Drug active transport into milk is a major concern for breastfeeding mothers and healthcare providers. Studies from the literature indicated that breast cancer resistance protein (ABCG2) plays an important role in drug transfer into milk. There has been limited study on stereoselective interactions with ABCG2. A mechanistic analysis of flux across cell monolayer model is a critical first step toward extrapolating in vitro results for predicting in vivo disposition (including distribution into milk), drug disposition or drug-drug interactions. The objectives of this thesis were (1) to establish a “Chemical knockout model” in rat for studying drug accumulation into milk, (2) to investigate the impact of stereoselective interaction between ABCG2/Abcg2 and pantoprazole on drug transport in milk, (3) to understand in vitro monolayer flux model using experimental data and a mechanistic mathematical model. Quantitive PCR, Western blotting and immunohistochemistry results indicated that Abcg2 was up-regulated during lactation and localized on apical side of epithelial cells in mammary gland. In vitro and in vivo experiments confirmed that Abcg2 is responsible for nitrofurantoin active transport in rat milk and GF120918 was established as a chemical knockout model. Abcg2 interacts stereoselectively with pantoprazole isomers. A significant different apical flux between two pantoprazole isomers was observed in Abcg2-MDCKII cell line. The milk to serum (M/S) ratio of (-)-pantoprazole was almost 3 times as that of (+)-pantoprazole in lactating rats. Administration GF120918 decreased M/S of (-)-pantoprazole (p<0.001) but not (+)-pantoprazole (p>0.05). A stably transfected ABCG2/Abcg2 overexpressing MDCKII cell line was successfully created and used to explore the theoretical relationships in a monolayer flux model. Based on the profiles of pantoprazole isomer transport, a simple three compartment model for drug transfer into breast milk incorporating the permeability-surface area products for passive diffusion (PSD), paracellular flux (PSPC) and apically efflux ABCG2 (PSA,E) transfection was developed. The mathematical model was developed to more fully understand the interplay of paracellular, passive diffusion, active transport, and flux kinetic parameters (Km, Vmax, IC50 and Ki). This model provided useful insights into the meaning and limitation of parameters obtained from monolayer flux.

Recent literature has established that breast cancer resistance protein (ABCG2) is upregulated during lactation and is responsible for the greater than predicted accumulation of many drugs in breast milk. The objectives of this project were (1) to investigate the role of this transporter in the reported apically-directed nitrofurantoin flux in the CIT3 cell culture model of lactation, (2) to develop a mathematical model for drug transfer into breast milk to relate initial flux rates, steady-state concentrations, efflux ratios, and in vivo milk to serum ratios (M/S) and (3) to identify xenobiotic transporters that are highly expressed, and therefore potentially important for drug accumulation during lactation in mice and humans. Expression, localization, and functional assays confirmed that Abcg2 is the molecular mechanism for the apically-directed nitrofurantoin flux in CIT3 cells despite an unchanged expression level following lactogenic hormone stimulation in this model. A simple three compartment model for drug transfer into breast milk incorporating the permeability-surface area products for passive diffusion (PSD), paracellular flux (PSPC), endogenous transporters (PSB,U, PSA,E, PSB,E, and PSA,U), and ABCG2 (PSA,E(ABCG2)) transfection was developed. A stably transfected ABCG2 overexpressing MDCKII cell line was successfully created and used to explore the theoretical relationships of this new model. Derivations and correlations presented herein show the relationships between the calculated efflux ratios, PSA,E(ABCG2), and M/S attributed to ABCG2. Six xenobiotic transporters (Abcg2, Slc22a1, Slc15a2, Slc29a1, Slc16a1, and Abcc5) were identified as upregulated during lactation in murine developmental datasets analyzed by microarray expression profiling. As existing methods were inadequate to obtain pure populations of luminal epithelial cells in sufficient numbers from human breast milk or reduction mammoplasty samples for microarray analysis, a new fluorescence activated cell sorting method was developed and validated. ABCG2, SLC15A2, SLC22A12, SLC6A14, and SLCO4C1 were significantly upregulated 164-, 70-, 41-, 8-, and 2-fold during lactation, respectively. ABCC10, SLC10A1, SLC16A1, SLC22A4, SLC22A5, SLC22A9, SLC28A3, SLC29A1, SLC29A2, and SLCO4A1 had an expression level similar to, or greater than, levels in the kidney or liver. The significant upregulation of SLCO4C1 with ABCG2 is a novel finding that suggests a coordinated vectorial pathway for substrate movement into breast milk.

Maternal folate intake and levels of folate in milk, serum and red cells were assessed in 57 healthy, lactating women, ages 22-38 years, throughout early (0-6 months) and later (7-23 months) lactation. Average maternal folate intake from diet alone was 212 µg/day or 78.5% RDA (1989) and mean total folate intake from diet and supplements was 314% RDA (878 µg/day) at 0-6 months and 238% RDA (620 µg/day) at >6 months. Human milk folate was sufficient to meet the RDA (1989) for infants. Milk folate was not related to maternal folate intake, maternal serum or red cell folate and was unaffected by extended lactation (7-23 months), perhaps at the expense of maternal folate stores. Compared with early lactation, serum folate decreased (p=0.0004) and red cell folate tended to decrease (p=0.08) in later lactation and were both increased by folate supplementation (p < 0.001).Level of folic acid supplementation appeared to predict red cell folate concentration. An average of 884 µg supplemental folate/day was associated with red cell folate levels >400 ng/mL, which have previously been reported as optimal for prevention of folateresponsive neural tube defects. The addition of an 880 µg/day folic acid supplement to the diet of lactating women may raise red cell folate concentrations of lactating women to protective levels. / Department of Family and Consumer Sciences

Darbo tikslas: Įvertinti įvairių pieninių veislių karvių reprodukcinių savybių, produktyvumo bei tešmens sveikumo rodiklius ir nustatyti jų tarpusavio ryšius. Darbo uždaviniai: 1. Įvertinti: a) karvių reprodukcines savybes; b) karvių produktyvumo rodiklius; c) karvių somatinių ląstelių skaičių piene; 2. Palyginti skirtingų karvių veislių reprodukcinių savybių, produktyvumo bei tešmens sveikumo rodiklius; 3. Nustatyti tirtų rodiklių koreliacijos koeficientus. Išvados: 1. Tirtos karvės buvo vidutiniškai 3,64±0,032 laktacijos, vidutinė laktacijos trukmė – 228,47±3,576 dienų, vidutinė veršingumo trukmė – 281,72±1,280 dienų, karvės vidutiniškai sėklintos 1,95±0,034 kartų, neveršingumo laikotarpis vidutiniškai truko 154,06±3,566 dienų ir laikotarpis tarp apsiveršiavimų – 448,76±3,156 dienų. Vidutinis pieno kiekis per laktaciją buvo 5046,8±73,817 kg, pieno riebumas – 4,35±0,016 proc., riebalų kiekis – 214,60 ±2,844 kg, baltymingumas – 3,23±0,007 proc., baltymų kiekis – 162,32±2,191 kg, SLS piene – 407,03±23,328 tūkst./cm3. 2. Išanalizavus atskirų veislių karvių reprodukcines savybes nustatyta, kad ilgiausia veršingumo trukmė buvo LŽ veislės karvių (285,14±2,040), trumpiausia – VJ veislės karvių (279,32±1,994). Mažiausiai kartų sėklintos VJ veislės karvės (1,80±0,197), daugiausiai – LŽ veislės karvės (2,02±0,086). Trumpiausias neveršingumo laikotarpis nustatytas LŽ veislės karvių (139,91±6,310), ilgiausias – SP veislės karvių (182,53±20,981). Ilgiausias laikotarpis tarp... [toliau žr. visą tekstą] / Aim of work: To evaluate the different breeds of dairy cow reproductive performance, productivity and udder health traits and to establish interrelationships between between them. Place of research: The work was carried out in the Departament of Animal Breeding and Genetics of Veterinary Academy and also in the dairy cow‘s farm during 2011 – 2013 years studies. The results of survey: Cows were on average 3.64 ± 0.032 lactation. The average duration of lactation - 228.47 ± 3,576 days. The average gestation period - 281,72 ± 1.280 days. Cows inseminated an average of 1.95 ± 0.034 times. The servis period lasted an average of 154.06 ± 3,566 days and the interval between calving - 448.76 ± 3.156 days. Average milk production per lactation was 5046.8 ± 73.817 kg, of milk fat - 4.35 ± 0.016 percent., fat - 214.60 ± 2.844 kg, protein - 3.23 ± 0.007 percent., protein - 162.32 ± 2.191 kg, milk SCC - 407.03 ± 23,328 thousand/cm3. The analysis of individual cows reproductive traits showed that the longest pregnancy lasted Lithuanian reds cows (285.14 ± 2.040), the shortest - German black & whites cows (279.32 ± 1.994). At least once inseminated German black & whites cows (1.80 ± 0.197), mainly - Lithuanian reds cows (2.02 ± 0.086). The servis period the shortest period of Lithuanian reds cows (139.91 ± 6.310), the longest - Simental cows (182.53 ± 20.981). The longest interval between calving the Holstein black & whites cows (496.14 ± 7.152), the shortest - Lithuanian black & whites... [to full text]

Endometritas – dažniausiai diagnozuojama karvių gimdos liga ir viena iš svarbiausių karvių nevaisingumo priežasčių. Endometritas apibrėžiamas kaip gimdos gleivinės (endometriumo) uždegimas, apimantis ne giliau nei Stratum spongiosum. Didelis (25,0–83,3 proc.) karvių sergamumas endometritu yra aktuali problema daugelyje šalių neišimtis ir Lietuva. Nustatyta, kad Lietuvos ūkiuose endometritu serga 11,0 proc., o kai kuriose stambiose fermose – net 33,2–48,6 proc. karvių. Beveik visoms susirgusioms karvėms ši liga pasireiškia panašiais klinikiniais požymiais, bet ligos etiologija ne visada būna aiški. Endometritas pagal tipą yra skirstomas į klinikinį ir subklinikinį arba kitaip dar vadinamas slaptos formos. Tiek klinikinis, tiek ir slaptasis endometritas, sukelia didelių ekonominių nuostolių, nes sumažėjo karvių reprodukcinė funkcija bei pieno primilžis, be to, neretai sergančias karves tenka išbrokuoti. Šio darbo tikslas – pagal klinikinius požymius ir reprodukcinius rodiklius nustatyti karvių endometritų gydymo efektyvumą priklausomai nuo karvės laktacijos ir produktyvumo. Tyrimai atlikti 2013 metais sausio-gegužės mėnesiais, Kelmės rajone esančiame X ūkyje. Tyrimo metu visos karvės buvo suskirstytos į dvi grupes: kliniškai sveikų ir endometritu sergančių karvių (kliniškai sveikų n=20, ir sergančių n=44). Karvių grupės buvo sudarytos remiantis gimdos išskyrų pobūdžiu, rektiniu tyrimu ir mikrobiologinio tyrimo rezultatais. Mikrobiologiniai karvių gimdos gleivių tyrimai buvo... [toliau žr. visą tekstą] / Endometritis − the most commonly diagnosed disease in the uterus of cows and one of the main causes of cows infertility. Endometritis is defined as the uterine lining (endometrium) inflammation, including no deeper than the stratum spongiosum. Large (25.0 to 83.3 %) cow incidence of endometritis is a pressing problem in many countries and Lithuania is no exception. It was found that in the Lithuanian farms from endometritis suffers 11.0 %, and in some large-scale farms – even 33.2 – 48.6 % of cows. Almost all of the sick cows have similar symptoms, but the disease etiology is not always clear. Endometritis is divided according to the type of clinical and subclinical or otherwise also known as a secret form. Both clinical and subclinical endometritis causes significant economic losses due to reduced reproductive performance of cows and milk yield, in addition, sick cows have to be culled often. The aim of this work – according to the clinical characteristics and reproductive traits to determine the effectiveness of treatment based on cow's lactation and productivity. Studies were carried out in 2013 January – May, in the X farm which is located in Kelmė district. During the study, all cows were divided into two groups: clinically healthy cows and cows infected with endometritis (clinically healthy n = 20 and sick n = 44). Cow groups were formed on the basis of uterine secretions nature, rectal examination and results of microbiological testing. Microbiological cow uterine... [to full text]

This study was designed to measure the prevalence and nutritional impact of dairy product restriction in both pregnant and lactating women. Dairy product (DP) restriction was assessed in 2091 prenatal class attendees. Subsamples of 279 healthy pregnant and 175 exclusively breastfeeding women provided repeat 24-hour dietary recalls which were used to estimate nutrient intake and to assess dietary adequacy using adjusted nutrient distributions. Pregnancy outcome was recorded. Pregnancy. DP restriction was reported by 13.2% (95% CI: 12.7%,14.1%) of pregnant women. Significant differences were found in the proportion of restrictors (Rs, 20.8%) below the calculated EAR for adjusted protein intakes vs. nonrestrictors (NRs, 9.2%, chi 2 = 6.78, p = 0.009). Intake distributions of calcium and vitamin D were compared to their respective Als for descriptive purposes and were lower for Rs vs. NRs but dietary inadequacy could not be assessed. Infants of NRs weighed on average 120 g +/- 468.9 g more than those of Rs but this difference was not statistically significant (p = 0.06). However, in multiple regression analysis where maternal weight gain, age, education and pregravid weight were controlled for, restriction predicted a comparable loss in birthweight of 122.5 g's (p = 0.048) and vitamin D intakes were positively associated with fetal growth. Lactation. Restriction (≤250 ml milk/day) was reported by 23% (95% CI; 16%--29%) of exclusively lactating women. Protein was lower than the EAR for 60% of Rs and 37.8% of NRs (chi 2 = 6.22, p = 0.025). The proportion of Rs and NRs < EAR from diet differed for thiamin (15% vs. 0%, chi2 = 0.97, p < 0.001), riboflavin (15% vs. 1.5%, chi2 = 12.93, p < 0.001) and zinc (65.0% vs. 34.8%, chi2 = 11.6, p < 0.041). From diet and supplement intake vitamin D and calcium remained lower for Rs compared to NRs but inadequacy could not be assessed or compared. DP restriction was not associated with greater weight loss amo

Substantial evidence supports a role of diet in glucose metabolism, but only a few reports have investigated the impact of diet during pregnancy on risk of gestational diabetes (GDM). Although metabolic hormones have been detected in milk, no studies have investigated the impact of maternal metabolic status assessed during pregnancy on insulin and adiponectin concentrations in human milk. The purpose of this thesis was to investigate the association of maternal macronutrient intakes with metabolic status during pregnancy and its subsequent impact on human milk hormones. Participating women (n=216) underwent a 3-hour oral glucose tolerance test at 30 (95% confidence interval [CI] 25, 33) weeks gestation, recalled their second trimester dietary intake, and donated early (the first week) and mature (3 months postpartum) milk. Higher vegetable and fruit fiber intake was associated with reduced insulin resistance (beta±SE -0.100±0.029, p=0.0008) and increased insulin sensitivity (0.029±0.012, p=0.01) among those with a family history of type 2 diabetes. Lower % carbohydrate and higher % total fat were associated with increased GDM risk (odds ratio 0.60 [95% CI 0.40, 0.90] and 1.61 [1.06, 2.44], respectively). Prenatal metabolic abnormalities including higher pregravid body mass index (beta±SE 0.053±0.014, p=0.0003), in addition to gravid hyperglycemia (0.218±0.087, p=0.01), insulin resistance (0.255±0.047, p<0.0001), lower insulin sensitivity (-0.521±0.108, p<0.0001), and higher serum adiponectin (0.116±0.029, p<0.0001) were associated with higher insulin in mature milk. Obstetrical measures including nulliparity (0.171±0.058, p=0.004), longer duration of gestation (0.546±0.146, p=0.0002), and unscheduled caesarean section (0.387±0.162, p=0.02) were associated with higher adiponectin in early milk. Holder pasteurization, a process recommended by the Human Milk Bank Association of North America before distributing human donor milk, reduced milk adiponectin and insulin concentrations by 32.8% and 46.1%, respectively (both p<0.0001). In conclusion, the distribution of macronutrient intakes during pregnancy was associated with risk for abnormal glucose metabolism later in pregnancy. In addition, maternal prenatal metabolic abnormalities were associated with high insulin concentrations in mature milk, while only obstetrical parameters were associated adiponectin concentrations in early milk. Our findings support the need for continued work to determine optimal prenatal nutritional strategies to prevent GDM and subsequently to improve infant nutrition.

Substantial evidence supports a role of diet in glucose metabolism, but only a few reports have investigated the impact of diet during pregnancy on risk of gestational diabetes (GDM). Although metabolic hormones have been detected in milk, no studies have investigated the impact of maternal metabolic status assessed during pregnancy on insulin and adiponectin concentrations in human milk. The purpose of this thesis was to investigate the association of maternal macronutrient intakes with metabolic status during pregnancy and its subsequent impact on human milk hormones. Participating women (n=216) underwent a 3-hour oral glucose tolerance test at 30 (95% confidence interval [CI] 25, 33) weeks gestation, recalled their second trimester dietary intake, and donated early (the first week) and mature (3 months postpartum) milk. Higher vegetable and fruit fiber intake was associated with reduced insulin resistance (beta±SE -0.100±0.029, p=0.0008) and increased insulin sensitivity (0.029±0.012, p=0.01) among those with a family history of type 2 diabetes. Lower % carbohydrate and higher % total fat were associated with increased GDM risk (odds ratio 0.60 [95% CI 0.40, 0.90] and 1.61 [1.06, 2.44], respectively). Prenatal metabolic abnormalities including higher pregravid body mass index (beta±SE 0.053±0.014, p=0.0003), in addition to gravid hyperglycemia (0.218±0.087, p=0.01), insulin resistance (0.255±0.047, p<0.0001), lower insulin sensitivity (-0.521±0.108, p<0.0001), and higher serum adiponectin (0.116±0.029, p<0.0001) were associated with higher insulin in mature milk. Obstetrical measures including nulliparity (0.171±0.058, p=0.004), longer duration of gestation (0.546±0.146, p=0.0002), and unscheduled caesarean section (0.387±0.162, p=0.02) were associated with higher adiponectin in early milk. Holder pasteurization, a process recommended by the Human Milk Bank Association of North America before distributing human donor milk, reduced milk adiponectin and insulin concentrations by 32.8% and 46.1%, respectively (both p<0.0001). In conclusion, the distribution of macronutrient intakes during pregnancy was associated with risk for abnormal glucose metabolism later in pregnancy. In addition, maternal prenatal metabolic abnormalities were associated with high insulin concentrations in mature milk, while only obstetrical parameters were associated adiponectin concentrations in early milk. Our findings support the need for continued work to determine optimal prenatal nutritional strategies to prevent GDM and subsequently to improve infant nutrition.

Two experiments were conducted to examine the interrelationships between body composition at parturition, herbage allowance during lactation and milk production. Detailed measurements of feed intake and changes in body composition allowed estimates of maintenance energy requirement and efficiency of use of energy for milk production. Dorset ewes were offered low (L-) or high (H-) herbage allowances during pregnancy to achieve post-partum body weights, respectively, of about 50 and 60-65 kg. During the first 6 weeks of lactation herbage allowances were approximately 2 (-L), 5 (-M and -Mm) and 8 (-H) kg DM/ewe/d and feed intake was determined using chromic oxide dilution. In the first experiment (Expt.A) ewes were machine-milked and in the second (Expt.B) they were suckled by twin lambs with a further group (-Mm) machine-milked. Samples of ewes were slaughtered during early pregnancy, post-partum, and after 6 weeks of lactation. Body weight differences post-partum between L- and H- groups were 10 and 15 kg in Expts.A and B respectively. During lactation body weight changes appeared erratic, particularly in Expt.B, owing to the effects of variation in gut fill. Daily feed intake of machine-milked ewes on -M and -H allowances reached a maximum (1.9-2.2 kg OM/ewe/d) 2-3 weeks after parturition but ewes rearing lambs on similar allowances showed maximum intake (2.5-2.8 kg OM/ewe/d) during the first week. Mean intakes during lactation were 58, 69 and 73 g DOM/kg W•⁷⁵/d respectively for -L, -M and -H groups machine-milked and 51, 75 and 83 g DOM/kg W•⁷⁵/d respectively for -L, -M and -H groups rearing twin lambs. In both experiments ewes in L- groups had approximately 14% greater mean intake (g DOM/kg W•⁷⁵/d) during lactation compared with those in H- groups. Mean daily milk production of ewes rearing lambs was 2.0, 2.5 and 2.7 kg/d respectively in -L, -M and -H groups and was 33-52% greater than that for machine-milked ewes offered similar herbage allowances. In both experiments L- ewes had 14% greater milk yield (g/kg W•⁷⁵/d) compared with H- ewes. Mean lamb growth rates in Expt.B averaged 209, 254 and 268 g/lamb/d in -L, -M and -H groups, respectively. Lambs in the H- group were approximately 11% heavier at birth than those in the L- group and their mean growth rate was 20 g/d greater. Body fat and energy content of live ewes at the start of lactation was predicted using regression relationships from ewes slaughtered post-partum. There was large variation in the energy content of body weight loss during lactation (-37 to +140 MJ/kg) owing to variation in weight of gut fill and changes in chemical composition of the empty body. In Expt.A the body fat content of ewes during early pregnancy was 16.9 kg. Predicted values post-partum were 10.0 and 15.7 kg in L- and H- groups, respectively. During lactation all groups showed body fat losses which ranged from 53 (LH) to 120 g/d (HL). Body fat content during early pregnancy in Expt.B was slightly greater (19.0 kg) than in Expt.A and predicted values post-partum were 11.5 (L-) and 19.7 kg (H-). Fat mobilization during lactation was greater than in Expt.A, and ranged from 157 (LH) to 287 g/d (HL). In both experiments there were losses in body protein during pregnancy in L- ewes (5 and 15 g/d in Expts.A and B respectively). During lactation, protein losses were most evident in H- ewes, being greatest in HL groups (26 and 43 g/d in Expts.A and B respectively). Water: protein ratio in the empty body showed a progressive increase during pregnancy and lactation in both experiments. Estimated maintenance energy requirement for machine-milked ewes tended to be greater for H- (.236 MJ ME/kg W/d) compared with L- ewes (.205 MJ ME/kg W/d) but was similar for both groups of ewe rearing lambs (average of.238 MJ ME/kg W/d). Efficiency of use of ME above maintenance for milk production (K₁) in L- and H- groups was, respectively, .69 and .95 in machine-milked, and .69 and .64 in suckled ewes. Energy from mobilized body tissues was utilized for milk production with respective efficiencies in L- and H- ewes of .23 and .35 in machine-milked, and .40 and .50 in suckled ewes. Efficiency of conversion of total energy available (i.e. ME above maintenance and mobilized tissue energy) to milk energy (K₁(t)) when compared among all groups in both experiments, tended to decrease (from .84 to .51) with increasing body energy mobilization. There was a positive relationship, however, between K₁(t) and the proportion of mobilized energy derived from body protein. Metabolizable energy intakes in the present experiments appeared to be 10-15% greater than calculated requirements from ARC (1980). Estimates of ME requirements for lactating ewes at pasture, in relation to body weight, level of milk production and body energy change, have been calculated based on the present data.

[Truncated abstract] In women, as in other mammals, the ability to successfully lactate depends on both complete functional development of the mammary gland and the stimulation of the milk ejection reflex to enable the suckling young to remove stored milk. Prior to my studies, Sir Astley Cooper’s carried out the most comprehensive investigation of the gross anatomy of the lactating human breast in 1840. I have used ultrasound to image the anatomy of the breasts of fully breastfeeding women (1-6 months, n=22) with particular emphasis on the distribution of the main milk ducts, glandular and adipose tissue. Scanning of the milk duct system demonstrated that the anatomy in the region of the areola and nipple is different to that depicted in standard anatomical textbooks. The main milk ducts were small (diameter, left: 1.9 ± 0.6 mm; right: 2.1 ± 0.7 mm), superficial (depth, left: 4.50 ± 1.98 mm; right: 4.74 ± 1.59 mm) and branched close to the nipple (within 8.20 ± 6.27 mm, left; 7.00 ± 3.98 mm, right) (mean ± SD). The lactiferous sinuses (described in current textbooks) were not observed and the number of main ducts detected at the base of the nipple was less than the quoted 15-20 (9.4, range 4-18). Quantitative descriptions of the morphology of either the lactating or, indeed, the non-lactating breasts have not been attempted using ultrasound. I developed a systematic approach to ultrasound imaging of the breast that provided a semi-quantitative description of the distribution of glandular and adipose tissues within the lactating breast. Approximately two thirds of the breast was comprised of glandular tissue. Intraglandular fat was identified as hypoechoic transects within the hyperechoic glandular tissue. Over 65% of the glandular tissue together with 50% of the intraglandular fat and 25% of the subcutaneous fat was located within a 30 mm radius of the base of the nipple. The absence of lactiferous sinuses and the arrangement of tissue within a 30 mm radius of the nipple suggested that the current conceptualisation of sucking dynamics of the infant requires revision. Successful milk removal depends on the stimulation of the milk ejection reflex and currently subjective assessments of milk ejection such as the mother’s sensations and an alteration in the infants sucking and swallowing are used clinically to confirm milk ejection whereas in research two stressful invasive procedures; changes blood oxytocin and intra-ductal pressure have been used. I have developed a non-invasive ultrasound technique to detect milk ejection in women