AN ABSTRACT OF THE THESIS OFMelanie Tharp, for the Master of Science degree in Food and Nutrition, presented on November 16, 2021, at Southern Illinois University Carbondale. TITLE: A SURVEY OF PHYSIOLOGICAL FACTORS ON BREASTFEEDING DURATION AND EXCLUSIVITYMAJOR PROFESSOR: Dr. Dawn NullBreastfeeding an infant is the current best recommendation for infant feeding, but only about a quarter of infants are breastfed until the suggested age of 6 months exclusively and 12 months with complementary foods. In this study, the objective was to find what determinants there are in women choosing to breastfeed and what barriers that exist to cause early cessation of breastfeeding. Current studies suggest that more research be done on the physiological factors such as weight and the presence of hormone problems that affect breastfeeding. The study focused on the physiological factors that may affect breastfeeding outcomes, in this study, duration and exclusivity. It was hypothesized that both duration and exclusivity would be lower in the women with the physiological risk factors. Participants were adult breastfeeding mothers. The data was collected through the use of an online survey exploring breastfeeding practices and physiological factors. A total of 507 participants started the survey with data from participants who did not complete at least 50% of the survey were excluded leaving 437 participants in the final sample. Overall, the sample had higher than average income, education, and age. There were no significant relationships found between the physiological factors studied and the duration of breastfeeding. However, there was a significant relationship found between formula use and exercise which suggests a lower formula use with more exercise. Suggestions for future studies include considering ethnicity and using a stronger study design to be able to find cause and effect relationships between physiological factors and breastfeeding outcomes.

The porcine mammary gland can be used for the production of recombinant proteins by directing a transgene to the mammary gland with a milk protein gene promoter. In order to determine whether or not the protein will be expressed, the animals must be maintained at least through their first lactation. An experiment was performed to determine if hormonal induction of lactogenesis in transgenic virgin pigs could be used as a method for identifying those gilts that are likely to express the recombinant protein during a natural lactation. Mammary development and lactogenesis were induced by administration of subcutaneous implants designed to release 7.1 mg of estradiol-17 beta and 18 mg of progesterone daily for 21 d. Histological analysis of tissue samples before and after the treatment period indicated that mammary secretory tissue underwent dramatic proliferation resulting in a greater degree of alveolar and individual epithelial cell differentiation. The presence of beta-lactoglobulin mRNA was detected in high levels in post-implant tissue samples, and minimally detected in samples cultured in media supplemented with insulin, hydrocortisone, and prolactin. However, protein expression was only detected in the post-implant samples, indicating that beta-lactoglobulin was not maintained well by in vitro culture. The transgene mRNA, recombinant human fibrinogen (A-alpha chain), was detected in all analyzed samples at varying levels. However, the corresponding protein was not detected in any sample, under either reduced or nonreduced conditions. These results indicate that lactogenesis was successfully induced using the hormonal implants. Also, the transgene was activated by the hormonal induction in vivo and in vitro, but the corresponding protein could not be detected. This study indicates that induction of lactogenesis can be used to detect the presence of transgene mRNA in mammary tissue of gilts. However, we cannot conclusively demonstrate that this procedure can be used to identify those gilts that are likely to express the recombinant protein during a natural lactation. / Master of Science

Clinical Issue Health Canada recommends exclusive breastfeeding for the first 6 months post birth and then the addition of complementary foods with breastfeeding extending to a minimum of two years. Breastfeeding initiation rates in Canada are currently at around 87% but, by one month, about 21% of women have stopped breastfeeding. Engorgement and edema in breast tissue can lead to breastfeeding challenges which may contribute to early weaning. Purpose The purpose of this pilot research study was to explore the relationship between intravenous (IV) fluids given to mothers during the peripartum period and postpartum breast or nipple swelling in the first ten days postpartum and determine if a larger study was warranted and feasible. The research question for this pilot study was, "What is the relationship between the amount of IV fluids given to labouring women and edema of the breast and areola complex experienced by breastfeeding women in the first 10 days postpartum?" Methods It is a prospective, longitudinal, observational cohort pilot study with repeated measures and a within-subjects design. Participants are first time mothers who planned to exclusively breastfeed and gave birth to a single, healthy newborn by means of a spontaneous vaginal birth, Mother and baby were discharged home together with no contraindications to exclusive unrestricted breastfeeding. Descriptive statistics are reported and linear regression analysis is used to model the relationship between IV therapy and postpartum breast edema. Results Women who received IV fluids during labour had higher levels of edema postpartum and rated their breasts as firmer as and more tender than women who did not receive IV fluids. Participants who had IV fluids appeared to be less aware of the fullness associated with lactogenesis II, and the pattern of fullness they described appeared to be related to edema noted. Participants who did not have IV fluids appeared to have unrelated patterns of fullness and edema, and therefore appeared more aware of the onset of lactogenesis II. The results support a larger study about the relationships between maternal perinatal IV fluids and breast or nipple changes.

Clinical Issue Health Canada recommends exclusive breastfeeding for the first 6 months post birth and then the addition of complementary foods with breastfeeding extending to a minimum of two years. Breastfeeding initiation rates in Canada are currently at around 87% but, by one month, about 21% of women have stopped breastfeeding. Engorgement and edema in breast tissue can lead to breastfeeding challenges which may contribute to early weaning. Purpose The purpose of this pilot research study was to explore the relationship between intravenous (IV) fluids given to mothers during the peripartum period and postpartum breast or nipple swelling in the first ten days postpartum and determine if a larger study was warranted and feasible. The research question for this pilot study was, "What is the relationship between the amount of IV fluids given to labouring women and edema of the breast and areola complex experienced by breastfeeding women in the first 10 days postpartum?" Methods It is a prospective, longitudinal, observational cohort pilot study with repeated measures and a within-subjects design. Participants are first time mothers who planned to exclusively breastfeed and gave birth to a single, healthy newborn by means of a spontaneous vaginal birth, Mother and baby were discharged home together with no contraindications to exclusive unrestricted breastfeeding. Descriptive statistics are reported and linear regression analysis is used to model the relationship between IV therapy and postpartum breast edema. Results Women who received IV fluids during labour had higher levels of edema postpartum and rated their breasts as firmer as and more tender than women who did not receive IV fluids. Participants who had IV fluids appeared to be less aware of the fullness associated with lactogenesis II, and the pattern of fullness they described appeared to be related to edema noted. Participants who did not have IV fluids appeared to have unrelated patterns of fullness and edema, and therefore appeared more aware of the onset of lactogenesis II. The results support a larger study about the relationships between maternal perinatal IV fluids and breast or nipple changes.

A series of trials was carried out to examine the role of prolactin (PRL) in the control of lactogenesis in New Zealand Romney x Border Leicester ewes. In addition, a study was made of differences in milk yields and plasma PRL concentrations between spring- and autumn-lambing ewes.Daily subcutaneous injections of 2 mg CB154 inhibited PRL secretion and delayed lactogenesis. There were no consistent effects on plasma progesterone or insulin concentrations. CB154 treatment was more effective in reducing milk yield in twin-bearing than in single-bearing ewes when used for 20 days than for 9 days prepartum. The differential effects on milk yield cannot be explained by corresponding effects on plasma PRL or insulin concentrations. Circulating PRL during the period 20 to 10 days prepartum may have an important effect on milk yield in twin- but not single-bearing ewes.Subcutaneous injections of 0.5 mg/kg live weight oPRL, administered on 2 consecutive days peripartum, to ewes treated with CB154 for 7 days prepartum, resulted in milk yields similar to those in control ewes and significantly (P<0.01) greater than those in ewes treated with CB154 alone. This indicated that oPRL prevented the CB154-induced reduction of milk yields and has established that the effect of CB154 on lactogenesis is mediated through suppression of PRL secretion and not by effects on some other hormone.Injection of 10 mg oPRL directly into one mammary gland (via the teat duct) increased milk yields relative to the contralateral, bicarbonate-treated gland in CB154-treated ewes. The intramammary oPRL injection did not raise circulating PRL concentrations. Furthermore, the milk yields of bicarbonate-treated glands in ewes treated with bicarbonate only, did not differ from those of bicarbonate-treated glands in ewes treated with oPRL in the contralateral gland, demonstrating that there were no effects of oPRL, transferred via the circulation from the treated gland, on the contralateral gland. Glands treated with oPRL produced 15% (P<0.05) more milk than the bicarbonate-treated glands during the first 8 days of lactation and the difference was maintained throughout the 8-week lactation period, indicating that the oPRL had effected a permanent change in the ability of the gland to produce milk. It is concluded that PRL acts directly on the mammary gland without the need for a putative intermediate hormone, and that intramammary PRL concentrations during lactogenesis may have long-lasting effects on lactation.The possibility was examined that dietary differences were responsible for seasonal differences in plasma PRL concentrations, milk yields, milk composition, lamb birthweight and lamb growth rate, observed in earlier trials. Mean plasma PRL levels were significantly (P<0.01) higher in spring- (192±38 ng/ml) than in autumn- (71±17 ng/ml) lambing ewes housed indoors under constant photoperiod (18L:6D) and fed the same diet. Milk yields were also significantly (P<0.05) higher in the spring- (2041±114 g/d) than in the autumn- (1563±109 g/d) lambing ewes over the 8 day lactation. Lamb growth rates (adjusted for birthweight, birthrank and sex of lamb) from birth to 8 weeks of age were significantly (P<0.001) higher in spring (282±12 g/d) than in autumn (225±15 g/d). The seasonal differences were confounded with corresponding differences in ewe live weight and it was not possible to determine whether dietary differences contributed significantly to the differences observed.Two routes of oPRL supplementation were used to test the effectiveness of elevating peripheral or local levels of PRL in autumn-lambing ewes which, based on previous results, were expected to have low plasma PRL concentrations and milk yields relative to spring-lambing ewes. Administration of 10 mg supplementary oPRL directly into the gland or subcutaneous injection of 0.5 mg/kg oPRL did not increase the milk yields, or change the composition of milk, compared to controls. These results suggest that the circulating level of PRL, and the intramammary concentration of PRL, in autumn-lambing ewes are not limiting lactogenesis. Because the plasma prolactin concentration in the ewes was unexpectedly high, it was not possible to reach firm conclusions regarding possible effects of supplementary oPRL in ewes with naturally low plasma PRL concentrations. Nevertheless, the results indicate that raising the intramammary concentration of PRL around the time of parturition, in ewes with circulating PRL levels characteristic of normal spring-lambing ewes, does not enhance lactogenesis.It is concluded that PRL is important to the complete initiation of lactogenesis in ewes, that it acts directly on the gland and that it is necessary for establishing the maximum potential of the gland to secrete milk.

The mammary explant culture model has been a major experimental tool for studying hormonal requirements for milk protein gene expression as markers of secretory differentiation. Experiments with mammary explants from pregnant animals from many species have established that insulin, prolactin, and glucocorticoid are the minimal set of hormones required for the induction of maximal milk protein gene expression. However, the extent to which mammary explants mimic the response of the mammary gland in vivo is not clear. Recent studies have used microarray technology to study the transcriptome of mouse lactation cycle. It was demonstrated that the each phase of mouse lactation has a distinct transcriptional profile but making sense of microarray results requires analysis of large amounts of biological information which is increasingly difficult to access as the amount of literature increases. / The first objective is to examine the possibility of combining literature and genomic analysis to elucidate potentially novel hypotheses for further research into lactation biology. The second objective is to evaluate the strengths and limitations of the murine mammary explant culture for the study and understanding of murine lactogenesis. The underlying question to this objective is whether the mouse mammary explant culture is a good model or representation to study mouse lactogenesis. / The exponential increase in publication rate of new articles is limiting access of researchers to relevant literature. This has prompted the use of text mining tools to extract key biological information. Previous studies have reported extensive modification of existing generic text processors to process biological text. However, this requirement for modification had not been examined. We have constructed Muscorian, using MontyLingua, a generic text processor. It uses a two-layered generalizationspecialization paradigm previously proposed where text was generically processed to a suitable intermediate format before domain-specific data extraction techniques are applied at the specialization layer. Evaluation using a corpus and experts indicated 86-90% precision and approximately 30% recall in extracting protein-protein interactions, which was comparable to previous studies using either specialized biological text processing tools or modified existing tools. This study also demonstrated the flexibility of the two-layered generalization-specialization paradigm by using the same generalization layer for two specialized information extraction tasks. / The performance of Muscorian was unexpected since potential errors from a series of text analysis processes is likely to adversely affect the outcome of the entire process. Most biomedical entity relationship extraction tools have used biomedical-specific parts-of-speech (POS) tagger as errors in POS tagging and are likely to affect subsequent semantic analysis of the text, such as shallow parsing. A comparative study between MontyTagger, a generic POS tagger, and MedPost, a tagger trained in biomedical text, was carried out. Our results demonstrated that MontyTagger, Muscorian's POS tagger, has a POS tagging accuracy of 83.1% when tested on biomedical text. Replacing MontyTagger with MedPost did not result in a significant improvement in entity relationship extraction from text; precision of 55.6% from MontyTagger versus 56.8% from MedPost on directional relationships and 86.1% from MontyTagger compared to 81.8% from MedPost on un-directional relationships. This is unexpected as the potential for poor POS tagging by MontyTagger is likely to affect the outcome of the information extraction. An analysis of POS tagging errors demonstrated that 78.5% of tagging errors are being compensated by shallow parsing. Thus, despite 83.1% tagging accuracy, MontyTagger has a functional tagging accuracy of 94.6%. This suggests that POS tagging error does not adversely affect the information extraction task if the errors were resolved in shallow parsing through alternative POS tag use. / Microarrays had been used to examine the transcriptome of mouse lactation and a simple method for microarray analysis is correlation studies where functionally related genes exhibit similar expression profiles. However, there has been no study to date using text mining to sieve microarray analysis to generate new hypotheses for further research in the field of lactational biology. Our results demonstrated that a previously reported protein name co-occurrence method (5-mention PubGene) which was not based on a hypothesis testing framework, is generally more stringent than the 99th percentile of Poisson distribution-based method of calculating co-occurrence. It agrees with previous methods using natural language processing to extract protein-protein interaction from text as more than 96% of the interactions found by natural language processing methods coincide with the results from 5-mention PubGene method. However, less than 2% of the gene co-expressions analyzed by microarray were found from direct co-occurrence or interaction information extraction from the literature. At the same time, combining microarray and literature analyses, we derive a novel set of 7 potential functional protein-protein interactions that had not been previously described in the literature. We conclude that the 5-mention PubGene method is more stringent than the 99th percentile of Poisson distribution method for extracting protein-protein interactions by co-occurrence of entity names and literature analysis may be a potential filter for microarray analysis to isolate potentially novel hypotheses for further research. / The availability of transcriptomics data from time-course experiments on mouse mammary glands examined during the lactation cycle and hormone-induced lactogenesis in mammary explants has permitted an assessment of similarity of gene expression at the transcriptional level. Global transcriptome analysis using exact Wilconox signed-rank test with continuity correction and hierarchical clustering of Spearman coefficient demonstrated that hormone-induced mammary explants behave differently to mammary glands at secretory differentiation. Our results demonstrated that the mammary explant culture model mimics in vivo glands in immediate responses, such as hormone-responsive gene transcription, but generally did not mimic responses to prolonged hormonal stimulus, such as the extensive development of secretory pathways and immune responses normally associated with lactating mammary tissue. Hence, although the explant model is useful to study the immediate effects of stimulating secretory differentiation in mammary glands, it is unlikely to be suitable for the study of secretory activation.

The goal of this dissertation was to evaluate genomic regulation during bovine mammary epithelial cell (BMEC) growth and differentiation. To accomplish this goal, a collagen gel cell culture system was developed that was capable of mimicking the prepartum stages of epithelial development and differentiation. In addition, a 4,600-cDNA bovine microarray was developed in order to profile gene expression. Analysis of BMEC in collagen cultures using various lactogenic conditions highlighted the critical importance of both hormonal and structural signals. The objective of the first study utilizing the microarray was to evaluate the contribution of the two prominent lactogenic factors in vitro, 1) prolactin and 2) gel release. Collectively, lactogenic stimulation appears to turn off genes associated with structural progression and morphogenesis, and turn on genes involved in alveolar MEC differentiation such as cell polarization, milk protein synthesis and ER/Golgi transport. The objective of the second study utilizing these resources was to evaluate the direct effects of thermal stress on BMEC growth and development. The structural response to thermal stress was characterized by morphogenic inhibition and dramatic regression of the ductal branches. Microarray analysis revealed an overall up-regulation of genes associated with stress response, DNA repair, protein degradation and cell death. In contrast, genes associated with cellular and MEC-specific biosynthesis, metabolism, and morphogenesis, were generally down-regulated. Subsequent to the analysis of BMEC differentiation was a targeted effort focusing on two small molecules hypothesized to be involved in regulating the BMEC secretory response: serotonin and prostaglandin E2. A pilot study suggested that serotonin is produced by bovine MEC and a model was proposed that describes serotonin's role as a feedback inhibitor during milk synthesis and secretion. A second pilot study demonstrated that PGE2 had a consistently positive influence on lumen diameter of alveolar structures in vitro. Overall, this dissertation provides new resources for studying bovine functional genomics, particularly within the mammary gland, and it provides a strong foundation for understanding genomic regulation of mammary epithelial structure and function. Furthermore, it establishes potential roles for local regulation of milk production by serotonin and PGE2.

Lactogenesis is constituted by two phases, called as lactogenesis I and lactogenesis II. The transition between those phases is characterized by increase in the lactose concentration. This study aimed to evaluate the interference of diabetes mellitus in the lactose concentration associated to transition between phases I and II of lactogenesis. It was studied 11 puerperal women with pre-gestational diabetes mellitus and 19 without diabetes. During the five first days after delivery, at each 24 hours, it was collected colostrums samples to evaluate its lactose concentration by reaction with picric acid. In both groups there were progressive increases in the lactose levels with time. The lactose concentrations in both groups exhibited statistically significant sigmoidal dependences with the time after delivery, with neat two-state transition between the phases I and II of lactogenesis. The analysis of those transitions revealed an 18 hours time delay in the beginning of lactogenesis II of diabetes carriers women with inadequate metabolic control in relation to puerperal females without diabetes. / A lactogênese é constituída por duas fases, designadas como I e II. Uma das características da transição entre estas fases é o aumento da concentração de lactose no colostro. Este estudo teve como objetivo avaliar a interferência do diabetes mellitus na transição da lactogênese I para a lactogênese II. Foram avaliadas 11 mulheres puérperas portadoras de diabetes mellitus tipo 1 pré-gestacional e 19 puérperas sem diabetes. Nos cinco primeiros dias após o parto, a cada intervalo de 24 horas, foram coletadas amostras do colostro das mães para análise de seu teor em lactose, por reação com ácido pícrico. Em ambos os grupos houve um aumento progressivo na concentração de lactose com o tempo. A dependência da concentração de lactose com o tempo, após o parto, foi ajustada a uma curva de crescimento sigmoidal que mostrou a transição da lactogênese I para a lactogênese II. A análise desta transição revelou um atraso de 18 horas para início da lactogênese II nas puérperas portadoras de diabetes mellitus com controle metabólico inadequado em relação às puérperas sem diabetes. / Mestre em Ciências da Saúde

La comprensione dei geni che controllano la crescita, lo sviluppo, e il metabolismo della ghiandola mammaria suina può rivelare potenziali vie metaboliche o di segnale per migliorare l'efficienza di sintesi del latte. Un microarray suino costituito da 13.263 oligonucleotidi (mer 70) è stato utilizzato per lo studio del profilo di trascrizione del tessuto mammario da 4.5 scrofe a -34, -14, -4, 0, 7, 14, 21, e 28 giorni rispetto alla data del parto. ANOVA (FDR ≤ 0.10) ha individuato 2664 geni differenzialmente espressi (DEG) in relazione allo stato fisiologico. L’analisi dei network e delle vie metaboliche ha identificato come funzioni molecolari più affette dallo stato fisiologico: crescita e proliferazione cellulare (548 geni) cellule di segnale(612 geni).La qPCR rimane il metodo migliore per la misurazione dell’ abbondanza mRNA ad alta precisione e per la validazione di dati array. Essenziale per assicurare l'affidabilità della qPCR è la normalizzazione dei dati con l’utilizzo di geni di controllo interno (ICG). Un analisi sulla stabilità dei geni ha identificato, tra i 19 potenziali ICG, API5, VABP, e MRPL39 come i più stabili ICG nel tessuto mammario suini e ha inoltre stabilito che l'uso di tali 3 geni è il più appropriato per il calcolo di un fattore di normalizzazione. I risultati sottolineano l'importanza di una corretta validazione dei controlli interni per qPCR ed evidenziano le limitazioni di utilizzo dell’assenza dell’effetto tempo come unico criterio per la selezione di CIG. / Elucidating genes controlling growth, development, and metabolism of swine mammary glands can reveal potential metabolic or signalling pathways that might help improve efficiency of milk synthesis. A swine microarray consisting of 13,263 oligonucleotides (70 mer) was used for transcript profiling of mammary tissue from 4-5 sows at -34, -14, -4, 0, 7, 14, 21, and 28 d relative to parturition. ANOVA (FDR ≤ 0.10) identified 2,664 differentially expressed genes (DEG) dueto physiological state. Gene network/pathway analysis revealed that cell growth and proliferation (548 genes) and cell signaling (612 genes) were among the most affected molecular functions due to physiological state in DEG. QPCR remains the chosen method for high-precision mRNA abundance analysis and for array data validation. Essential for reliability of qPCR data is normalization using appropriate internal control genes (ICG). Gene stability analysis identified , among 19 potential ICG, API5, VABP, and MRPL39 as the most stable ICG in porcine mammary tissue and indicated that the use of those 3 genes was most appropriate for calculating a normalization factor. Results underscore the importance of proper validation of internal controls for qPCR and highlight the limitations of using absence of time effects as the criteria for selection of appropriate ICG.