No abstract

The role the mammary epithelial circadian clock plays in gland development and lactation is unknown. We hypothesized that mammary epithelial clocks function to regulate mammogenesis and lactogenesis, and propose the core clock transcription factor BMAL1:CLOCK regulates genes that control mammary epithelial development and milk synthesis. Our objective was to identify transcriptional targets of BMAL1 in undifferentiated (UNDIFF) and lactogen differentiated (DIFF) mammary epithelial cells (HC11) using ChIP-seq. Ensembl gene IDs with the nearest transcriptional start site to ChIP-seq peaks were explored as potential targets, and represented 846 protein coding genes common to UNDIFF and DIFF cells and 2773 unique to DIFF samples. Genes with overlapping peaks between samples (1343) enriched cell-cell adhesion, membrane transporters and lipid metabolism categories. To functionally verify targets, an HC11 line with Bmal1 gene knocked out (BMAL1-KO) using CRISPR-CAS was created. BMAL1-KO cultures had lower cell densities over an eight-day growth curve, which was associated with increased (p<0.05) levels of reactive oxygen species and lower expression of superoxide dismutase 3 (Sod3). RT-qPCR analysis also found lower expression of the putative targets, prolactin receptor (Prlr), Ppara, and beta-casein (Csn2). Findings support our hypothesis and highlight potential importance of clock in mammary development and substrate transport.

Circadian, metabolic, and reproductive systems are inter-regulated. Excessive fatness and circadian disruption alter normal physiology and the endocrine milieu, including cortisol, the primary stress hormone. Our aim was to determine the effect feeding a high fat diet to female ICR mice had on diurnal feeding pattern, weight gain, body composition, hair corticosterone levels and circadian patterns of fecal corticosterone. Prepubertal (~35d of age) ICR mice were assigned to control (CON; 10% fat) or high fat (HF; 60% fat) diet and fed for 4 wk to achieve obesity under 12h light and 12h of dark. Feed intake was measured twice daily to determine diurnal intake. Mice were weighed weekly. After 4 wk on diets hair was collected to measure corticosterone, crown-rump length was measured to calculate body mass index (BMI), and body composition was measured with EchoMRI to determine percent fat. HF mice weighed more (P<0.05) after week two, BMI and percent body fat was greater (P<0.05) in HF than CON at the end of wk 4. HF mice consumed more during the day (P<0.05) than CON mice after 1 week on diets. Hair corticosterone was higher in HF mice than in CON (P<0.05). Fecal circadian sampling over 48hr demonstrated that HF mice had elevated basal corticosterone, attenuated circadian rhythms, and a shift in amplitude. High fat feeding for diet induced obesity alters circadian eating pattern and corticosterone rhythms, indicating a need to consider the impact of circadian system disruption on reproductive competence.

Survival, feed efficiency, growth and fertility of swine are dependent on colostrum intake in the first 24 h after birth. This study determined the effects of three doses of a homogeneous colostrum sample on 24 h body weight, rectal temperature (RT), immunocrit, and growth and survival to postnatal day (PND) 7. Three female piglets were selected from 8 litters (n = 24 piglets) at birth, removed from their litter, and bottle-fed 10% (COL10, n = 8), 15% (COL15, n = 8) or 20% (COL20, n = 8) colostrum based on birth weight over 12 bottle feedings every 2 h. At 24 h, piglets were weighed, RT recorded and blood was collected to measure immunocrit. Piglets were returned to litter of origin and weight was measured daily until PND 7. Colostrum dose had an overall effect on weight gain at 24 h, RT, immunocrit, and growth to PND 7 (P&lt;0.05). Piglets in the 20% BrW colostrum group had greater weight gain, RT and immunocrit at 24 h than COL10 piglets (P&lt;0.05), but these variables were not different between COL15 and the other treatments. Despite no difference in average daily gain after being returned to their litters, the greater weight (P&lt;0.05) in COL20 compared to COL10 and COL15 was sustained over 7 days. Seven piglets in each treatment survived to PND 7. This model using standardized doses of a homogeneous colostrum sample enables controlled studies aimed at understanding the role of 24 h colostrum intake on piglet development.

Low colostrum intake relates to poorer health and infertility in swine. We previously connected vaginal lipid profiles at weaning to fertility of sows. We hypothesized vaginal lipidome varied with colostrum intake. Our objective was to determine whether indicators of colostrum intake, immunocrit (IM) and weight gain 24 h postnatal (PN), related to vaginal lipids at d21 PN. Gilts (n=60) were weighed and blood sampled to measure IM. On d21 PN vaginal swabs were taken and lipids measured using multiple reaction monitoring. Abundance of multiple lipids differed (P&lt;0.05) between gilts categorized as high versus low IM and high versus low 24 h gain. The abundance of multiple lipids correlated with IM and 24 h gain. Phosphatidylcholine PC(36:3), PC(36:2), and arachidonic acid (C20:4) positively (P&lt;0.05) correlated with IM. The ether lipid PCo(38:6) and multiple cholesteryl esters negatively (P&lt;0.05) correlated with IM. ROC analysis indicated arachidonic acid and docosanoic acid (C22:0) may serve as excellent biomarkers that distinguish between high and low IM. Similar to gilts found to be infertile, lipid profiles of low colostrum intake animals had greater abundance of very long chain fatty acids, lipids with high levels of unsaturation, and cholesteryl esters, which are metabolized in peroxisomes indicating their potential dysfunction.

Circadian clock disruption decreased mammary development and impaired lactation in late gestation cows and mice. Transcriptional targets of the circadian clock genes BMAL1 and CLOCK include the cell-cell and cell-ECM proteins e-cadherin (CDH1) and zonula occludens-1 (ZO-1). The polarization and lumen formation of milk-producing acini is dependent on these junctional proteins. We hypothesized that if BMAL1 and CLOCK were disrupted, mammary epithelial cells (HC11) will have a reduced ability to form acini. Our objective was to measure the effect of BMAL1 gene deletion (BMAL1-KO) and CLOCK protein reduction (shCLOCK) in HC11 cells on the formation of acini and expression of CDH1 and ZO-1 in three-dimensional (3D) cultures. Cells were plated on Matrigel, embedded, and cultured to create 3D structures. ImageJ software was used to quantify the acini and found that the BMAL1-KO and shCLOCK lines formed fewer and smaller acini (p < 0.05). Confocal microscopy revealed that CDH1 was located along lateral membranes and ZO-1 was apically located in all three lines. Levels of CDH1 and ZO-1 were greater (p < 0.05) per unit volume of cell in the shCLOCK cell line. Live/dead staining of cells found little to no cell death across three lines. Transmission electron microscopy (TEM) of acini indicated less polarization of cells within BMAL1-KO and shCLOCK acini. Polarization of cells is required for formation of 3D structures and requires coordination of temporal-spatial cues. Disruption of circadian clocks results disturbs temporal organization of cellular processes and causes a reduced ability of cells to polarize and form acini.

Colostrum intake potentiates piglet survival by transfer of immune factors. Additionally, colostrum also contains nutritional and bioactive molecules which could promote piglet’s growth and development. The objective of this study was to determine the effect of colostrum intake amount during the first 24h on plasma insulin, glucose, protein, and amino acid (AA) concentrations. Two female piglets were selected from 8 litters (n = 16) at birth and fed 10% (COL10) or 20% (COL20) colostrum based on birth weight over 12 bottle feedings occurring every 2h. Colostrum was collected from multiple sows and mixed to create a homogeneous pool. At birth and 24h, blood sample were collected, plasma separated and used to measure insulin, glucose, total protein, and AA concentrations. Analysis of the variance (PROC MIXED, SAS v.9.4) was used to evaluate significant (P≤0.05) differences between colostrum dose and time (birth vs. 24h). Colostrum intake did not influence plasma insulin at 24h (P=0.54). Glucose was higher at 24h (P&lt; 0.001), but did not differ between COL10 and COL20 piglets (P=0.74). Total protein was greater at 24h (P&lt; 0.001) as well as in COL20 compared to COL10 (P=0.006). Non-essential AAs (Ala, Asn, Asp, Cys, Gly and Glu) were reduced at 24h compared to birth (P&lt; 0.03), while essential AAs (His, Ile, Met, Phe, Trp and Val) were increased after 24h of colostrum feeding (P&lt; 0.05). At 24h, COL20 piglets had greater Ile and Met (P&lt; 0.03) and tended (P=0.08) to have greater Leu and Phe. In conclusion, colostrum intake increases glycemia as well as total circulating proteins, and the dose of colostrum is related with greater circulation of total protein and essential AAs.

The first milk sows synthesize is colostrum, and is only available to the neonate right after birth. Colostrum provides immunity, nutrients, energy, and bioactive factors which are essential for the survival, growth, and development of newborn piglets. The first few days after birth the gastrointestinal (GI) tract undergoes developmental changes and rapid growth in response to bioactive factors in milk. We hypothesized that amount of colostrum a neonate consumes the first 24 h postnatal effects the development of the GI tract. The objectives of this study were to measure the histomorphic growth of the jejunum between birth (day 0, D0) and postnatal day 7 (D7) and to determine the effect of ingesting a high versus low amount of colostrum. Gilts were identified at birth and immediately euthanized (D0, n = 6) or bottle fed a 24 h colostrum dose of 10% (COL10, n = 7) or 20% (COL20, n = 7) of birth body weight. Colostrum fed neonates were returned to birth sows after 24 h and allowed to nurse naturally until postnatal (D7), when gilts were euthanized. Gilts were dissected and jejunum was removed, weighed and placed in buffered formalin for preparation of histological sections. Five µm sections were mounted on glass slides and stained with hematoxylin and eosin (H&E). Images were captured using light microscopy at 10X magnification. ImageJ was used to measure villi length, width, stromal area, epithelial area, and crypt length. T-test analysis indicated that there was no difference between COL10 and COL20 in any of the morphological features (P &gt; 0.05), however between D0 and D7 villi width, epithelial area, and crypt length increased (P &lt; 0.05). Differences in histomorphology across the first week postnatal was not affected by level of colostrum intake in the first 24 h postnatal.