Species-specific cell-matrix interactions are essential for differentiation of alveoli like structures and milk gene expression in primary mammary cells of the Cape fur seal (Arctocephalus pusillus pusillus)

Few models are in place for analysis of extreme lactation patterns such as that of the fur seals which are capable of extended down regulation of milk production in the absence of involution. During a 10–12 month lactation period, female fur seals suckle pups on shore for 2–3 days, and then undertake long foraging trips at sea for up to 28 days, resulting in the longest intersuckling bouts recorded. During this time the mammary gland down regulates milk production. We have induced Cape fur seal (Arctocephalus pusillus pusillus) mammary cells in vitro to form mammospheres up to 900 μm in diameter, larger than any of their mammalian counterparts. Mammosphere lumens were shown to form via apoptosis and cells comprising the cellular boundary stained vimentin positive. The Cape fur seal GAPDH gene was cloned and used in RT-PCR as a normalization tool to examine comparative expression of milk protein genes (αS2-casein, β-lactoglobulin and lysozyme C) which were prolactin responsive. Cape fur seal mammary cells were found to be unique; they did not require Matrigel for rapid mammosphere formation and instead deposited their own matrix within 2 days of culture. When grown on Matrigel, cells exhibited branching/stellate morphogenesis highlighting the species-specific nature of cell–matrix interactions during morphological differentiation. Matrix produced in vitro by cells did not support formation of human breast cancer cell line, PMC42 mammospheres. This novel model system will help define the molecular pathways controlling the regulation of milk protein expression and species specific requirements of the extracellular matrix in the cape fur seal.

Variation in maternal provisioning by lactating Antarctic fur seals: response to experimental manipulation in pup demand

An experiment involving the supplementary feeding of pups was conducted on Antarctic fur seals to investigate the factors influencing maternal foraging-attendance cycles and the differential use of nutritional resources for growth, maintenance and storage by pups. For 40% of the lactation period, male pups were given a supplement mimicking the chemical composition of Antarctic fur seal milk at a dose equivalent to 35% of the normal mass-specific milk energy intake for the species. Milk consumption, body composition and growth rates were monitored during and after the supplementary feeding period and maternal foraging-attendance cycles were monitored throughout lactation. During the supplementary feeding period, treatment pups (n=8) grew 32% faster and deposited greater adipose tissue stores than controls (n=8) but consumed the same amount of maternal-delivered milk. When supplementary feeding was stopped (timed to coincide with peak maternal milk yield in this species), treatment pups lost mass whereas control group pups continued to grow. Treatment pups weaned at a younger age (109 days) than control pups (116 days) but at the same mass (13 kg). Maternal attendance durations did not differ between the treatment and control groups throughout lactation. However, mothers of treatment pups had significantly shorter foraging trip durations (3.74 days) than mothers of control pups (4.74 days) during the period of supplementary feeding (there were no significant differences throughout the rest of lactation). These findings are in accordance with predictions of a marginal-value model of fur seal lactation behaviour.

Historical dynamics of the fur seal population : evidence of regulation by man?

The Australian fur seal (Arctocephalus pusillus doriferus) was severely over-exploited in the 18th and 19th centuries and until relatively recently its population had remained steady at well below estimated presealing levels. However, the population is now increasing rapidly (6%–20% per annum) throughout its range and there is a need to understand its dynamics in order to assess the potential extent and impact of interactions with fisheries. Age distribution (n = 156) and pregnancy rate (n = 110) were determined for adult females collected at a breeding colony on Seal Rocks, southeast Australia, in 1971–1972. Mean ± SE and maximum observed ages were 9.37 ± 0.41 and 20 years (n = 1), respectively. A stochastic modelling approach was used to fit an age distribution to the observed age-structure data and calculate rates of recruitment and adult survival. Annual adult female survival and recruitment rates between 1954 and 1971 were 0.478 ± 0.029 (mean ± SE) and 0.121 ± 0.007, respectively, suggesting that the population was experiencing a decline during the 1960s. The pregnancy rate increased from 78% at 3 years of age to an average of 85% between 4–13 years of age before significantly decreasing in older females (the oldest was 19 years of age). There was no significant effect of body mass or condition on the probability of a female being pregnant (P > 0.5 in both cases) and the nutritional burden of lactation did not appear to affect pregnancy rates or gestational performance. These findings suggest that the low survivorship was due to density-independent effects such as mortality resulting from interactions with fishers, which are known to have been common at the time. The recent increase in the population is consistent with anecdotal evidence that such interactions have decreased as fishing practices have changed.

Milk consumption, body composition and pre-weaning growth rates of Australian fur seal

Pre-weaning growth rates, body composition, milk consumption and mass gain efficiency were measured in Australian fur seal Arctocephalus pusillus doriferus pups born in two consecutive breeding periods. Australian fur seals have the highest birth mass of any fur seal species (male 8.3 kg; female 7.2 kg). While their absolute pre-weaning growth rate (male 62 g·day⁻¹; female 53 g·day⁻¹) is similar to that of other temperate latitude fur seals, they have the longest birth-mass doubling time of any otariid species (134–136 days). Daily milk consumption increased from 400 g·day−1 (5 MJ·day−1) after birth to 675 g·day⁻¹ (13.7 MJ·day⁻¹) at age 210 day. However, mean mass-specific milk consumption (41 g·kg⁻¹) is substantially lower than in other otariid species (58–70 g·kg⁻¹) and, combined with a low mass gain efficiency (0.12 g·g⁻¹), contributes to the low mass-specific growth rates observed. There were no significant differences in either absolute or mass-specific milk consumption between the sexes. Significant differences, however, were found between the sexes in the body composition of pups with females generally having larger body lipid stores than males for any given mass. Peak milk yield by Australian fur seal females is estimated at 0.60 MJkg−0.75, substantially less than in Antarctic fur seals. The low level of maternal energy transfer in Australian fur seals may reflect the relatively low marine productivity of their foraging areas.

Dive behaviour, foraging locations and maternal-attendance patterns in Australian fur seals (Arctocephalus pusillus doriferus)

The dive behaviour, foraging locations, and colony-attendance patterns of female Australian fur seals (Arctocephalus pusillus doriferus) from Kanowna Island (39°10'S, 146°18'E) in Bass Strait, southeastern Australia, were determined throughout lactation during 1997–1999. Foraging-trip durations increased as lactation progressed, being shortest in summer (3.71 ± 0.24 days; mean ± 1 SE) and longest in winter (6.77 ± 0.57 days, P < 0.05), but maternal-attendance periods did not differ in duration (1.70 ± 0.10 days, P > 0.5). Individual mean attendance periods and trip durations were positively correlated (r² = 0.21, P < 0.005). Diving commenced shortly after seals left the colony (2.6 ± 0.4 h), was continuous for long periods (up to 36 h), occurred mostly during daylight hours, and lacked regular diel variation in depth. The majority of dives (78%) were typically U-shaped and reached depths corresponding to the prevailing depths in Bass Strait (65–85 m), indicating that these animals forage mostly on the benthos of the shallow continental shelf in this region. Such behaviour is unusual for fur seals but is reminiscent of that of some sea lion species. Mean dive durations varied between 2.0 and 3.7 min (maximum 8.9 min) and the theoretical aerobic dive limit (3.91–4.26 min) was exceeded on 17.3% of dives. Dive frequency (8.3 ± 0.6/h) and the proportion of time at sea spent diving (40.7 ± 2.1%) were weakly negatively related to the duration of the foraging trip (r²= 0.07, P < 0.004, and r² = 0.13, P < 0.0001, respectively). Data from at-sea locations showed that lactating females forage almost exclusively within Bass Strait during all seasons.

Temporal structure of diving behaviour in sympatric Antarctic and Subantarctic fur seals

Lactation is considerably briefer (4 vs. 10 mo) and daily pup energy expenditure higher in Antarctic (AFS) than in subantarctic fur seals (SFS), even in sympatric populations of both species, where their foraging locations and diets are similar. Therefore, lactational demands may be higher for AFS females. We investigated whether sympatric lactating AFS and SFS females differ in their physiological or behavioural diving capacities, and in the temporal structure of foraging behaviour. Mean dive depth and duration were greater in SFS, but dives below 140 m were performed only by AFS. An index of activity level during the bottom phase of dives, when fur seals are thought to capture prey, was higher in SFS. Despite these differences, SFS females showed a steady increase in the minimum postdive interval following dives lasting longer than 250 s, compared to the steady increase following dives lasting longer than only 150 s in AFS. These results suggest that physiological constraints on diving behaviour are stronger on AFS females, and that the behavioural aerobic dive limit is greater for SFS. Assuming that dive bouts reflect foraging in prey patches, AFS females exploited more patches per unit time, and remained in them for briefer periods of time, compared to SFS females. Dive bout structure did not differ between overnight and long foraging trips. Our data suggest that AFS females spend greater foraging effort, but may gain access to prey patches of better quality, which may help them cope with higher lactational demands.

Copper is taken up efficiently from albumin and α2-macroglobulin by cultured human cells by more than one mechanism

Ionic copper entering blood plasma binds tightly to albumin and the macroglobulin transcuprein. It then goes primarily to the liver and kidney except in lactation, where a large portion goes directly to the mammary gland. Little is known about how this copper is taken up from these plasma proteins. To examine this, the kinetics of uptake from purified human  albumin and α2-macroglobulin, and the effects of inhibitors, were measured using human hepatic (HepG2) and mammary epithelial (PMC42) cell lines. At physiological concentrations (3–6 µM), both cell types took up copper from these proteins independently and at rates similar to each other and to those for Cu-dihistidine or Cu-nitrilotriacetate (NTA). Uptakes from   α2-macroglobulin indicated a single saturable system in each cell type, but with different kinetics, and 65–80% inhibition by Ag(I) in HepG2 cells but not PMC42 cells. Uptake kinetics for Cu-albumin were more complex and also differed with cell type (as was the case for Cu-histidine and NTA), and there was little or no inhibition by Ag(I). High Fe(II) concentrations (100–500 µM) inhibited copper uptake from albumin by 20–30% in both cell types and that from {alpha}2-macroglobulin by 0–30%, and there was no inhibition of the latter by Mn(II) or Zn(II). We conclude that the proteins mainly responsible for the plasma-exchangeable copper pool deliver the metal to mammalian cells efficiently and by several different mechanisms.α2-Macroglobulin delivers it primarily to copper transporter 1 in hepatic cells but not mammary epithelial cells, and additional as-yet-unidentified copper transporters or systems for uptake from these proteins remain to be identified.

Lack of functional alpha-lactalbumin prevents involution in Cape fur seals and identifies the protein as an apoptotic milk factor in mammary gland involution

The mammary gland undergoes a sophisticated programme of developmental changes during pregnancy/lactation. However, little is known about processes involving initiation of apoptosis at involution following weaning. We used fur seals as models to study the molecular process of involution as these animals display a unique mammary gland phenotype. Fur seals have long lactation periods whereby mothers cycle between secreting copious quantities of milk for 2 to 3 days suckling pups on land, with trips to sea alone to forage for up to 23 days during which time mammary glands remain active without initiating apoptosis/involution.

Support needs of parents of sick and/or preterm infants admitted to a neonatal unit

Background: The sudden unexpected delivery of a preterm infant is a shock to parents with studies confirming the importance of providing them with nursing support. The purpose of this descriptive study was to identify the type and level of support that nurses provided to the parents of sick and/or preterm infants. It also investigated whether parents were satisfied with the support provided by nursing staff.
Method: A convenience sample of 112 parents in an Australian tertiary neonatal unit were invited to complete the ‘Nurse Parent Support Tool’ (NPST), which is a 21-item questionnaire. The NPST consists of four domains: emotional, informational, appraisal and instrumental support. A second tool was used to assess parents’ satisfaction with the nurse to parent support.
Results: Instrumental support had the highest mean score for both nurse to parent support at 4.51 (out of 5) and 4.36 (out of 5) for satisfaction. The mean score for nursing support was 4.21 followed by parental satisfaction with nurse to parent support which was 4.16. In contrast, emotional support had the lowest mean score for both nurse to parent support at 3.94 and 3.97 for satisfaction. Parents indicated that they needed further support with lactation, breastfeeding and assistance with parenting skills.
Conclusion: Overall, the results from the study indicated that parents’ perception of nursing support was positive and that parents were highly satisfied with the nursing support provided in the neonatal unit. However, some attention needs to be given to providing more support and information on breastfeeding and parenting skills.

Bovine Muc1 is a highly polymorphic gene encoding an extensively glycosylated mucin that binds bacteria

The bovine Muc1 protein is synthesized by mammary epithelial cells and shed into milk as an integral component of the milk fat globule membrane; however, the structure and functions of this mucin, particularly in relation to lactation, are poorly defined. The objectives of this investigation were to investigate the Muc1 gene and protein structures in the context of lactation and to test the hypothesis that Muc1 has a role in innate immune defense. Polymerase chain reaction analysis of genomic DNA from 630 cattle revealed extensive polymorphism in the variable number of tandem repeats (VNTR) in the bovine Muc1 gene. Nine allelic
variants spanning 7 to 23 VNTR units, each encoding 20 AA, were identified. Three alleles, containing 11, 14, and 16 VNTR units, respectively, were predominant. In addition, a polymorphism in one of the VNTR units has the potential to introduce a unique site for N-linked glycosylation. Statistical analysis indicated weak associations between the VNTR alleles and milk protein and fat percentages in a progeny-tested population of Holstein-Friesian dairy cattle. No association with somatic cell count could be demonstrated. Bovine Muc1 was purified from milk fat globule membranes and characterized. The protein was highly glycosylated, primarily with O-linked sialylated T-antigen [Neu5Ac(α2–3)-Gal(β1–3)-GalNAcα1] and, to a lesser extent, with N-linked oligosaccharides, which together accounted for approximately 60% of the apparent mass of Muc1. Purified bovine Muc1 directly bound fluorescently labeled Escherichia coli BioParticles (Invitrogen, Mount Waverley, Australia) and inhibited their binding to bovine mammary epithelial cells grown in vitro.

Support needs of parents of sick and/or preterm infants admitted to a neonatal unit

The sudden unexpected delivery of a preterm infant elicits many emotional responses from parents, with studies confirming the importance of providing parents with nursing support. The purpose of this study was to measure parents' perception of the type and level of support that nurses provided in an Australian neonatal unit, and to determine the extent to which parents were satisfied with the support currently provided. A convenience sample of 112 parents in an Australian tertiary neonatal unit were invited to complete the nurse parent support tool (NPST), which is a 21-item questionnaire used to measure the level of nursing support. The NPST consists of four domains -emotional, informational, appraisal and instrumental support. A second tool was used to assess parent satisfaction with nurse-to-parent support. A total of 62 parents completed the NPST and satisfaction tool.

Results showed that instrumental support had the highest mean score for both nurse-to-parent support 4.51 (out of 5) and 4.36 for satisfaction. The mean score for nursing support was 4.21 followed by satisfaction with nurse-to-parent support at 4.16. In contrast, emotional support had the lowest mean score for both nurse-to-parent support at 3.94 and 3.97 for satisfaction. Parents indicated that they needed further support with lactation, breastfeeding and assistance with parenting skills. Overall, results indicated that parent perception of nursing support was positive and that parents were highly satisfied with the nursing support provided in the neonatal unit. However, some attention needs to be given to providing more support and information on breastfeeding and parenting skills.

No evidence of expression of two classes of natural antibiotics (cathelicidins and defensins) in a sample of platypus milk

Marsupial neonates are born without a fully functioning immune system, and are known to be protected in part by natural antimicrobial peptides present in their mother's milk. Monotreme neonates hatch at a similar stage in development, and it has been hypothesised that their survival in a non-sterile burrow also relies on the presence of natural antibiotics in their mother's milk. Here we review the field of monotreme lactation and the antimicrobial peptide complement of the platypus (Ornithorhynchus anatinus). Using reverse transcriptasepolymerase chain reaction of milk cell RNA from a sample of platypus milk, we found no evidence for the expression of cathelicidins or defensins in the milk. This was unexpected. We hypothesise that these natural antibiotics may instead be produced by the young platypuses themselves.

A novel approach identified the FOLR1 gene, a putative regulator of milk protein synthesis

This study has utilised comparative functional genomics to exploit animal models with extreme adaptation to lactation to identify candidate genes that specifically regulate protein synthesis in the cow mammary gland. Increasing milk protein production is valuable to the dairy industry. The lactation strategies of both the Cape fur seal (Artocephalus pusillus pusillus) and the tammar wallaby (Macropus eugenii) include periods of high rates of milk protein synthesis during an established lactation and therefore offer unique models to target genes that specifically regulate milk protein synthesis. Global changes in mammary gene expression in the Cape fur seal, tammar wallaby, and the cow (Bos taurus) were assessed using microarray analysis. The folate receptor α (FOLR1) showed the greatest change in gene expression in all three species [cow 12.7-fold (n = 3), fur seal 15.4-fold (n = 1), tammar 2.4-fold (n = 4)] at periods of increased milk protein production. This compliments previous reports that folate is important for milk protein synthesis and suggests FOLR1 may be a key regulatory point of folate metabolism for milk protein synthesis within mammary epithelial cells (lactocytes). These data may have important implications for the dairy industry to develop strategies to increase milk protein production in cows. This study illustrates the potential of comparative genomics to target genes of interest to the scientific community.

Characterisation of monotreme caseins reveals lineage-specific expansion of an ancestral casein locus in mammals

Using a milk-cell cDNA sequencing approach we characterised milk-protein sequences from two monotreme species, platypus (Ornithorhynchus anatinus) and echidna (Tachyglossus aculeatus) and found a full set of caseins and casein variants. The genomic organisation of the platypus casein locus is compared with other mammalian genomes, including the marsupial opossum and several eutherians. Physical linkage of casein genes has been seen in the casein loci of all mammalian genomes examined and we confirm that this is also observed in platypus. However, we show that a recent duplication of β-casein occurred in the monotreme lineage, as opposed to more ancient duplications of α-casein in the eutherian lineage, while marsupials possess only single copies of α- and β-caseins. Despite this variability, the close proximity of the main α- and β-casein genes in an inverted tail-tail orientation and the relative orientation of the more distant kappa-casein genes are similar in all mammalian genome sequences so far available. Overall, the conservation of the genomic organisation of the caseins indicates the early, pre-monotreme development of the fundamental role of caseins during lactation. In contrast, the lineage-specific gene duplications that have occurred within the casein locus of monotremes and eutherians but not marsupials, which may have lost part of the ancestral casein locus, emphasises the independent selection on milk provision strategies to the young, most likely linked to different developmental strategies. The monotremes therefore provide insight into the ancestral drivers for lactation and how these have adapted in different lineages.

Influences on fecundity in Australian fur seals (Arctocephalus pusillus doriferus)

Recovery of the Australian fur seal (Arctocephalus pusillus doriferus) population has been slower than that of other fur seals, perhaps due to nutritional constraints on fecundity. However, the population doubled in recent decades, suggesting that reproductive rates may have changed. Pregnancy rates and birthrates were estimated and the influences of maternal age, size, condition, and lactation status were investigated over 3 consecutive breeding seasons (2003–2005). Mid-gestation pregnancy rate, estimated from plasma progesterone concentrations, was 84% ± 3% (mean ± SE) and was negatively influenced only by age (P< 0.001). Birthrate, estimated from observations of females with newborn offspring during the breeding season, was 53% 6 3% (mean ± SE) and was influenced negatively by lactation status (P = 0.035). The difference between pregnancy rates and birthrates suggests that a high degree of late-gestation abortion occurs, especially in younger females. Lactation status was not influenced by any variables, and the high rate (86% ± 2%; mean ± SE) suggests that many offspring are suckled beyond the typical weaning period. Although relationships between reproductive rates and body condition were not found, the results are consistent with a nutritional stress hypothesis. The recent increase in population growth is not due to increased fecundity, which remains low compared to other otariids. Paradoxically, high pregnancy rates were observed in young females (3 years), suggesting that rapid maturity is adaptive for benthic foragers.