important in mammary gland growth, initiation of lactation, and maintenance of an established lactation (Cowie et al., 1980). Furthermore, PRL has extensive involvement in both adipose and mammary metabolism (Bauman and Elliot, 1983). However, exogenous PRL has not proven successful in enhancing milk production in lactating dairy cows (see review by Bauman and McCutcheon, 1986; Plaut et al., 1987). The possible involvement of ST and(or) PRL in the regulation of nutrient partitioning for milk synthesis in the pig has received only limited attention. It is possible that one or both play a key role, and that endogenous secretion is not adequate to support maximum milk output in lactating sows under normal conditions.

Endocrine Enhancement of Milk N u trimt Secretion. The study by Harkins et al. (1989) was the first to demonstrate that recombinant porcine ST could significantly increase milk nutrient output and consequently progeny weight gain. This demonstrates that milk secretion can be enhanced in lactating sows by elevating blood ST, but also embodies experimental


12 Lactating Sow

11 ~

E~ Q

E 9~

P X I 01

2i 10'

8

7 9,lO 16 22 28 9 , l O 16 22 28

Day of Lactation

Figure 5. Effect of treatment with porcine somatotropin (ST) on milk yield in sows and difference (ST - control) in the growth rate of progeny. Pretreatment milk yields were determined on d 9 and 10 of lactation by the weigh- suckle-weigh method. Treatment with recombinant ST (8.2 mg ST sc injection per d per sow) or excipient occurred on d 12 through d 29 of lactation. Means represent data from eight litters/treatment with litters standardized to 10 pigs each. Pooled CV was 13% and 12% for milk yield and difference in litter weight gain, respectively (adapted from Harkins et al., 1989).

considerations that we believe are critical to this type of research: First, a ST dose that increased average blood ST levels by 2.5 to 3.0 times was administered (8.2 mg per sow per day or approximately 42 p e g BW). This relative increase was chosen with the knowledge that a daily injection of approximately 60 pg of STkg BW is required to elicit maximal stimulation of milk secretion in dairy cows (Eppard et al., 1985). Second, injections began on d 12 of lactation, so an increase in milk secretion was not premature relative to the needs of the litter (see Figure lb) . Premature stimulation could conceivably be counterproductive if incomplete milk removal occurred and caused premature involution of alveoli. Allied with this was litter standardization to 10 vigorous pigs, which was thought to be the minimum challenge (10 t o 14 preferable). Third, the authors did not begin treatment with ST until peak milk production was expected (> 90%) because administration of bovine ST prior to or near peak milk yield in dairy cattle elicited only small increases in milk yield (Bauman and Vernon, 1993). Fourth, the potential increase in milk output by mammary glands of ST- treated sows was projected, and the nutrient concentration of the diet adjusted accordingly. Finally, the ST preparation was highly purified to minimize foreign proteins and endotoxins. There was no indication of toxicity based on body temperature or anaphylactic response to injected ST. This would

presumably compromise a test of physiological response to the exogenous hormone.

The response in milk secretion and pig weight gain with ST treatment is summarized in Figure 5 (Harkins et al., 1989). Exogenous ST altered the shape of the lactation curve. Milk composition was not affected, so more milk nutrients were available early (d 16) and output increased throughout lactation commensurate with the expanding needs of growing progeny (Figure lb) . An enhanced growth rate of the nursing pigs paralleled the increase in milk output (+3.50 kg/lO pigs a t d 28). The basis for increased milk is not known, but mechanisms by which ST elicits an increase have been investigated more extensively in lactating cattle (see reviews by McDowell, 1991; Bauman and Vernon, 1993). The literature contains little information about direct effects of ST on mammary metabolism. However, Dodd et al. (1994) recently showed that porcine ST treatment of mammary explants has no effect on milk protein accumulation.

The experiment by Harkins and co-workers ( 1989) also illustrates problems that could preclude response to treatments designed to elevate blood levels of ST or other agents postulated to enhance milk nutrient secretion. They projected dietary amino acid needs by assuming 10.5 kg of milWd output and that feed intake would be at least equal to that of the control group. Feed intake progressively increased during

48 BOYD ET AL.

Table 4. Comparison of energy and lysine balance to milk nutrient output for lactating sows: control vs somatotropin (STY

Nutrient balance Milk nutrient output

Item Control ST Nutrient ST advantage

Energy Intake, Mcal MWd Expenditure, Mcal ME/db Balance, Mcal MEM

Lipid mobilization, g/dc

Total intake, g/d Metabolizable, g/dd Milk secretion, g/de Balance, g/d

Lysine

20.5 16.4 Energy, McaYd +1.56 23.6 26.6 Lactose, g/d +135 -3.1 -10.2 Fat, g/d +150

Protein, gJd 4 0 388 1,278

52 42 32 26 39 42 -7 -16

Protein mobilization, g/df 116 265

aData derived from d 22 to 28 of lactation (Harkins et al., 1989). bMaintenance and milk secretion assumptions using literature cited by Harkins et al. (1989).

Metabolizable energy assumed to be used with 65% efficiency for maintenance and milk production. ‘Body lipid mobilization to meet energy deficit. Body lipid assumed to have 9.39 kcaVg and used with

85% efficiency for maintenance and milk production (Verstegen et al., 1985). dAssumes 82% digestibility and a partial efficiency of 75% for conversion of metabolizable (digestible)

lysine to milk protein. eAmount of lysine secreted as milk protein. Assumed milk protein has 7.5 g lysind100 g protein. fAssumed 6. 7 g of lysind100 g of body protein (Krick et al., 1993) and 90% utilization for milk protein

synthesis

lactation for control sows; however, sows receiving ST failed to increase intake after d 15. This resulted in an average intake of 6.0 and 4.7 kg/d for control and ST- treated sows, respectively, during the 16-d treatment period. Sows treated with ST lost twice as much weight (13.6 vs 7.0 kg) and three times as much backfat (3 .7 vs 1.2 mm) as the control group. Comparison of actual energy and amino acid intake with output in milk suggests that the increase in milk yield could not have occurred without ST-treated sows utilizing substantial quantities of protein and lipid reserves (Table 4 ) . This is illustrated by calculations during the period of greatest response to ST (22 to 28 d). An additional 7 Mcal/d deficit in metabolizable energy balance required more than a threefold increase in lipid mobilization (1,280 vs 390 g/d). Dietary lysine intake was 42 g/d for sows receiving ST (vs 52 g/d, control), which increased the lysine deficit relative to controls (16 vs 7 g/d) and necessitated mobilization of more than twice as much body protein to accommodate milk lysine secretion (265 vs 116 g/ d). This assumes that ST does not improve the efficiency of amino acid utilization. However, it is known that ST treatment of growing pigs improves the efficiency of amino acid utilization (Krick et al., 1993), but this possibility has not been explored in lactating sows. It is also important to consider that mobilization of body protein may need to be greater if valine uptake (relative to lysine) by mammary tissue is as portrayed in Table 3. Body valine content relative to lysine is lower, thereby requiring more body protein mobilization than would be required to

meet the deficit for lysine. This scenario exemplifies the homeorhetic nature of ST. It stimulates the mammary gland for greater milk nutrient synthesis while controlling the partition of nutrients from adipose and perhaps protein “reserves.”

The intake response in ST-treated sows is discon- certing in that the use of body reserves is extensive but the mechanism is unclear. Feed intake is a component of homeorhetic control that may be driven indirectly by sensors that monitor energy balance and the status of body reserves. This is best illustrated with ST treatment of lactating dairy cows, in which feed intake gradually increases over several weeks to match the increased milk energy secretion. In the study of Harkins et al. (1989), blood glucose was chronically elevated in ST-treated sows (+19% on d 29) . This was also observed by Spence (1984) with pituitary ST and may reflect antagonism of insulin action in adipose tissue as shown for the growing pig (Walton and Etherton, 1986; Dunshea et al., 1992). Elevation of blood glucose suggests that this pre- eminent nutrient is not limiting for milk synthesis; however, this is paradoxical because the ST-treated sow is in extreme negative energy balance and is apparently mobilizing considerable lipid compared with control females (Table 4 ) . We have no explana- tion for this, but limitations in amino acid intake in the ST-treated sow probably represent the greatest nutritional constraint to milk synthesis. Glucose uptake by mammary tissue may then be limited to the level of milk protein synthesis allowable. The result- ing increase in glucose pool size may be due to a


combination of limited amino acid availability (for milk protein synthesis), increased gluconeogenesis from mobilized glycerol, and perhaps glucose sparing from increased fatty acid oxidation in mammary tissue. Thereafter, a reduction in the glucose pool could occur through greater uptake by adipose and(or) a reduction in DE intake. Decreased sensitivity to insulin would preclude residual glucose uptake and result in elevated blood glucose concentration. The level achieved may have been high enough to prevent feed intake from increasing during lactation. This scenario is similar to that observed for the growing pig (Dunshea et al., 1992).

Reconciliation with other Papers on Somatotropin. Use of exogenous ST (or GRF) to enhance milk production in sows has not enjoyed the success reported in lactating dairy cows. The report by Harkins et al. (1989) represents the most successll attempt, but four other studies have been reported. None has completely duplicated the results of Harkins and co-workers. In some instances, adverse responses (anaphylaxis, ulceration) and death were observed (Spence, 1984; Kervagas et al., 1986; Smith et al., 1991). The reason for the ill effects are unclear but the lactating sow presents special problems not previously observed in the dairy cow.

Two papers report the effects when GRF is used to elevate blood ST in lactating sows (Dubreuil et al., 1990; Farmer et al., 1992). Use of a ST secretogogue has the challenge of increasing circulating ST to a sufficient magnitude and duration to be effective. A possible advantage is stimulation of endogenous ST, thereby ensuring biologically active ST. In the study by Dubreuil et al. (1990), 20 pg of GRFkg BW was injected into lactating sows twice daily from d 5 t o 25 (litter size 8 to 9) . This resulted in only a brief elevation in plasma ST (approximately 2 h ) after each injection and no increase in weight gain of the nursing pigs. The authors concluded that the small increase in ST could explain the lack of effect, but diet fortifica- tion was not considered, and this might also have precluded an effect. Farmer et al. (1992) subcutane- ously injected 60 pg of GRFkg BW into lactating sows three times daily from d 3 to 29. Litters were standardized to nine pigs each, and the diet was fortified to support anticipated needs for an increase in milk synthesis (.go% lysine, 18.3% protein). Increased GRF dose and frequency resulted in a twofold increase in ST for at least 6 h with a significant increase in IGF-I. No adverse effects were reported. Milk volume and litter size were maintained (numerically greater but not significantly improved) for GRF-treated sows despite the fact they consumed less feed. Greater utilization of body lipid to support milk synthesis is suggested by greater fat loss. Either body protein reserves were mobilized or dietary protein was utilized more efficiently because amino acid intake was reduced.

Two groups used daily injections of recombinant ST in lactating sows. Smith et al. (1991) conducted a dose-response study between d 7 to 20 of lactation with litters of 9 to 11 pigs. They observed a significant growth response in nursing pigs from ST-treated sows, but this was confined to heavier litters (based on d 7 BW) in which nursing stimulus may have been greatest. In concert with other studies involving exogenous pituitary and recombinant porcine ST (Spence, 1984; Kervagas et al., 1986; Cromwell et al., 19921, but in contrast to Harkins et al. (1989) and studies with GRF (Dubreuil et al., 1990; Farmer et al., 1992), Smith et al. (1991) observed mortality in sows due to ST administration. A detailed evaluation showed death to be dose-related and the result of hemorrhaging ulcers. They observed that a pST dose high enough to elicit 100% mortality in lactating sows (16 mg/d) had no adverse effect in growing pigs. This suggests a difference in sensitivity due to physiological state and(or) age. Cromwell et al. (1992) did not observe an enhancement of milk secretion with exogenous recombinant ST injections ( 6 mg/d). The number of sows and diets used were good, but administration from d 108 of pregnancy through d 24 of lactation might have been counterproductive for reasons discussed earlier. Their second experiment involved weekly injections of ST (70 mg/sow), but we would not expect this to elicit a response based on our understanding of the mechanisms of action.

Therefore, stimulating milk nutrient secretion by elevating blood ST has not been conclusively realized. The lack of response in some studies might have been due to inadequacies in experimental protocol; some ST preparations seemed to elicit adverse health effects. These have not been reported with GRF, which might eliminate questions of biological activity and endotox- icity. It is also conceivable that other endocrine factors are first-limiting or co-limiting to milk synthesis. Our knowledge is sufficiently inadequate that unsuccessful attempts should not lead to gloom.

Possible Role of Prolactin in Homeorhetic Regulation. Prolactin’s involvement in the onset of lactation and peripartum changes in blood concentration of sows was reviewed by Hartmann and Holmes (1989). However, its role in maintenance and possible enhancement of an established lactation is unclear. Inhibiting PRL release (by injection of specific drugs) near parturition in ruminants impairs mammary differentiation of key biochemical and cytological events involved in milk synthesis in cattle (Johke and Hodate, 1978; Akers et al., 1981a,b) and pigs (Whitacre and Threlfall, 198 1). Abnormally low levels of PRL in the anterior pituitary gland and circulation have been reported in sows showing spontaneous lactation failure (Threlfall et al., 1974). This may explain why Dusza et al. (1991) observed an 8% improvement in litter weight gain in gilts (but not sows) with a single injection of purified porcine PRL on d 1 of lactation.

50 BOYD ET AL.

Prolactin is released with each nursing or milking, and the magnitude of the release corresponds to suckling intensity in rats and level of milk production in cows (Tucker, 1974). This may be a mechanism for maternal coordination in response to progeny needs (e.g., variation in number) and is apparently medi- ated by the intensity and duration of nursing stimulus (Tucker, 1974; Algers et al., 1991). Suppressing PRL release during an established lactation by injection of specific drugs had no effect on milk production in either cows or goats (Karg et al., 1972; Hart, 1974; Smith et al., 1974). In contrast, blocking PRL release caused cessation of lactation in nonruminant species (Fluckiger and Wagner, 1968; Mayer and Schutze, 1973; Taylor and Peaker, 1975). These results are not entirely comparable, however, because studies with nonruminants were conducted in early lactation and those with ruminants at later stages (post-peak). They nevertheless suggest that PRL is essential to maintenance of milk secretion during early lactation in nonruminant species studied to date, including the rat, dog, and rabbit.

We are aware of one report on short-term inhibition of PRL ( d 25 of lactation or after) in lactating sows (Benjaminsen, 1981). The author indicated that there was no effect on progeny growth. However, they stated that the dose of the PRL inhibitor, bromocryptine, was relatively low and did not result in “complete” suppression (i.e., < 10%) of PRL levels in all sows. Further, sows nursed 5 to 10 pigs, and there was no indication of whether supplemental feed was available to the progeny (significant effector of progeny gain after 21 d). The results of an unpublished study of milk yield in relation to PRL inhibition in sows are shown in Figure 6 (post-peak, d 29 to 30). A marked reduction in both plasma PRL (from 40 to 50 to 2 to 5 ng/mL) and milk yield (50%) was observed in each sow. This suggests that progeny weight gain would have been reduced if PRL inhibition were chronic. This preliminary study was limited by lack of a treatment group involving simultaneous replacement with exogenous PRL to confirm the specificity of inhibitor. It nevertheless agrees with previously cited work in lactating rats, dogs, and rabbits that suggests that PRL is important to the level of milk yield during lactation.

The potential to enhance milk secretion by exogenous PRL or stimulation of endogenous PRL release has received relatively little attention. En- hancing PRL release by administration of a PRL secretogogue increased milk yield in lactating ewes by more than 50% (Bass et al., 19741, but short-term administration of highly purified exogenous PRL to dairy cows failed to stimulate milk secretion (Plaut et al., 1987). In addition to the previously cited work of Dusza et al. (19911, there are two preliminary reports in which the effects of exogenous PRL on milk production of sows were investigated. Crenshaw et al.

(1989) treated sows with porcine PRL from d 107 of pregnancy until d 2 of lactation and reported no effect on milk production on either d 4 or d 21 of lactation. No milk yield data for the first two days of lactation were reported. King et al. (1993a) treated sows, which were fed a protein-deficient diet, with porcine PRL twice daily from d 102 of pregnancy through d 22 of lactation. They observed lower protein and higher fat levels in colostrum of PRL-treated sows than in the similarly fed control animals, which suggests that the process of lactogenesis was accelerated in the PRG treated sows. They also reported a trend toward reduced milk yields in treated sows by d 19 to 22 of lactation. It is possible that premature involution of secretory units occurred in the sows from both studies given that the PRL treatments were initiated well in advance of the arrival of pigs. We re-emphasize that using exogenous hormonal treatments to stimulate additional milk secretion will likely be effective only if we impose these treatments when the pigs have the capacity to consume the additional milk.

A systematic study is needed to clearly ascertain the involvement of PRL in maintenance of milk yield and the extent to which elevation of endogenous PRL levels could lead to increased rates of milk synthesis. The mode of administration needs careful considera- tion because a precedent has not been established for pattern and minimum effective dose. The time of PRL treatment should probably be during a stage of lactation when nursing pigs have the potential to take advantage of additional milk nutrients. Finally, concerns of bioactivity and purity could be minimized by stimulation of endogenous PRL secretion.

Glucose and Amino Acid Economy for ”Average” vs Optimized Milk Nutrient Output

Theoretical Estimates of Spec@ Nutrient Needs. Finally, we have used the pioneering work by Linzell et al. (1969) and Spincer and co-workers (1969) to estimate the requirements for glucose and two key indispensable amino acids for present vs near-optimum milk nutrient secretion (Table 5 1. Optimizing milk secretion would require greater dietary nutrient input to the sow, greater mammary uptake and synthesis of milk components, and possibly greater use of energy, protein, and mineral reserves. Supplies of glucose and amino acids represent the most critical factors limiting milk production. The quantitative glucose requirement for lactation has not been defini- tively established but can be approximated from data on mammary uptake and knowledge of glucose utilization by non-mammary tissues (Linzell et al., 1969). We estimated the proportion of glucose utilization by lactating mammary glands according to production level (present vs optimum). Predictably, mammary uptake accounts for 69 to 80% of glucose entry, so 1.27


60

50

E 40

C

C

0

0

v

.- ,w

i! 30

p' Q

f 20 (II

n

LM

MY d 29 10 -

Y

1 I 236

Day 29

120

Day 31 1 MY d 31 -

Time of Day

Figure 6. Effect of an inhibitor of prolactin (PRL) secretion on plasma PRL and milk yield of four multiparous sows. Litters were standardized to 10 pigs each by d 2 and maintained through the treatment period. Milk yields were determined by the weigh-suckle-weigh method on d 29 and 31 of lactation (see Harkins et al., 1989) and plasma PRL concentration was determined in samples acquired from venous cannulas every 2 d from d 29 through 31. Prolactin secretion was inhibited by administration of lergotrile mesylate (LM, .50 mg i.m./kg BW) twice on d 30. The impact of PRL inhibition on milk yield (MY) is shown on d 31 (unpublished data from Scott, Butler, and Boyd).

Table 5. Estimated uptake of selected nutrients by the mammary glands of lactating sows: Present vs optimized milk yield

Mammary gland uptakea Milk nutrient outputb

Milk yield, kg/dC Milk yieldC

Nutrient 9.0 17.5 Nutrient 9.0 17.5

Glucose uptake, g/d 1,270 2,470 Energy, McaVd 10.2 19.9 Glucose, whole-body Lactose, g/d 405 788 utilization, %d 69 80 Fat, gld 630 1,225

Lysine uptake, g/d 38 74 Protein, g/d 504 980 TGFA uptake, g/d 243 472 Lipid mobilization, g/de 297 504

aTheoretica1 estimates of nutrient uptake made using values presented in Table 1 (g/dL of milk). Lysine uptake estimated assuming lysine content of milk is 7.5% and that 100% of a mammary lysine uptake is used for lysine deposition in milk protein.

bComposition of milk assumed: lactose, 4.5%; fat, 7.0% protein, 5.6%. Energy density of milk, 1.14 Mcal of gross energykg of milk (Noblet and Etienne, 1987).

'Milk yield levels represent present (9.0 kg/d) and maximum need (17.5) based on milk yield of sows between d 13 to 15 of lactation for Noblet and Etienne (1987) and artificial rearing data from Figure lb, respectively. Ten pigs per litter assumed.

dGlucose entry used for maintenance was 2.21 mg glucosemin-l.kg BW-' (Linzell et al., 1969). eEstimated using assumptions described in Table 4, footnote c. Also assumed feed intake was 90% of

the requirement to achieve energy balance. Lipid mobilization makes up the residual.

52 BOYD ET AL.

to 2.47 kg of glucoseld are required. This is compared to approximately 3 kg/d, which is the estimated need for lactating dairy cows yielding 30 kg of milWd (Bauman and Elliot, 1983). Given the strong relationship between glucose availability (entry rate) and energy intake in cattle, it is virtually certain that a reduction in glucose availability would accompany reduced feed intake and have a negative effect on the rate of milk synthesis. However, we cannot predict that point with certainty because of inadequate knowledge of the metabolic interrelationships among various precursors.

The quantitative requirement for amino acids, like that for glucose, is substantial and increases dramati- cally with increased milk production (Table 5 ) . The mammary gland needs a specific array of amino acids, and this differs from the milk pattern (Table 3). We estimated lysine uptake by mammary tissue but expect valine (and branched-chain amino acids in general) to become increasingly important as milk production increases. The dietary pattern is expected to increasingly reflect the pattern of mammary uptake with increasing levels of milk production, but mobilization of “labile” protein is probably needed. The amount of body protein available is unclear, but this reserve pool in dairy cows is estimated a t approximately 25% (Botts et al., 1979). Their studies suggest, however, that approximately one-half of this could be used without adverse effects. Estimating the need for mobilizable substrate is complicated by the fact that the pattern of amino acids mobilized does not match the apparent need of the mammary gland. This would necessitate greater mobilization to meet the most limiting amino acid. The most noteworthy example is valine, which is approximately 60% of lysine in whole-body protein (Krick et al., 1993).

Evidence of a g1ucose:amino acid relationship has been provided by Tokach et al. (1992). This is similar to the energy-dependent relationship for protein deposition that was established earlier in growing pigs (Whittemore, 1986; Campbell, 1988) and lambs (Black and Griffiths, 1975). Thus, the amount of amino acids that are utilizable for milk production may depend on glucose availability and, thus, feed intake.

Znformation Needed to Model Mammary Metabolism More Accurately. Critical studies are required on mammary metabolism in lactating sows before the effects of milk production rate on the pattern of nutrient need can be reliably predicted. Information needed includes quantitative information on precursor-product relationships, interrelationships among milk precursors, kinetic relationships between blood levels and mammary uptake, and kinetics of metabolic pathways. A quantitative description should include information allowing one to model inputs from supportive tissues such as adipose and liver. Mammary transport of nutrients is also an important aspect that is likely regulated by endocrine signals. Mammary

tissue may be among the most important tissues for glucose and amino acid transport because the flux of both is tremendous. An understanding of the regulation of synthetic

capacity of the mammary gland is required if porcine milk secretion is to match the growth potential of the nursing pig. We believe that nutrition (energy, amino acid level) may constrain presently accessible synthetic potential by the porcine mammary gland, but endocrine deficiency is proposed to be the greatest constraint because potential milk output has been shown to be much greater than realized under normal conditions.

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R’ D’

Scand. 28:41. Discussion

Brian Bequette: I would first like to make a comment which You can sort of take as a question. I wonder whether or not there actually is a Supply

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limitation to the mammary gland. Because if you look at your extractions, if there was a limitation, there wouldn’t be any venous concentrations of amino acids. Now, those extractions are based upon net uptake. This leads to my next comment, which concerns your suggestion that valine might be limiting. Now we’ve shown in the goat, and I think a lot of other people have shown as well, that there’s extensive oxidation of the branched-chain amino acids. Have you determined in the sow whether or not there is a requirement for oxidation in the mammary gland?

R. Dean Boyd: Is your question in regard to valine and other amino acids?

Brian Bequette: For instance, we’ve just done a study where we altered the supply of leucine to the mammary gland and changed oxidation and uptake of leucine without changing milk protein output. So it suggested to us that there wasn’t a requirement for oxidation by the mammary gland.

R. Dean Boyd: Let me respond to the first issue raised regarding amino acid limitation and venous concentration. There are studies with lactating pigs where a dose response was demonstrated between amino acid intake and milk output. They also show that total milk protein output is dose-related. Venous levels of the amino acid are low, but clearly present; they rise sharply upon attainment of the dietary amino acid intake that maximizes milk output. Example amino acids are lysine and valine. We don’t know how mammary extraction and metabolism of the amino acid is altered, but net uptake is presumably less. We need to know how mammary uptake (arterio-venous difference) varies with circulating amino acid concentrations. A nice summary of this was provided for ruminants in a review by Baldwin and Kim (1993) but is non-existent for the lactating pig.

The question of whether there is a certain requirement for amino acid oxidation in the mammary gland of the sow is a good one. Unfortunately, we do not have the data in pigs to address this. For ruminants, I understand that at least 10 to 11% of the CO2 produced in the mammary gland comes from two of the branched chain amino acids. Valine is one of them. It is unclear to me if this is requisite in ruminants, let alone pigs. In my opinion, valine would be an important amino acid to study.

I raise a final point relative to valine. Disregarding the issue of oxidation, the A R C and NRC have very different standards for the dietary amount relative to lysine. The NRC places valine and lysine in a 1:l relationship. This is considerably higher than the ARC, but our presentation points out that mammary uptake vs milk output suggests that the ratio could be higher. It could be as high as 1.2 va1ine:l.O lysine.

There are preliminary studies from Kansas State University and the University of Minnesota that also suggest a requirement in excess of the 1:l NRC relationship. The challenge is to document the dietary need using both milk output and body protein conservation; thereafter, to determine the extent to which valine is oxidized and whether it is obligatory.

Brian Bequette: With respect to arginine, I know there is a paper coming out soon suggesting that arginine might be limiting. Any comments on that and its requirement for conversion to proline in the mammary gland?

R. Dean Boyd: It is known that arginine is converted to proline in mammary tissue of the goat by a pathway involving the formation of ornithine and urea. Output of proline in milk seems to exceed its uptake by the mammary gland of ruminants and pigs. It is conceivable that arginine could be limiting if the total uptake of citrulline and arginine fail to meet obligatory needs for this “dispensable” amino acid. Our calculations for proline balance (mammary uptake vs output) are based on very limited information but suggest that a large amount of proline precursor is needed because milk proline content is so high. Rate of intramammary synthesis of proline or other amino acids could limit milk output unless compensated for by increased mammary uptake. We need a more definitive study on mammary uptake and output to estimate the extent to which mammary synthesis may be required.

John Metcalf: To follow on from your graph of uptake to output derived from Jim Linzell’s data, you had at least three indispensable amino acids, as you labeled them, which did not account for their output requirement. Where do you think they’re coming from?

R. Dean Boyd: The data that you refer to represents all that is available from the literature, but there was only one sow and a t one point in time. Thus, the possibility for error in quantitative estimation is great. Consequently, my interpretation was limited to the observation that the lactating sow appears to be qualitatively similar to the lactating cow. In the sow, the uptake of indispensable amino acids appears to be close to or in excess of mammary need for output. Uptake of some “dispensable” amino acids appears to be significantly below their output. This is similar to the dairy cow. Our figure differs from the lactating dairy cow in that two amino acids are approximately 25% below the need for output. Lysine is one of the amino acids. I expect that lysine is, in fact, taken up at or above the need for milk lysine output. On the other hand, it is possible that a nutritionally indispensable amino acid can be synthesized to some extent. Lysine is not a good example but arginine is a possibility. There is enough non-mammary synthesis

56 BOYD ET AL.

to support the needs for pregnancy but not lactation in pigs.

John Metcult I see what you’re getting at. Trust me, I don’t believe it, but we will talk later! The number of amino acids that are taken up at below the output requirement seem to be rather too many, and the differences seem to be too great to be accounted for by conversion of amino acids within the mammary gland. You showed that for suckled versus artificially raised pigs, the artificially fed piglets grew faster. Are there any data on their body composition? Was i t all fat? Did they have an increased level of protein?

R. Dean Boyd: We did not determine body composition at any point in the growth curve for artificially reared pigs. They were allowed to achieve 110 kg and then slaughtered to determine if they differed in carcass fat depth and loin eye area. They were not different from sow-reared controls. We did not determine rates of protein and lipid deposition.

James Pettigrew: First a comment with regard to valine. We have found that the valine requirement is higher than we expected it to be for the heavily lactating sow and, in fact, in some cases, we believe it may actually be the first-limiting amino acid in a corn- soy diet. Second, I was impressed with the growth rates that you were showing for your artificially reared pigs. I have been convinced that the pig has quite a lot of capacity for growth, and that milk production is limiting that growth. I wonder though if in this situation, is it strictly a matter of nutrient supply? If you look at the work coming out of Iowa

State and some from Minnesota and other places on segregated early weaning of pigs, we’re seeing very dramatic increases in growth rates that in some cases we interpret as coming from an improvement in health status. Now I wonder if it’s possible that what we are seeing in the dramatic difference you show may be a combination of nutrient supply and health status.

R. Dean Boyd: The point is well taken. We have given thought to that and agree that there is a possibility that artificially reared pigs could have had less of a pathogenic challenge. They were reared in a separate room between d 2 and 23 but recombined with sow- reared pigs in a common nursery-grower building for the remainder of the study. Although we can’t sort out the effect of segregated rearing for 21 d of growth, I am inclined to believe that the impact was not too dramatic. We began this work with an artificial rearing system placed within the same room as sow- reared pigs. Twenty-one-day end-weights have typically averaged at least 8.4 kg/pig or more.

Nathalie Trottier: Dr. Boyd, to what extent can radioactive tracers be used to better understand amino acid and energy substrate metabolism across the mammary gland?

R. Dean Boyd: Some of the points that I made in relation to glucose were the result of tracer technique. Again, data with ruminants are instructive in that mammary uptake, ability to quantitatively trace metabolism, and kinetics of arterial concentration and uptake are important to an understanding of precursor-product relationships.

Nutrient Uptake and Endocrine Regulation of Milk Synthesis by Mammary Tissue of Lactating Sows'

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