Mammary extraction of plasma triglycerides in the cow during lactogenesis

• 1.1. In control cows, extraction of triglycerides from the circulation by the mammary gland increased abruptly and markedly, and the concentration of triglycerides in arterial plasma fell, on the day of parturition.
• 2.2. In cows that had fluid regularly removed from the mammary glands before parturition, these changes occurred at varying times from more than 4 days before parturition in an animal that secreted triglyceride copiously to 24 hr before parturition in an animal that secreted little triglyceride pre-partum
• 3.3. In all animals, the time when triglyceride extraction increased was close to the time when K⁺ concentration in the mammary secretion became maximal.

     

The onset of mammary extraction of plasma triglycerides and circulating levels of progesterone during lactogenesis in the cow and goat

In untreated cows and goats, the onset of mammary extraction of plasma triglyceride was sometimes detected several hours before parturition, prior to removal of secretion from the mammary glands by offspring and when circulating progesterone concentrations were low. In cows and goats that had secretion regularly removed from the mammary glands late in gestation, the onset of triglyceride extraction occurred up to several days before parturition, when circulating progesterone concentrations were moderately high (up to 3.9 ng/ml in the cow and 6.2 ng/ml in the goat).     

The effect of pre-partum milking on the transfer of immunoglobulin into mammary secretion of cows.

The significance of local effects associated with mammary involution on transfer of immunoglobulin and especially on the selective transfer of IgG1 into mammary secretion of cows approaching parturition has been determined. This was carried out by measuring the changes in the concentration of IgG1, IgG2, IgM and IgA in serum and mammary secretion of 5 cows in which two mammary glands were milked continuously (twice daily) during the period preceding parturition, while the other two glands were allowed to undergo normal involution. In the secretion of unmilked glands of all cows there was a substantial increase in the concentration of IgG1 as cows approached parturition. In contrast, the increases in the concentration of IgG1 and in the selective index for IgG1 of milked glands were either virtually non-existent (1 cow) or generally reduced in magnitude and delayed in time of onset (4 cows). It is clear from the results that continued milking of a mammary gland throughout pregnancy tends to maintain milk production in the milked gland and at the same time reduces the massive selective transfer of IgG1 into secretion of that gland.     

Lactogenesis in the rat: an ultrastructural study of the initiation of the secretory process.

Three specimens were taken from mammary glands of rats killed on the 18th and 21st days of pregnancy and on the 1st day of lactation. Ultrastructural features of the tissue were compared among rats within and between the two stages of development. The similarity among specimens from the same rats made feasible a comparison of serial biopsies obtained every 4 hr, starting on the afternoon of the 21st day of pregnancy. From the 18th to the 21st days of pregnancy, a marked increase in the amount of rough endoplasmic reticulum occurred. The alveolar cells of rats killed on both days and in biopsies obtained at 17 and 13 hr before partuirtion contained abundant small lipid droplets and vacuoles containing many protein granules with little clear fluid (stasis vacuoles). Alveolar lumina were distended with secretion by 17 hr before parturition. Between 8 and 12 hr. before parturition, the accumulated protein and lipid were rapidly extruded from the alveolar cells despite evidence of continued biosynthesis. It is suggested that active transport processes are initiated independently of milk synthesis before parturition.     

Hormonal and immunological changes in blood and mammary secretion in the sow at parturition

The purpose of the present study was to record possible variations of estradiol-17 beta (E2) and cortisol concentrations, and parameters related to granulocyte phagocytosis in mammary secretions from healthy sows at parturition. The study was comprised 8 primiparous sows (Landrace x Yorkshire). Blood and mammary secretion samples were collected twice daily from 3 d before (only blood) until 3 d after farrowing. Estradiol-17 beta and cortisol concentrations were determined in plasma and in cell-depleted skimmed mammary secretions. Phagocytic capacity of polymorphonuclear cells (PMN) was assessed in whole blood and in cell suspensions derived from mammary secretions. Opsonic activity was assessed in serum and in cell-depleted skimmed mammary secretions. The 2 assays were based on chemiluminescence. Estradiol-17 beta concentration in plasma decreased (P < 0.001) directly after parturition. In skimmed secretions, the highest E2 concentration was recorded in the first sample after parturition and decreased (P < 0.01) thereafter. The highest cortisol concentration in plasma was recorded in the evening before parturition (P < 0.01). In skimmed secretions, there was no significant variation in cortisol concentration. The concentrations of both steroid hormones were lower in mammary secretions than in plasma. The phagocytic capacity of PMN in blood and mammary secretion, expressed as peak chemiluminescence per PMN, showed no significant change. This was also true for the opsonic activity in serum. In skimmed secretions the opsonic activity increased (P < 0.01) after parturition. These data emphasize the differences between plasma and mammary secretion concentrations of steroid hormones as well as between systemic and mammary gland immune competence. Regarding the phagocytosis process in mammary secretions, the part directly related to the PMN function seemed not to be altered at parturition compared with later on in lactation, whereas the part related to opsonic activity seemed to be impaired at parturition. The latter may play a role in the development of coliform mastitis at this time.     

Acyl specificity in triglyceride synthesis by lactating rat mammary gland.

We have studied the specificity of the acyl-CoA:diglyceride acyltransferase reaction in lactating rat mammary gland to provide a rational explanation at the enzyme level for the nonrandom distribution of fatty acids in milk fat triglycerides. Acyl-CoA:diglyceride acyltransferase activity was measured using various diglyceride and radioactive acyl-CoA substrates; products were identified as triglycerides by thin-layer and gas-liquid chromatography. Most of the enzymatic activity was located in the microsomal fraction and showed a broad specificity for the acyl donors tested C10, C12, C14, C16, C18, and C18:1 CoA esters). The acyltransferase activity was highly specific for sn-1,2-diglyceride enantiomers; rac-1,3- and sn-2,3-diglycerides were relatively inactive. The acyl-CoA specificity was not affected by the type of 1,2-diglyceride acceptor offered, although dilaurin was the best acceptor and sn-1,2-dilaurin greater than sn-1,2-dimyristin greater than sn-1,2-dipalmitin greater than sn-1,2-distearin. We have previously shown that in the microsomal fraction from lactating rat mammary gland, the acyltransferase activities concerned with the conversion of sn-glycero-3-phosphate to diacylglycerophosphate show a very marked specificity for long chain acyl-CoA's. Therefore, we conclude that the predominant localization of long chain fatty acids in the 1 and 2 positions, and of shorter chain fatty acids in the 3 position of the glycerol backbone, results at least in part from the specificities of the mammary gland acyltransferases.     

The epithelial glucocorticoid receptor is required for the normal timing of cell proliferation during mammary lobuloalveolar development but is dispensable for milk production

Glucocorticoids have been shown to influence mammary gland function in vivo and to stimulate milk protein gene expression in vitro. Here, we describe the generation and analysis of a mouse model to study glucocorticoid receptor (GR, NR3C1) function in mammary epithelial cells. Using the Cre-loxP system, mutant mice were obtained in which the GR gene is specifically deleted in epithelial cells during lobuloalveolar development, leading to a complete loss of epithelial GR at the onset of lactation. Mice harboring the mammary-epithelial-specific GR mutation are able to nurse their litters until weaning. During pregnancy, however, GR deficiency delays lobuloalveolar development, leading to an incomplete epithelial penetration of the mammary fat pad that persists throughout lactation. We identified a reduced cell proliferation during lobuloalveolar development as reason for this delay. This reduction is compensated for by increased epithelial proliferation after parturition in the mutant glands. During lactation, GR-deficient mammary epithelium is capable of milk production and secretion. The expression of two milk proteins, namely whey acidic protein and beta-casein, during lactation was not critically affected in the absence of GR. We conclude that GR function is not essential for alveolar differentiation and milk production, but influences cell proliferation during lobuloalveolar development.     

Evidence for opioidergic inhibition of oxytocin release in periparturient mares

In the horse mare, the onset of parturition is associated with an increase in oxytocin secretion, and it has been suggested that the onset of parturition may be triggered by endogenous oxytocin release. To test the hypothesis that oxytocin secretion is regulated by endogenous opioids in the periparturient period, we have 1) characterized oxytocin secretion in response to vaginocervical stimulation and 2) determined the effect of naloxone, an opioid antagonist, on oxytocin secretion induced by vaginocervical stimulation in prepartum mares and in postpartum mares at estrus and diestrus. During the last 2 months of pregnancy, the first diestrus and subsequent estrus post partum, a total of 66 vaginocervical stimulations were performed. Mares were pretreated with naloxone (0.5 mg/kg i.v.) or saline, administered 20 min before vaginocervical stimulation on subsequent days, using a randomized switchback design in which mares served as their own controls. Plasma was collected from 30 min before until 30 min after stimulation and was analyzed for oxytocin concentrations. Vaginocervical stimulation resulted in a significant increase in oxytocin secretion in all mares. Between Days 30 and 20 prepartum, the total amount of oxytocin secreted (calculated as area under the curve for 0 to 10 min after vaginocervical stimulation) was significantly greater in naloxone-treated than in saline-treated mares. From Day 20 prepartum until parturition, the differences between naloxone and saline-treated mares tended to decrease with approaching parturition, and were no longer statistically different. Peak plasma oxytocin concentrations were greater in naloxone-treated mares than in saline-treated mares during the entire prepartum period. During the postpartum period, total amount of oxytocin secreted following vaginocervical stimulation tended to be greater than during the prepartum period, and stimulated oxytocin secretion was significantly greater in naloxone-treated mares than in saline-treated mares. In conclusion, these data suggest that endogenous opioids suppress oxytocin secretion pre and post partum. It appears that opioid inhibition is not limited to the prepartum period, tends to decrease gradually towards parturition and is reinstated after foaling.     

Effect of essential fatty acid deficiency on release of triglycerides by the perfused rat liver

Studies are reported on release of triglycerides during perfusion of livers of male Sprague-Dawley rats fed a fat-free diet or diets containing hydrogenated coconut oil or corn oil. Perfusions were carried out with Krebs-Ringer bicarbonate buffer containing albumin with and without infusion of oleate or linoleate. Infusion with sodium oleate or linoleate caused an accumulation of triglycerides in the livers of the corn oil-fed animals and stimulated the release of triglycerides into the perfusing medium. In similar experiments with essential fatty acid-deficient animals, which were fed fat-free diets or diets containing hydrogenated coconut oil, there was no increase in secretion of triglycerides into the perfusate, and the amount of triglyceride which accumulated in the liver was greater than in the livers of the control (corn oil-fed) animals. Tracer experiments with oleate-1-(14)C or linoleate-1-(14)C also showed that with livers of essential fatty acid-deficient animals, secretion of triglyceride into the perfusate was not stimulated by infusion of fatty acids into the perfusing medium. It is concluded that impairment of the secretion of triglycerides is a factor in the accumulation of fat in the livers of essential fatty acid-deficient animals.     

Progesterone and corticosteroids in the initiation of lactation in the sow

The concentration of lactose in the mammary secretion from individual glands of two sows increased significantly (P less than 0.01) between 0 and 24 h after parturition. In six sows studied during the perinatal period there was a negative correlation (r = -0.80; P less than 0.02) at parturition between the concentration of progesterone in the blood and the concentration of lactose in the mammary secretion. Furthermore, the increase in concentration of lactose in the mammary secretion after parturition was related to the timing of the decline of plasma progesterone to low levels. The results indicate that the initiation of lactation occurs within 24 h of parturition in most sows, and the results are consistent with the hypothesis that progesterone withdrawal acts as the 'trigger'. Neither the changes in corticosteroid binding globulin nor the changes in total corticosteroids were temporally related to the initiation of lactation. However, a circadian rhythm was observed for total corticosteroids in the blood of three out of nine lactating and pregnant sows, whereas no circadian rhythm was observed in progesterone of the four pregnant sows. The results are discussed in relation to the disease complex mastitis-metritis-agalactia.     

Changes in mammary development and composition of secretion during late pregnancy in the mare.

Small samples of mammary secretion were taken for analysis from Thoroughbred mares during the last 3 weeks of pregnancy up to the time of foaling. The concentrations of sodium and chloride decreased while those of lactose, potassium, citrate, phosphate, calcium, magnesium and protein increased. The time-course of these changes showed marked variation between animals. The concentration of whey proteins began to increase about 10 days before parturition. The appearance of the secretion and the size of the mammary glands increased in the last few days of pregnancy. It is suggested that the concentration of calcium in mammary secretion could provide the basis of a test for impending parturition in this species.     

The relationship between the transfer of immunoglobulins, sodium and potassium into mammary secretion of the parturient ewe.

The experiment comprised two sections. First, radiotracer techniques were used to study the metabolism of IgG1 and IgG2 in 5 non-pregnant and 4 pregnant ewes. In the pregnant ewes, the rates of synthesis for IgG1 and IgG2 were similar to the rates observed in non-pregnant animals. However, the irreversible loss of IgG1 was significantly greater than IgG2 in pregnant ewes and IgG1 in non-pregnant ewes. Additionally, it was found that most of the IgG1 and virtually all of the IgG2 in mammary secretion was serum derived. Secondly, the levels of sodium, potassium and lactose and the selective index for IgG1 in mammary secretions of 5 pregnant ewes were monitored over the parturient period. The values of all the measures remained relatively constant until one day before parturition. From one day pre-partum, the levels of potassium and lactose in mammary secretion began to increase and had risen 2-3 fold by 5 days post-partum. Over the same period, the selective index for IgG1 decreased 20 fold, wehreas the level of sodium fell from approximately 32 mmol/l to 18 mmol/l. The concentration of IgG1 in plasma slowly declined from approximately 23 g/l to 15 g/l over the last 10 days of pregnancy. During the parturient period, the decline in plasma IgG1 levels, in comparison with IgG2, without alteration in the rates of synthesis of either immunoglobulin, supports the hypothesis that selective transport of IgG1 into mammary secretion occurs without degradation. The results also indicate that the transport of sodium and potassium into mammary secretions are altered over the parturient period.     

Human lactation: maternal transfer of dietary triglycerides labeled with stable isotopes

A stable isotope tracer method was utilized to measure quantitatively the secretion of diet-derived fatty acids (FA) into human milk. A mixture of [2H6]tripalmitin, [2H18]-triolein, and [2H12]trilinolein was administered to three healthy, lactating women 22 to 30 years of age. Milk and blood samples were collected sequentially for 72 hr. The FA composition and concentration of total plasma, lipoprotein, and milk triglycerides were determined by gas-liquid chromatography (GLC) and the isotopic enrichment was determined by gas-liquid chromatography-mass spectrometry (GLC-MS). There were no statistically significant differences in mammary secretion of the individual fats, either by a single individual or between subjects. The mean secretion of fat by one breast was 5.11 +/- 1.26% of the dose (CV = 25%). There was a significant 6.0-hr delay between peak occurrence of the tracer in plasma and its occurrence in milk. The lipids are transported to the mammary gland primarily by the chylomicron and very low density lipoprotein triglycerides.     

Initiation of fatty acid synthesis in rat mammary glands.

The rate of fatty acid synthesis from [6-14C]glucose in mammary tissue remained low until parturition at 22 days of gestation and increased 10-fold at 1 day post partum. Administration of progesterone on days 20 and 21 or removal of pups at parturition abolished this increase. In the latter case, administration of prolactin, corticosterone or oxytocin had no stimulatory effect; tissue from suckled glands in which the ducts had been ligated at parturition also showed no increased rate in 24 h. Foetoplacentectomy on day 18 did not stimulate fatty acid synthesis but subsequent suckling by foster pups did. Whereas lactose synthesis is initiated by withdrawal of progesterone from the circulation, a further stimulus related to removal of milk by suckling is required to initiate fatty acid synthesis.     

Induction of parturition in snow skinks: can low temperatures inhibit the actions of AVT?

The influence of environmental factors on the timing of parturition has not been investigated in viviparous squamates. We investigated the interaction between temperature and parturition in two viviparous skink species, the southern snow skink (Niveoscincus microlepidotus) and the metallic skink (N. metallicus). In these species, the timing of parturition is separated from the completion of embryonic development; the delay is attributed to their cool and variable habitats. We examined whether the neurohypophyseal hormone arginine vasotocin (AVT) stimulated parturition in southern snow skinks with late stage embryos in autumn (approximately 6-7 months prior to parturition) and in metallic skinks with late stage embryos in summer (approximately 2-3 weeks prior to parturition). The experiments were conducted at a range of environmentally relevant temperatures (6 degrees C, 15 degrees C, 22 degrees C, and 28 degrees C). AVT induced parturition in both species at all temperatures; time until birth, however, occurred more quickly at warmer temperatures (22 degrees and 28 degrees C), whereas cooler temperatures delayed parturition. We hypothesize that if cool temperatures are preventing parturition, then temperature must act at some level within the brain to prevent or slow the secretion of AVT. Future experiments will need to determine how temperature influences AVT production. Further research is also required to determine how the timing of parturition is influenced by interactions between temperature, photoperiod, and seasonal hormone patterns.     

Interleukin-2 secretion by bovine mammary gland mononuclear cells during the nonlactating period

Bovine blood and mammary gland mononuclear cells were isolated from five primiparous Holstein cows at 14-18 and 28-32 days of involution and 7-13 days prior to parturition and used in a bioassay to determine if bovine mammary mononuclear cells produced interleukin-2 in culture. Interleukin-2 production by mononuclear cells was stimulated in 24 hr cultures using concanavalin A. Bovine interleukin-2 dependent cytotoxic T-lymphocytes were used as target cells. Interleukin-2 production by mammary gland and blood mononuclear cells was comparable. Interleukin-2 production by mammary gland mononuclear cells did not vary significantly over the three time periods evaluated. However, blood mononuclear cells isolated at 28-32 days of involution produced significantly greater amounts of interleukin-2 compared to blood mononuclear cells isolated during the early nonlactating period and the prepartum period. These data suggest that bovine mononuclear cell hyporesponsiveness to mitogens during the nonlactating period is not due to a deficiency in interleukin-2 production.     

Impact of maternal dietary n-3 and n-6 fatty acids on milk medium-chain fatty acids and the implications for neonatal liver metabolism

Levels of n-6, n-3, and medium-chain fatty acids (MCFA) in milk are highly variable. Higher carbohydrate intakes are associated with increased mammary gland MCFA synthesis, but the role of unsaturated fatty acids for milk MCFA secretion is unclear. This study addressed whether n-6 and n-3 fatty acids, which are known to inhibit hepatic fatty acid synthesis, influence MCFA in rat and human milk and the implications of varying MCFA, n-6, and n-3 fatty acids in rat milk for metabolic regulation in the neonatal liver. Rats were fed a low-fat diet or one of six higher-fat diets, varying in 16:0, 18:1n-9, 18:2n-6, 18:3n-3, and long-chain (LC) n-3 fatty acids. Higher maternal dietary 18:2n-6 or 18:3n-3 did not influence milk MCFA, but lower maternal plasma triglycerides, due to either a low-fat or a high-fat high-LC n-3 diet led to higher milk MCFA. MCFA levels were inversely associated with 18:1n-9, 18:2n-6, and 18:3n-3 in human milk, likely reflecting the association between dietary total fat and unsaturated fatty acids. High LC n-3 fatty acid in rat milk was associated with lower hepatic Pklr, Acly, Fasn, and Scd1 and higher Hmgcs2 in the milk-fed rat neonate, with no effect of milk 18:1n-9, 18:2n-6, or MCFA. These studies show that the dietary fatty acid composition does not impact MCFA secretion in milk, but the fatty acid composition of milk, particularly the LC n-3 fatty acid, is relevant to hepatic metabolic regulation in the milk-fed neonate.     

Long-term effects of cis and trans monounsaturated (18:1) and saturated (16:0) fatty acids on the synthesis and secretion of apolipoprotein A-I- and apolipoprotein B-containing lipoproteins in HepG2 cells

The objective of this study was to compare the long-term effects of oleic (cis 18:1), elaidic (trans 18:1), and palmitic (16:0) acids on hepatic lipoprotein production, using HepG2 cells as an experimental model. The net accumulation in the medium of apolipoprotein A-I (apoA-I) was not significantly altered by fatty acids, whereas that of apoB was increased with oleic and elaidic acids. Oleic acid, and to a lesser extent elaidic and palmitic acids, increased the mass of triglycerides in the medium and the incorporation of [(3)H]glycerol into secreted triglycerides. The incorporation of [(14)C]acetate into cellular and secreted total cholesterol was stimulated by 96% and 83%, respectively, with elaidic acid but was not significantly modified by oleic or palmitic acid. Relative to oleic acid, the secretion of (14)C-labeled phospholipids and triglycerides was decreased 28% to 31% with elaidic and palmitic acids whereas that of free cholesterol and cholesteryl esters was enhanced 93% and 73%, respectively, with elaidic acid but remained unchanged with palmitic acid. Compared with oleic acid, elaidic acid stimulated the secretion of very low density lipoprotein cholesterol (VLDL-Chol), low density lipoprotein cholesterol (LDL-Chol), and high density lipoprotein cholesterol (HDL-Chol) by 43%, 70%, and 34%, respectively, whereas palmitic acid decreased VLDL-Chol but had no significant effect on LDL-Chol and HDL-Chol. The ratios of total cholesterol to HDL-Chol were 3.17, 3.60, and 3.25 with oleic, elaidic, and palmitic acids, respectively; the corresponding ratios of LDL-Chol to HDL-Chol were 0.87, 1.10, and 0.93, respectively. Compared with oleic and palmitic acids, the LDL and HDL particles secreted in the presence of elaidic acid contained higher levels of free cholesterol and cholesteryl esters and a lower content of phospholipids. The phospholipid-to-total cholesterol ratios of HDL were 1.05, 0.40, and 0.76 with oleic, elaidic, and palmitic acids, respectively.Our results indicate that in comparison with cis monounsaturated and saturated fatty acids, trans fatty acids have more adverse effects on the concentration and composition of lipoproteins secreted by HepG2 cells.