A novel dynamic impact approach (DIA) for functional analysis of time-course omics studies: validation using the bovine mammary transcriptome

The overrepresented approach (ORA) is the most widely-accepted method for functional analysis of microarray datasets. The ORA is computationally-efficient and robust; however, it suffers from the inability of comparing results from multiple gene lists particularly with time-course experiments or those involving multiple treatments. To overcome such limitation a novel method termed Dynamic Impact Approach (DIA) is proposed. The DIA provides an estimate of the biological impact of the experimental conditions and the direction of the impact. The impact is obtained by combining the proportion of differentially expressed genes (DEG) with the log2 mean fold change and mean -log P-value of genes associated with the biological term. The direction of the impact is calculated as the difference of the impact of up-regulated DEG and down-regulated DEG associated with the biological term. The DIA was validated using microarray data from a time-course experiment of bovine mammary gland across the lactation cycle. Several annotation databases were analyzed with DIA and compared to the same analysis performed by the ORA. The DIA highlighted that during lactation both BTA6 and BTA14 were the most impacted chromosomes; among Uniprot tissues those related with lactating mammary gland were the most positively-impacted; within KEGG pathways 'Galactose metabolism' and several metabolism categories related to lipid synthesis were among the most impacted and induced; within Gene Ontology "lactose biosynthesis" among Biological processes and "Lactose synthase activity" and "Stearoyl-CoA 9-desaturase activity" among Molecular processes were the most impacted and induced. With the exception of the terms 'Milk', 'Milk protein' and 'Mammary gland' among Uniprot tissues and SP_PIR_Keyword, the use of ORA failed to capture as significantly-enriched (i.e., biologically relevant) any term known to be associated with lactating mammary gland. Results indicate the DIA is a biologically-sound approach for analysis of time-course experiments. This tool represents an alternative to ORA for functional analysis.     

Bovine platelet-activating factor acetylhydrolase (PAF-AH) activity related to fertility

Plasma platelet-activating factor acetylhydrolase (PAF-AH), the enzyme characterized by the association with plasma lipoproteins, degrades platelet-activating factor (PAF) as well as PAF-like oxidatively fragmented phospholipids produced during oxidative stress. Apart from pro-inflammatory properties, PAF is also related to reproductive processes and successful fertility. In order to get a better insight into the involvement of PAF-AH in the fertility of cows, the aim of the study was to determine the PAF-AH activity as well as the C-reactive protein, cholesterol and high density lipoprotein-cholesterol (HDL-C) in the serum of dairy cows throughout the pregnancy and lactation, as well as in infertile cows. The results showed that serum PAF-AH activity changes throughout pregnancy and lactation with a lower level during periparturient period. It is also found higher PAF-AH activity in lactating cows with reproductive disorders compared to high lactating cows without reproductive disorders. Strong correlation between PAF-AH activity and HDL-C concentration indicates that HDL could have considerable influence on PAF-AH activity in bovine plasma. CRP concentration was also lower during transition period suggesting that lactation might stimulate CRP synthesis in bovine. A higher CRP concentration in cows with reproductive disorders compared to fertile cows at the peak of lactation, demonstrates that milk production is not the only factor influencing CRP in cows. A significant correlation between PAF-AH activity and CRP level shows that both parameters could be influenced by reproductive status of dairy cows.     

Proteomic analysis of physiological function response to hot summer in liver from lactating dairy cows

Lactation performance of dairy cattle is susceptible to heat stress. The liver is one of the most crucial organs affected by high temperature in dairy cows. However, the physiological adaption by the liver to hot summer conditions has not been well elucidated in lactating dairy cows. In the present study, proteomic analysis of the liver in dairy cows in spring and hot summer was performed using a label-free method. In total, 127 differentially expressed proteins were identified; most of the upregulated proteins were involved in protein metabolic processes and responses to stimuli, whereas most of the downregulated proteins were related to oxidation–reduction. Pathway analysis indicated that 3 upregulated heat stress proteins (HSP90α, HSP90β, and endoplasmin) were enriched in the NOD-like receptor signaling pathway, whereas several downregulated NADH dehydrogenase proteins were involved in the oxidative phosphorylation pathway. The protein–protein interaction network indicated that several upregulated HSPs (HSP90α, HSP90β, and GRP78) were involved in more interactions than other proteins and were thus considered as central hub nodes. Our findings provide novel insights into the physiological adaption of liver function in lactating dairy cows to natural high temperature.     

Comparative analysis of gene expression profiles in ruminal tissue from Holstein dairy cows fed high or low concentrate diets

Cattle are often fed high concentrate (HC) diets to increase productivity, although HC diets cause changes in ruminal environment such as pH reduction. Despite those well-documented changes in cattle fed HC diets, there is currently a paucity of data describing the molecular events regulating the ruminal environment. Our objective was to gain an understanding of which genes are differentially expressed in ruminal tissue from Holstein cows fed a HC comparing to low concentrate (LC) diet using microarray analysis using a bovine 24 k microarray. A total of 5,200 differentially expressed genes (DEG) were detected for cows fed HC relative to LC. The DEG were firstly annotated with gene ontology (GO) and Kyoto encyclopedia of Genes and Genomes (KEGG), indicating that the DEG were associated with catalytic activity and MAPK pathway, respectively. Further characterization using GeneCodis identified patterns of interrelated annotations for the DEG to elucidate the relationships among annotation groups revealed that a cAMP-dependent protein kinase A catalytic subunit beta (PRKACB), may be associated with ruminal tissue maintenance. The results contributed to understanding of the regulatory mechanisms at the mRNA level for Holstein cows fed at different concentrate ratio diets.     

Effects of continuous lactation and short dry periods on mammary function and animal health

The dry period is required to facilitate cell turnover in the bovine mammary gland in order to optimize milk yield in the next lactation. Traditionally, an 8-week dry period has been a standard management practice for dairy cows based on retrospective analyses of milk yields following various dry period lengths. However, as milk production per cow has increased, transitioning cows from the nonlactating state to peak milk yield has grown more problematic. This has prompted new studies on dry period requirements for dairy cows. These studies indicate a clear parity effect on dry period requirement. First parity animals require a 60-day dry period, whereas lactations following later parities demonstrate no negative impact with 30-day dry period or even eliminating the dry period when somatotropin (ST) is also used to maintain milk yields. Shortened dry periods in first parity animals were associated with reduced mammary cell turnover during the dry period and early lactation and increased numbers of senescent cells and reduced functionality of lactating alveolar mammary cells postpartum. Use of ST and increased milking frequency postpartum reduced the impact of shortened dry periods. The majority of new intramammary infections occur during the dry period and persist into the following lactation. There is therefore the possibility of altering mastitis incidence by modifying or eliminating the dry period in older parity animals. As the composition of mammary secretions including immunoglobulins may be reduced when the dry period is reduced or eliminated, there is the possibility that the immune status of cows during the peripartum period is influenced by the length of the dry period.     

Differentiation dynamics of mammary epithelial stem cells from Korean holstein dairy cattle under ECM-free conditions

The “stem cells” are commonly defined as “cells capable of self-renewal through replication and differentiating into specific lineages”. The mammary gland contains functional stem/progenitor cells. The current study was planned with the objectives to study the differentiation dynamics of Korean Holstein mammary epithelial stem cells (KHMESCs) under the optimum culture conditions. Lineage negative KHMESCs isolated from mammary tissue of lactating cows have shown the typical differentiation dynamics with formation of lobulo–alveolar structures in in vitro culture. This suggests the existence of bipotential mammary epithelial stem cells in the mammary gland. The strong mRNA expression of pluripotency factors indicates stemness, whereas expression of milk protein genes and epithelial cell-specific gene indicate their differentiation capabilities. Further, immunostaining results have shown the differentiation capabilities of KHMESCs into both luminal and basal lineages under the extracellular matrix (ECM, matrigel) free environment. However, under matrigel, the differentiation process was comparatively higher than without matrigel. Immunostaining results also suggested that differentiated cells could secrete milk proteins such as β-casein. To our knowledge, these data represent the first report on the differentiation dynamics and establishment of mammary epithelial stem cells from Korean Holstein with typical stemness properties. It was observed that isolated KHMESCs had normal morphology, growth pattern, differentiation ability, cytogenetic and secretory activity even without ECM. Therefore, it is concluded that established KHMESCs could be used for further studies on Korean Holstein dairy cows related to lactation studies, as non-GMO animal bioreactors and stem cell-based management of bovine mastitis including post-mastitis damage.     

Saturated fatty acids stimulate and insulin suppresses CIDE-A expression in bovine mammary epithelial cells

Cell death-inducing DNA fragmentation factor-alpha-like effector A (CIDE-A) was first identified by its sequence homology with the N-terminal domain of DNA fragmentation factor (DFF). CIDE-A negatively regulates the activity of uncoupling protein 1 (UCP1) in brown adipose tissue. CIDE-A and UCP1 mRNA were detected by RT-PCR in cloned bovine mammary epithelial cells (bMEC) and lactating bovine mammary glands. Physiological concentrations of saturated fatty acids (stearate and palmitate), but not unsaturated fatty acids (oleate and linoleate) induced up-regulation of CIDE-A mRNA in bMEC. Treatment with insulin (5-10 ng/ml) induced down-regulation of CIDE-A and UCP1. The expression levels of CIDE-A and UCP1 mRNA in bovine mammary glands at various stages of the lactation cycle were determined by quantitative RT-PCR analysis. CIDE-A mRNA expression at peak lactation (2 months after parturition) was significantly higher than at dry off and non-pregnancy but not late lactation. These results suggest that CIDE-A and UCP1 are regulated by insulin and/or fatty acids in mammary epithelial cells and lactating mammary glands, and thereby play an important role in lipid and energy metabolism.     

Effect of timing of first clinical mastitis occurrence on lactational and reproductive performance of Holstein dairy cows

Objectives of this study were to determine the influence of timing of first clinical mastitis case occurrence on lactational and reproductive performance in high producing lactating dairy cows during the first 320 days in milk (DIM). Holstein cows, 1001, from two commercial dairy farms in California were retrospectively divided into four treatment groups according to timing of first clinical mastitis case caused by environmental pathogens: control with no recorded clinical cases of mastitis (C; n=501); first clinical mastitis prior to first postpartum AI (MG1; n=250); first clinical mastitis between first postpartum AI and pregnancy diagnosis (MG2; n=147); and first clinical mastitis after diagnosed pregnant (MG3; n=103). Clinical cases of mastitis were identified at every milking by the herd personnel based on abnormal milk or swelling of the mammary gland. A fore sample of milk was aseptically collected from every clinical case for microbiological culture. Mastitis decreased yields of milk, 3.5% fat-corrected milk, and milk components, but the effect was only observed for MG1 and MG2. Cows in the control group had lower linear somatic cell count (SCC) score throughout the lactation. Culling was increased by mastitis, and cows in the mastitis groups left the study earlier than controls. Conception rate at first postpartum AI and pregnancy rate at the end of the study were both decreased by mastitis prior to or after first AI, and MG1 and MG2 cows had extended days open. Furthermore, cows experiencing mastitis during lactation had a higher incidence of abortions. The negative effects of mastitis on reproduction were observed regardless of clinical case being caused by either Gram positive or negative bacteria. Mastitis either prior to or after first postpartum AI impairs lactation performance, increases culling, and decreases reproductive efficiency in high producing Holstein dairy cows.     

Alterations in the relative abundance of Haptoglobin (Hp) transcripts in total milk somatic cells during different stages of lactation cycle in high yielding cross-bred cows

The expression profile of Haptoglobin (Hp) gene in total milk somatic cells (SCC) of high-yielding cross-bred Karan Fries (KF) was studied during early, mid, and late lactation cycle. Milk samples (200 ml/animals) were collected from 10 high-yielding and 10 low-yielding cows throughout the lactation cycle (from day 7 to day 300) with an interval of one month. Relative mRNA expression profiles of Hp by RT polymerase chain reaction was studied in high-yielding cows, whereas low-yielding cows were taken as control. The folds of induction of Hp was significantly (p < 0.001) downregulated by a mean factor of 0.207 in milk SCC during early lactating cows. Whereas, it was significantly (p < 0.01) upregulated by a mean factor of 20.888 during mid lactation. The expression was unaltered during the late lactation. The study demonstrates that Hp is synthesized within the mammary gland and significantly upregulated during mid-lactation period compared to other stages of lactation cycle.     

Epidemiological study on factors influencing fertility indices in Israeli dairy herds

The objective of this study was to determine feeding factors connected with differences in the fertility of Israeli Kibbutz dairy herds. In an epidemiological case-control survey, data from 30, low-fertility Kibbutz dairy herds having a mean overall conception rate of 35% in multiparous cows were compared with the data from 30, high-fertility Kibbutz dairy herds having a mean overall conception rate of 48% in multiparous cows. Nutritional factors accounted for 67% of the differences between low-fertility and high-fertility herds in the overall conception rate of multiparous cows, while only 4.8% could be related to the body condition during the dry period. Among the factors which occurred more frequently in the low-fertility than in the high-fertility herds were 1) a higher average protein density and lower energy/protein ratio was fed during lactation and 2) a single feeding group was maintained for all lactating cows. 3) There were phytoestrogens in the silage or alfalfa hay, fed during lactation. 4) Faulty dry period was instituted, which was defined as the presence of at least one of the following three practices: a) the daily feed was above 3 kg of high lactation mix; b) more than 15 Mcal of net energy per day was given during the first part of the dry period; c) more than 30% of the cows were obese during the dry period. Three or more risk factors were found in one high-fertility herd and in 20 low-fertility herds. This finding emphasizes the importance of identifying and removing risk factors as a possible means for improving the reproductive performance of herds.     

Microarray analysis of gene expression profiles in the bovine mammary gland during lactation

Mammary glands undergo functional and metabolic changes during virgin, lactation and dry periods. A total of 122 genes were identified as differentially expressed, including 79 up-regulated and 43 down-regulated genes during lactation compared with virgin and dry periods. Gene ontology analysis showed the functional classification of the up-regulated genes in lactation, including transport, biosynthetic process, signal transduction, catalytic activity, immune system process, cell death, and positive regulation of the developmental process. Microarray data clarified molecular events in bovine mammary gland lactation.     

The intake and digestion of maize silage-based diets by dairy cows and sheep

Six lactating cows, 6 dry cows and 6 wether sheep were fed ad libitum on diets of maize silage, maize silage plus lucerne, or maize silage plus lucerne plus wheat. Faeces and urine collections allowed for the determination of digestibility of dry matter, organic matter and nitrogen, and balances of nitrogen and water.

Voluntary feed intakes were highest and digestibility values were lowest in lactating cows. The addition of lucerne reduced organic matter digestibility in dry cows, but not in lactating cows or sheep. The addition of wheat decreased intake in dry cows and sheep, but not in lactating cows. Production of milk, protein, solids-not-fat and total solids increased with dietary quality, but there was a depression in milk fat content as a result of wheat supplementation.

The ranking of the 3 diets on the basis of feed intake differed with each class of livestock, but lactating cows and sheep gave the same ranking on the basis of organic matter digestibility.