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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.

Circadian disruption increased insulin resistance and decreased mammary development in late gestation, nonlactating (dry) cows. The objective was to measure the effect of circadian disruption on transcriptomes of the liver and mammary gland. At 35 days before expected calving (BEC), multiparous dry cows were assigned to either control (CON) or phase-shifted treatments (PS). CON was exposed to 16-h light and 8-h dark. PS was exposed to 16-h light to 8-h dark, but phase of the light-dark cycle was shifted 6 h every 3 days. On day 21 BEC, liver and mammary were biopsied. RNA was isolated ( n = 6 CON, n = 6 PS per tissue), and libraries were prepared and sequenced using paired-end reads. Reads mapping to bovine genome averaged 27 ± 2 million and aligned to 14,222 protein-coding genes in liver and 15,480 in mammary analysis. In the liver, 834 genes, and in the mammary gland, 862 genes were different (nominal P < 0.05) between PS and CON. In the liver, genes upregulated in PS functioned in cholesterol biosynthesis, endoplasmic reticulum stress, wound healing, and inflammation. Genes downregulated in liver function in cholesterol efflux. In the mammary gland, genes upregulated functioned in mRNA processing and transcription and downregulated genes encoded extracellular matrix proteins and proteases, cathepsins and lysosomal proteases, lipid transporters, and regulated oxidative phosphorylation. Increased cholesterol synthesis and decreased efflux suggest that circadian disruption potentially increases the risk of fatty liver in cows. Decreased remodeling and lipid transport in mammary may decrease milk production capacity during lactation.

Milk is an easily digestible source of nutrients and bioactive factors, its composition reflects the neonate’s needs, and changes from colostrum to transitional and mature milk. Our objective was to measure milk fat, lactose, total carbohydrate, and protein content in parallel with global proteome of homogenate milk samples to characterize changes across the three phases of swine lactation. Milk samples were collected from multiparous sows (n = 9) on postnatal day 0 (D0; colostrum), 3 (D3; early transitional), 7 (D7; late transitional), and 14 (D14; mature). On D3, percent fat (16 ± 2.1) and lactose (3.8 ± 0.3) were higher (P < 0.05) than on D0 (10 ± 3.9 and 1.5 ± 0.3, respectively). Levels of fat and lactose were not different between D3 and D14. Percent total protein decreased (P < 0.05) between D0 (11 ± 2.1) and D3 (5 ± 0.7), but there was no significant change in percent protein between D3 and D14. Total carbohydrates increased (P < 0.05) between D3 (944 ± 353 µg/mL) and D14 (1,150 ± 462 µg/mL). Quantitative proteomic analysis using liquid chromatography tandem mass spectrometry (LC-MS/MS) of homogenate D0, D3, and D14 milk samples (n = 6) identified 772 protein groups which corresponded to 501 individual protein-coding genes. A total of 207 high confidence proteins were detected in n = 3 sows/day. Of the high confidence proteins, 81 proteins were common among all 3 days of lactation. Among the proteins that decreased between the days (false discovery rate; FDR < 0.05) were multiple apolipoproteins and XDH which decreased between D0 to D3. Proteins that increased across the days (FDR < 0.05) were complement factors and 14-3-3 proteins (YWHAQ, YWHAE). Our data provide a good characterization of milk proteome changes that likely reflect mammary function as well as the neonate’s phase-specific developmental needs. This data may be useful in developing approaches to enhance the health and welfare of swine.

Perinatal nutrition affects future milk production. The number of mammary epithelial cells affect milk production capacity. Therefore, it was hypothesized that the level of colostrum intake affects the proliferation rate and the total number of mammary epithelial cells in the gland. The ratio of newly synthesized protein to newly synthesized DNA reflects the relative amount of cellular differentiation to cell division. The study objective was to determine the relationship between the level of colostrum intake and 24 h-level of circulating amino acid, glucose and insulin with mammary parenchyma histological features, cell division and protein synthesis over the first week postnatal. One of two standardized doses of a homogenate colostrum sample, 10% (n = 8) and 20% (n = 8) of birth bodyweight, was fed to gilts over the first 24 h postnatal. Gilts were administered deuterium oxide immediately after birth and daily to label newly synthesized DNA and proteins. Gilts were euthanized on postnatal day seven, and DNA and protein were isolated from mammary parenchyma. DNA and protein fractional synthesis (f) and fractional synthetic rate (FSR) were calculated using mass isotopomer distribution analysis. The ratio of protein f and FSR to DNA f and FSR were calculated and used to indicate the relative amounts of differentiation to cell division. Mammary morphological development was also analyzed by measuring the parenchymal epithelial area and the stromal and epithelial proliferation index on postnatal day seven. Colostrum dose was not related to any of the variables used to evaluate mammary development. However, plasma lysine levels at 24 h postnatal were positively related to average daily gain (ADG, r = 0.54, p = 0.05), DNA f (r = 0.57, p = 0.03) and DNA FSR (r = 0.57, p = 0.03) in mammary parenchyma. Plasma lysine was inversely related to the ratio of protein to DNA f and FSR (r = −0.56, p = 0.04). ADG was related to the parenchymal epithelial area and DNA and protein f and FSR (p &lt, 0.05). These relationships support the idea that the nutritional environment affects early mammary development and that higher lysine levels in the perinatal period favored a greater degree of cell division versus differentiation in mammary of neonatal pigs and thus, warrant further investigations.

Swine colostrum intake within the first 24 hours after birth plays a large role in determining survivability, feed efficiency, growth, and fertility. This study tested the effects of feeding three doses of a homogeneous colostrum sample on BW over 24h, RT, immunocrit, and growth through PND7. Three female piglets were selected from eight litters at birth (n = 24 piglets total; n = 3/liter), and bottle-fed 10% (COL10, n = 8), 15% (COL15, n = 8) or 20% (COL20, n = 8) BW colostrum over 24h. Piglet birth weights were similar between treatments (P = 0.838). Piglets were weighed, RT recorded and immunocrit measured at 24h postnatal, then returned to their litter of origin. BW was recorded daily through PND7. At 24h, COL20 had an ADG of 136.3g, COL15 86.3g, and COL10 8.8g, and an average RT of 39.30⁰C, 38.84⁰C and 38.81⁰C respectively. Immunocrit levels varied from 0.028, 0.024 and 0.017 at 24h. SAS MIXED procedure was used to perform statistical analysis. Post-hoc analysis was performed using Tukey’s test. Colostrum dose had an effect on 24h weight gain (P < 0.001), RT (P = 0.007), immunocrit (P = 0.026), and growth through PND7 (P < 0.05). Treatment, day and interaction were determined as main effects of BW and ADG through PND7. Post-hoc analysis showed COL20 had higher ADG (136.3±54.8g), RT (39.30±0.11°C) and immunocrit (0.028±0.010) at 24h than COL10 (ADG 8.8±24.9g, RT 38.81±0.26°C, immunocrit 0.017±0.004). COL15 ADG (86.3±85.2g) differed from COL10 (P < 0.001), and RT (38.84±0.45) differed from COL20 (P = 0.017). There was no statistical difference in ADG between groups through PND7 (P = 0.874). One piglet within each treatment was crushed before PND7. This model permits controlled studies intended to understand the level of 24h colostrum intake on piglet growth and development using standardized, homogenous colostrum doses.

Metabolic, circadian, sleep, and reproductive systems are integrated and reciprocally regulated, but the understanding of the mechanism is limited. To study this integrated regulation, the circadian timing system was disrupted by exposing late pregnant nonlactating (dry) cows to chronic shifts in the light-dark phase, and rhythms of body temperature and circulating cortisol (CORT), progesterone (P4), serotonin (5HT), melatonin (MEL), and growth hormone (GH) concentrations were measured. Specifically, across 2 identical studies (1 and 2), at 35 d before expected calving (BEC) multiparous cows were assigned to control (CON; n = 24) and exposed to 16 h light and 8 h dark or phase shift (PS; n = 24) treatments and exposed to 6-h light-dark phase shifts every 3 d until parturition. All cows were exposed to control lighting after calving. Blood samples were collected in the first study at 0600 h on d 35 BEC, d 21 BEC, and 2 d before calving, and d 0, 2, 9, 15, and 22 postpartum (PP). A subset of cows (n = 6/group) in study 1 was blood sampled every 4 h over 48 h beginning on d 23 BEC, 9 BEC, and 5 PP. Body temperature was measured every 30 min (n = 8-16/treatment) for 48 h at 23 BEC and 9 BEC in both studies; and at 14 PP and 60 PP only in study 2. Treatment did not affect levels of CORT, GH, or P4 at 0600 h, but overall level of 5HT was lower and MEL higher in PS cows across days sampled. A 2-component versus single-component cosinor model better described [coefficient of determination (R

Milk fats are vital to neonate survival and development, but vary highly by diet, maternal metabolic state and stage of lactation. To gain a better understanding of changes in lipid composition of sow milk across lactation, milk was collected from nine multiparous sows on days 0, 3, 7, and 14, relative to birth and lipids were profiled using multiple reaction monitoring (MRM) profiling. Percent fat was determined by creamatocrit, and found to be different (P

Historically, sows have been induced to farrow using prostaglandin followed by an injection of oxytocin 24 h later. Benefits of induction can include decreased rate of stillbirths, dystocia, and postnatal mortality along with increasing the likelihood of farrowings being attended. Several studies have indicated that oxytocin administration may negatively impact fetal oxygen supply during parturition, potentially from umbilical cords breaking prior to birth, resulting in increased preweaning mortality. Therefore, the objective of this study was to determine if various induction protocols impact umbilical cord breakage and fetal blood parameters at birth. Fifty-eight primiparous and multiparous sows were assigned to one of three treatments: no induction (NO; n = 24) or 2 cc prostaglandin administered on day 114 of gestation followed by either 1 cc of oxytocin 24 h later (OXY24; n = 13) or 0.5 cc of oxytocin at 6 and 12 h after prostaglandin (OXY6; n = 21). Details of the farrowing process were recorded, and umbilical cord blood was collected from piglets at birth and evaluated on an iSTAT machine using an Abbott EC8+ test cartridge. There were no differences in total born, number born alive, stillborns, mummies, or assistance needed during farrowing. Induced sows were more likely to farrow by day 115 compared to naturally farrowing sows (P = 0.02). Sows in the OXY24 treatment tended to have longer farrowings when compared to both NO and OXY6 (4.8 vs. 3.6 vs. 3.9 h; P = 0.09). Colostrum from OXY6 sows tended to have a greater amount of lactose present than NO and OXY24 (P = 0.05). Colostrum from sows with longer gestation lengths had a higher percentage of fat (P = 0.03). Piglets born from NO sows had higher base excess, total carbon dioxide, and glucose, which suggests that these piglets had prolonged moments of asphyxiation (P < 0.01). OXY24 piglets had the lowest blood pH which is indicative of hypoxic birthing conditions (P < 0.01). Preweaning mortality was driven largely by a low birth weight coupled with low colostrum intake (P = 0.03). All piglets, regardless of treatment, displayed signs of stress during farrowing. Induction did not influence preweaning mortality but has the potential to decrease the incidence by increasing attended farrowings.

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 eight 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 the 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 < 0.05). Piglets in the 20% BrW colostrum group had greater weight gain, RT, and immunocrit at 24 h than COL10 piglets (P < 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 < 0.05) in COL20 compared to COL10 and COL15 was sustained over 7 d. 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.

Sow milk fat content is crucial to neonatal survival, as it is utilized for thermogenesis and nutrition. However, fat is the most variable component of milk in concentration and lipid species. Characterizing lipid changes across the course of a sow’s lactation may help identify molecules or systems to target to help enhance milk fat quality and quantity for neonatal survival and growth. Percent fat variation is greatest in colostrum, the first milk. Little is known regarding colostrum synthesis, other than it accumulates in the gland beginning in mid-late pregnancy, which is prior to the initiation of fatty acid synthesis in lactocytes. The objective of this study was to characterize changes in lipid composition of milk across the course of lactation and determine if there was a relationship between fat percent and lipid species in colostrum and mature milk. Milk was collected from 9 multiparous sows on days 0, 3, 7, and 14 relative to birth. Percent fat was determined by creamatocrit, and found to be different (p< 0.05) between day 0 (12.36 ± 5.90%) and day 3 (16.22 ± 3.65%) but not between day 7 (13.13 ± 2.19%) and 14 (12.13 ± 2.45%). Fat was extracted from milk using the Bligh-Dyer method and profiled using multiple reaction monitoring. Amounts of lipid species were calculated relative to standards and data analysis was performed using Metaboanalyst 4.0. Principle component analysis revealed lactation day had a significant effect on distribution of fats. Triacylglycerides (TAG), phosphatidylglycerol (PG), and plasma membrane lipids were modified from colostrum to mature milk, with a significant increase in PGs and TAGs across the course of lactation. Correlation analysis of percent fat with lipid concentration indicated strong relationships (P < 0.05; |r| >0.80) with eight lipids. No differences are found in the abundance of plasma membrane phospholipids, sphingomyelin, or cholesterol esters across lactation days.

It is known that sperm cells will settle out of extender over time which could impact the concentration per dose within a batch. The objective of this study to investigate the effects of mixing prior to packaging on concentration of packaged insemination doses. At a commercial boar stud, three ejaculates were collected, extended, pooled and assigned to one of three treatment groups: pour-mix (PM) where ejaculates were immediately packaged after combining the three ejaculates, stir-mix (SM) where three ejaculates were combined and stirred thoroughly then packaged, or no mixing (NM) where pooled ejaculates were allowed to sit for 10 min then packaged. Semen was packaged with 35ml per tube creating ~350 individual doses of semen. Every fifth dose was labeled with consecutive numbers (~70/treatment) and overnighted to Purdue University. Concentration of each dose was determined using the NucleoCounter® SP-100TM. The entire study was repeated over 6 weeks. Within treatment, concentration did not differ among doses for the PM and SM treatments. However, NM doses were less concentrated (P< 0.001) in doses >300 compared to all others and doses 251–300 were less concentrated than doses 1 to 50. NM samples 1–250 averaged 36.12 million cells/ml whereas samples 251 to the end of the batch averaged 34.68 million/ml. This results in the last 20 doses having ~50.4 million less cells/dose. Across treatments, NM had lower concentrations (P=0.02) from doses >200 compared to the SM treatment. PM appeared to be an intermediate as it was numerically lower than SM, but not statistically different from either NM or SM. The difference in concentrations between the NM and SM >200 was 1.85 million/ml equaling 64.75 million fewer cells. Overall, allowing semen to settle and not mixing prior to packaging can result in 50–64 million cells fewer per dose in the last 50–100 doses packaged.

Breast milk plays an essential role for offspring development; however, there lacks evidence of how specific milk components like nucleic acids mechanistically function to regulate neonate development. Previously, we found that maternal high-fat diet (HFD) not only significantly affected mRNA and miRNA content of the secreted milk transcriptome in mice but also affected the duodenal proteome of suckling pups. Here, we hypothesized that nucleic acids differentially expressed in milk of HFD fed dams are related to differentially abundant proteins in offspring duodenum nursed by HFD dams. We tested this hypothesis by analyzing one-to-one relationships in RNA-seq data of milk transcriptomes from control (10% kcal fat) and HFD (60% kcal fat) fed mice and liquid chromatography–tandem mass spectrometry (LC-MS/MS) duodenal proteome data from pups exposed to milk. Ten percent of differentially abundant duodenal proteins between controls and HFD-exposed pups had predicted upregulation or downregulation based on differential milk RNA content. Of these, 76% were targets of upregulated miRNA, and linear regression analysis indicated relationships (p < 0.05) between multiple milk miRNA counts and duodenal protein abundance. Duodenal proteins that were potential targets of milk miRNA enriched Gene Ontology (GO) terms and KEGG pathways related to cytoskeletal structure and neural development, suggesting potential regulation of pup enteric nervous system. One-to-one relationships between milk miRNA content and protein abundance in neonate duodenum support the potential for milk miRNAs regulating neonate development. Identification of milk miRNAs that changed in response to maternal diet will enable design of mechanistic studies that test effects on neonate.

Maternal obesity increases the risk of offspring to become obese and develop related pathologies. Exposure to maternal high-fat diet (HFD) only during lactation increases the risk of obesity-related diseases, suggesting that factors in milk affect long-term health. We hypothesized that prepregnancy obesity induced by HFD alters milk lipidome, and in turn, alterations may affect neonate serum lipidome. The objective of this study was to determine the effect of prepregnancy obesity induced by HFD on circulating lipids in dams and neonates and in milk. Female mice were fed an HFD (60% kcal fat) or control diet (CON, 10% kcal fat) beginning 4 weeks before breeding. On postnatal day 2 (PND2), pups were cross-fostered to create pup groups exposed to HFD during pregnancy, lactation, or both or exposed to CON. On PND12, dams were milked and then euthanized along with pups to collect blood. Serum and milk were processed for multiple reaction monitoring (MRM) lipidomics profiling to quantify the relative expression of lipid classes. Lipidome of HFD dam serum and milk had increased proportion of C18:2 free fatty acid and fatty acyl residues in all lipid classes. Lipidome of serum from pups exposed to maternal HFD during lactation was similarly affected. Thus, maternal HFD induced redistribution of fatty acyl residues in the dam's circulation, which was associated with modification in milk and suckling neonate's lipidome. Further studies are needed to determine if increased circulating levels of C18:2 in neonate affects development and predisposes offspring to obesity and metabolic syndrome.Graphical AbstractSummary SentenceMaternal prepregnancy obesity induced by high-fat diet significantly impacts circulating lipids and similarly modifies the lipid composition of milk and circulating lipid of suckling neonates.

Irregular rest-activity patterns can disrupt metabolic and hormonal physiology and potentially lead to disease. Little is known regarding rest-activity patterns during gestation and their association with hormonal rhythms and health in pregnant women. We conducted a pilot study to determine if 24 h rest-activity was related to saliva cortisol rhythms and maternal-fetal health in an economically disadvantaged population. Primiparous women wore a wrist actigraphy device for a week to record activity during gestational weeks 22 (G22; n = 50) and 32 (G32; n = 46) and postpartum week one (PPW1; n = 39). Participants collected saliva samples every 4 hr over a 24 hr period during G22 (n = 22), G32 (n = 20) and 24–48 hr postnatal (n = 20), and cortisol concentrations were measured with ELISA. Circadian rhythmicity was assessed using autocorrelation coefficient (r24) and cosinor analysis. Blood glucose levels, body mass index (BMI), gestational disease data, and gestational age of infant at birth were abstracted from medical charts. Time of cortisol peak (acrophase) during G22 was related with acrophase of activity (r = 0.66; p = 0.001) and blood glucose levels (r = 0.58; p = 0.006). During G22, minutes of wake after sleep onset was positively related to cortisol mesor and AUC (p

Periparturient dairy cattle are susceptible to metabolic disease due to immense physiological stress caused by calving and the onset of lactation. Susceptibility to disease is related to the cow’s ability to maintain metabolic homeostasis and mount a proper immune response, despite high demand for glucose to support fetal growth and milk synthesis, and as a fuel for increased circulating levels of white blood cells (WBC). This study was conducted to determine if there was a correlation among white blood cell percentages, blood glucose, and the levels of the ketone betahydroxybutyrate (BHBA) on the days immediately around parturition, and to determine if the variables exhibited circadian rhythms. We hypothesized that glucose levels would vary inversely with WBC, and BHBA levels would vary directly with glucose levels. Blood was sampled from the tail vein of multiparous Holstein cows (n=12) at 0600 from 4 days prepartum to 4 days postpartum, and every 4 hr over a 24-hr period (n=6) starting at 0600 on postpartum day 3. Blood glucose and BHBA levels were measured using a CentriVet™, and WBC percentage was calculated by measuring packed cell volume. Glucose was higher (p

Maintaining metabolic balance is a key factor in the health of dairy cattle during the transition from pregnancy to lactation. Little is known regarding the role of the circadian timing system in the regulation of physiological changes during the transition period. We hypothesized that disruption of the cow's circadian timing system by exposure to chronic light-dark phase shifts during the prepartum period would negatively affect the regulation of homeostasis and cause metabolic disturbances, leading to reduced milk production in the subsequent lactation. The objective was to determine the effect of exposure to chronic light-dark phase shift during the last 5 wk prepartum of the nonlactating dry period on core body temperature, melatonin, blood glucose, β-hydroxybutyric acid (BHB) and nonesterified fatty acid (NEFA) concentrations, and milk production. Multiparous cows were moved to tiestalls at 5 wk before expected calving and assigned to control (CTR; n = 16) or phase-shifted (PS; n = 16) treatments. Control cows were exposed to 16 h of light and 8 h of dark. Phase-shifted cows were exposed to the same photoperiod; however, the light-dark cycle was shifted 6 h every 3 d until parturition. Resting behavior and feed intake were recorded daily. Core body temperature was recorded vaginally for 48 h at 23 and 9 d before expected calving using calibrated data loggers. Blood concentrations of melatonin, glucose, BHB, and NEFA were measured during the pre- and postpartum periods. Milk yield and composition were measured through 60 DIM. Treatment did not affect feed intake or body condition. Cosine fit analysis of 24-h core body temperature and circulating melatonin indicated attenuation of circadian rhythms in the PS treatment compared with the CTR treatment. Phase-shifted cows had lower rest consolidation, as indicated by more total resting time, but shorter resting period durations. Phase-shifted cows had lower blood glucose concentration compared with CTR cows (4 mg/mL decrease), but BHB and NEFA concentrations were similar between PS and CTR cows. Milk yield and milk fat yield were greater in PS compared with CTR cows (2.8 kg/d increase). Thus, exposure to chronic light-dark phase shifts during the prepartum period attenuated circadian rhythms of core body temperature, melatonin, and rest-activity behavior and was associated with increased milk fat and milk yield in the postpartum period despite decreased blood glucose pre- and postpartum. Therefore, less variation in central circadian rhythms may create a more constant milieu that supports the onset of lactogenesis.

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< 0.001), but did not differ between COL10 and COL20 piglets (P=0.74). Total protein was greater at 24h (P< 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< 0.03), while essential AAs (His, Ile, Met, Phe, Trp and Val) were increased after 24h of colostrum feeding (P< 0.05). At 24h, COL20 piglets had greater Ile and Met (P< 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 > 0.05), however between D0 and D7 villi width, epithelial area, and crypt length increased (P < 0.05). Differences in histomorphology across the first week postnatal was not affected by level of colostrum intake in the first 24 h postnatal.

Circadian clocks influence virtually all physiological processes, including lactation. Here, we investigate the role of the CLOCK gene in regulation of mammary epithelial cell growth and differentiation. Comparison of mammary morphology in late-pregnant wild-type and ClockΔ19 mice, showed that gland development was negatively impacted by genetic loss of a functional timing system. To understand whether these effects were due, in part, to loss of CLOCK function in the gland, the mouse mammary epithelial cell line, HC11, was transfected with short hairpin RNA that targeted Clock (shClock). Cells transfected with shClock expressed 70% less Clock mRNA than wild-type (WT) HC11 cultures, which resulted in significantly depressed levels of CLOCK protein ( P < 0.05). HC11 lines carrying shClock had four-fold higher growth rates ( P < 0.05), and the percentage of cells in G1 phase was significantly higher (90.1 ± 1.1% of shClock vs. 71.3 ± 3.6% of WT-HC11) following serum starvation. Quantitative-PCR (qPCR) analysis showed shClock had significant effects ( P < 0.0001) on relative expression levels of Ccnd1, Wee1, and Tp63. qPCR analysis of the effect of shClock on Fasn and Cdh1 expression in undifferentiated cultures and cultures treated 96 h with dexamethasone, insulin, and prolactin (differentiated) found levels were reduced by twofold and threefold, respectively ( P < 0.05), in sh Clock line relative to WT cultures. Abundance of CDH1 and TP63 proteins were significantly reduced in cultures transfected with sh Clock. These data support how CLOCK plays a role in regulation of epithelial cell growth and differentiation in the mammary gland.

Canarian goats are well adapted to extended milkings. To gain more information on this adaptation, 32 dairy goats in mid lactation belonging to Majorera and Palmera breeds were subjected to milk stasis of 10, 14, 24 (the normal routine), 28 and 32 h; and milk volumes, milk composition, concentrations of Na + and K + in milk and blood plasma and plasma lactose were measured at each interval. The major findings were: i. Milk stasis did not induce changes in milk fat and milk protein secretion rates. In contrast, the extension of the interval between milkings was associated with a decrease of milk lactose secretion rate, ii. Comparison of the relative changes of Na + and K + in milk with blood and plasma lactose content indicates that the tight junction (TJ) permeability to lactose was much higher than to monovalent cations. It was concluded that an important element in the adaptation of Canarian dairy goats to extended milking intervals may be related to maintenance TJ resistant to paracellular leakage, particularly to passage of monovalent ions. Thus, the present study increases the understanding on adaptation of small ruminant to extended milking interval, which has been related so far to their high cistern storage capacity.

Offspring nutrition depends on the mother during gestation and lactation; thus, maternal nutrition and metabolism can affect their development. We hypothesized that maternal exposure to high-fat (HF) diet affects neonate's gastrointestinal tract development. Our objective was to determine the effect of maternal HF diet during gestation and lactation on neonate's duodenum histomorphology and proteome. Female mice were fed either a control (C, 10% kcal fat) or an HF (60% kcal fat) diet for 4 weeks and bred. On postnatal day 2, half the pups were cross-fostered to dams fed on different diet, creating 4 treatments: C-C, C-HF, HF-C, and HF-HF, indicating maternal diet during gestation-lactation, respectively. On postnatal day 12, pups' duodenum was excised and prepared for histology and liquid chromatography-tandem mass spectrometry analysis of proteome. Villi were significantly longer in HF-HF pups, and crypt cell proliferation rate was not different among treatments. Between C-C and HF-HF, HF-C, or C-HF, 812, 601, or 894 proteins were differentially expressed (Tukey adjusted P .05), respectively. Functional analysis clustered proteins upregulated in HF-HF vs C-C in fat digestion and absorption, extracellular matrix, cell adhesion, immune response, oxidation-reduction processes, phagocytosis, and transport categories. Proteins downregulated were classified as RNA splicing, translation, protein folding, endocytosis, and transport. There was evidence for a carryover effect of exposure to HF diet during gestation to the postnatal period. Alterations in proteome relative to HF exposure potentially reflect long-term changes in the functioning of the duodenum.

Serotonin receptors (5-HTR) are present in the mammary tissue of mouse, humans, cows, and rats. In these species, serotonin is important for the mammary gland function and lactation performance. The mammary gland expression of 5-HTR in small dairy ruminants has yet to be described. In the present study, primer sequences were developed to amplify 5-HTR (1A, 1D, 1E,1B, 1F, 2A, 2B, 2C, 3a, 4, 5a, 6, and 7) using real-time quantitative PCR for the detection of mRNA expression in mammary tissue of dairy sheep, goats, and cows. The distribution of commonly expressed 5-HTR between the 3 species (1B, 1E, 2A, 2B, 4, and 7) was analyzed in the mammary tissue of late-lactation and dried-off sheep, goats, and cows using immunohistochemical staining. Real-time quantitative PCR analysis showed that the 3 studied species expressed receptors 5-HTR1B, 1E, 2A, 2B, 4, and 7. Goats and sheep expressed 5-HTR1D and 5a; 5-HTR1A and 1F were expressed only in sheep. The mammary epithelial cells were positively stained for all the studied receptors by immunohistochemistry (5-HTR1B, 1E, 2A, 2B, 4, and 7). The endothelial cells of blood vessels were positively stained for 5-HTR1B, 2A, 2B, and 7 in all the species. Additionally, 5-HTR1E was present in cow endothelium. The myoepithelial cells stained positively for 5-HTR1E in all the species, and 5-HTR4 myoepithelial staining was present only in cows and sheep. Between the lactating and dried-off mammary glands, the location of 5-HTR in the epithelial cells changed from a cytoplasmic reaction in lactating udders to a reaction in the apical region in dry udders. These results showed that the distribution of 5-HTR subtypes in the mammary gland of dairy ruminants vary among species, tissue type, and stage of gland development. These findings warrant future studies aimed at understanding whether the differences in 5-HTR subtype expression and location accounts for the differences in milk secretion and lactocyte activity among cows, goats, and sheep.

The aim of this study was to investigate the BW and immune status of lambs reared under natural conditions or under artificial conditions fed two different colostrum amounts. In this study, 60 lambs were randomly divided into groups according to treatment. Twenty lambs remained with their dams (natural rearing (NR) group). Forty lambs were removed from their dams at birth. Lambs were bottle-fed with a pool of sheep colostrum, receiving either 4 g of IgG/kg of BW at birth (C4 group) or 8 g of IgG/kg of BW at birth (C8 group). The total colostrum amount was equally divided into three meals at 2, 14 and 24 h after birth. After this period, lambs were bottle-fed a commercial milk replacer. Blood plasma sample analysis and BW recordings were carried out before feeding at birth and then at 1, 2, 3, 4, 5 and 20 days after birth. Another blood sample analysis and BW recording was carried out when animals reached 10 kg of BW. During weaning (30 days), sampling was carried out every 5 days. Blood plasma was used to determine the concentrations of IgG and IgM and the complement system activity - total and alternative pathways. The NR group showed greater BW than the C4 and C8 groups during milk feeding period, whereas the C4 and C8 groups had greater BW than the NR group at the end of weaning period. The C8 and NR groups had greater plasma IgG and IgM concentrations than the C4 group during milk feeding period. In addition, C4 and C8 groups showed similar IgG concentrations and greater IgM concentrations than the NR group at the end of the weaning period. Complement system activity was greater in the NR group than in the C4 and C8 groups during the first 3 days after birth. In conclusion, lambs fed amounts of colostrum equivalent to 8 g of IgG/kg of BW showed similar immune variables compared to lambs reared under natural conditions, obtaining a greater BW at the end of the weaning period. Nevertheless, this study shows that not only the colostrum amount but also the management during the milk feeding and weaning period, such as stress produced by dam separation, milk quality and suckling frequency, can affect the final immune status of lambs.

Tissue percentages (secretory, connective, ductal and vascular tissues) and the number and size of the alveoli in the udders of three dairy goat breeds under two milking frequencies (once- vs. twice-daily milking) were studied. Nine dairy goats, three of each breed studied (Majorera, Tinerfena and Palmera), were milked during 6 weeks beginning at the tenth week of lactation. The right udder half was milked twice daily, and the left udder half was milked once daily. Moreover, during the experimental period, morphological udder data, milk yield, milk fractioning and milk composition were recorded. The goats were sacrificed and two samples for each gland were taken for the histological study. The statistical analyses revealed that the histological parameters were not influenced by the milking frequency, and that the breed determines different percentage of tissue components. Correlations between morphological parameters of the udder and milk-yielding parameters were high and determined the greater importance...

Since lactation is one of the most metabolically demanding states in adult female mammals, beautifully complex regulatory mechanisms are in place to time lactation to begin after birth and cease when the neonate is weaned. Lactation is regulated by numerous different homeorhetic factors, all of them tightly coordinated with the demands of milk production. Emerging evidence support that among these factors are the serotonergic and circadian clock systems. Here we review the serotoninergic and circadian clock systems and their roles in the regulation of mammary gland development and lactation physiology. We conclude by presenting our hypothesis that these two systems interact to accommodate the metabolic demands of lactation and thus adaptive changes in these systems occur to maintain mammary and systemic homeostasis through the reproductive cycles of female mammals.

The aim of this study was to investigate the histochemical parameters of muscle fibre in a Spanish autochthonous pig breed. Due to its fatty meat and high prolificacy, this pork is revered among local consumers. However, the aforementioned breed has become endangered. A total of 11 native pigs (Black Canary Pig) were evaluated. Longissimus lumborum muscle samples were collected in order to evaluate contractile and metabolic characteristics of the varying fibre types by histochemical staining techniques (nicotinamide adenine dinucleotide tetrazolium reductase [NADH-TR] and myosin adenosine triphosphatase activity [mATPase]). Cross-sectional area, diameter and percentage of the fibres were calculated. The oxidative fibre percentage was elevated (33.63%) which in turn can improve the colour, tenderness and juiciness of the meat, hence the glycolytic fibre percentage being moderate (66.37%). The size of three fibre types was higher than the values provided by other pig breeds. A higher percentage of IIB fibre...

Pre-print The effects of Crypthecodinium cohnii (Cryp.), Chlorela spp. (Chlo.) and Isochrysis galbana (Iso.) addition to milk replacer on goat kids and lambs growth were evaluated. About 80 Majorera goat kids (males and females) and 80 Canarian sheep lambs were randomly assigned into four different groups (by specie) according to diet. Control groups were fed with a commercial milk replacer at 16% (w/w); Cryp. groups received a commercial milk replacer (15.1% w/w) supplemented with 9 g of a paste of C. cohnii; Chlo. groups received a commercial milk replacer (15.1% w/w) supplemented with 9 g of a paste of Chlorela spp.; Iso. groups received a commercial milk replacer (15.1% w/w) supplemented with 9 g of a paste of I. galbana. After colostrum period, animals were individually bottle-fed twice daily (8 am and 8 pm) ad libitum with the corresponding diet until day 60 of life. Animals were weighted every week at 8 am and liquid diet intake was recorded weekly. No effects of microseaweed addition were observed, neither growth nor milk replacer intake. Yes

The importance of small ruminants to the dairy industry has increased in recent years, especially in developing countries, where it has a high economic and social impact. Interestingly and despite the fact that the mammary gland is the specialised milk production organ, very few authors studied the modifications occurring in the mammary gland through the lactation period in production animals, particularly in the small ruminants, sheep (Ovis aries) and goat (Capra hircus). Nevertheless, understanding the different mammary gland patterns throughout lactation is essential to improve dairy production. In addition, associating these patterns with different milking frequencies, lactation number or different diets is also of high importance, directly affecting the dairy industry. The mammary gland is commonly composed of parenchyma and stroma, which includes the ductal system, with individual proportions of each changing during the different periods and yields in a lactation cycle. Indeed, during late gestation, as well as during early to mid-lactation, mammary gland expansion occurs, with an increase in the number of epithelial cells and lumen area, which leads to increment of the parenchyma tissue, as well as a reduction of stroma, corresponding macroscopically to the increase in mammary gland volume. Throughout late lactation, the mammary gland volume decreases owing to the regression of the secretory structure. In general, common mammary gland patterns have been shown for both goats and sheep throughout the several lactation stages, although the number of studies is limited. The main objective of this manuscript is to review the colostrogenesis and lactogenesis processes as well as to highlight the mammary gland morphological patterns underlying milk production during the lactation cycle for small ruminants, and to describe potential differences between goats and sheep, hence contributing to a better description of mammary gland development during lactation for these two poorly studied species.