Mono(2-ethylhexyl) phthalate (MEHP) induces transcriptomic alterations in oocytes and their derived blastocysts
. Toxicology 2019
Mono(2-ethylhexyl) phthalate (MEHP), the main di(2-ethylhexyl) phthalate (DEHP) metabolite, is a known reproductive toxicant. Residual levels of 20 nM MEHP have been found in follicular fluid aspirated from IVF-treated women and DEHP-treated animals. The current study examined whether these residual MEHP levels have any effect on the follicle-enclosed oocyte or developing embryo. Bovine oocytes were matured with or without 20 nM MEHP for 22 h. Microarray analysis was performed for both mature oocytes and 7-day blastocysts. A proteomic analysis was performed on mature oocytes (n = 200/group) to reveal a possible direct effect on the oocyte proteomic profile. Transcriptome analysis revealed MEHP-induced alterations in the expression of 456 and 290 genes in oocytes and blastocysts, respectively. The differentially expressed genes are known to be involved in various biological pathways, such as transcription process, cytoskeleton regulation and metabolic pathway. Among these, the expression of 9 genes was impaired in both oocytes exposed to MEHP (i.e., direct effect) and blastocysts developed from those oocytes (i.e., carryover effect). In addition, 191 proteins were found to be affected by MEHP in mature oocytes (Data are available via ProteomeXchange with identifier PXD012092). The study explores, for the first time, the risk associated with exposing oocytes to low concentration (i.e., environmentally relevant concentration) of MEHP to the maternal transcripts. Although it was the oocytes that were exposed to MEHP, alterations carried over to the blastocyst stage, following embryonic genome activation, implying that these embryos are of low quality.
168 Melatonin slightly alleviates the effect of heat shock on nuclear maturation of the bovine oocyte
. Reproduction, Fertility and Development 2019
, 209 - 209. Publisher's VersionAbstract
Exposing bovine cumulus-oocyte complexes (COC) to heat shock during in vitro maturation (IVM) reduces oocyte developmental competence. Melatonin has been reported to improve cleavage of heat-shocked oocytes after fertilization. The current study examined whether protective activity of melatonin is associated with nuclear or cytoplasmic maturation events, or both. The COC were aspirated from Holstein ovaries collected at an abattoir and randomly divided into 3 experimental groups. The control COC were matured (22 h) in standard oocyte maturation medium (OMM) at 38.5°C and 5% CO2; the treatment COC were matured (22 h) in OMM at 41.5°C and 6% CO2 (HS) without or with 10−7 M melatonin (HS+M). Data are presented as least squares means ± s.e.m. To examine nuclear maturation, COC were denuded of cumulus cells at the end of maturation, fixed in 2% (vol/vol) paraformaldehyde, and labelled with 10 μg mL−1 4′,6-diamidino-2-phenylindole (DAPI) (200 COC, 3 replicates). The meiotic status of denuded oocytes was evaluated under inverted fluorescence microscope using Nis Elements software (Nikon, Tokyo, Japan). The proportion of oocytes that resumed meiosis and reached the metaphase II (MII) stage was lower in the HS group than in the control (13.7 v. 55%, respectively; chi-squared, P < 0.05); melatonin slightly alleviated this effect, as indicated by a higher proportion of MII-stage oocytes in HS+M than in the HS group (32 v. 13.7%, respectively; chi-squared, P < 0.05). Cytoplasmic maturation was evaluated according to the distribution pattern of cortical granules (CG). Matured COC were denuded and the oocytes were stained with 100 μg mL−1 fluorescein isothiocyanate-conjugated Arachis hypogaea agglutinin (FITC-PNA) and counterstained with 10 μg mL−1 DAPI. Oocytes were examined under an inverted fluorescence microscope and classified as 1 of 3 types according to their cortical distribution pattern (3 replicates; 200 COC). The proportion of oocytes classified as type 3, which is associated with cytoplasmic maturation, did not differ between the experimental groups, either when analysed for total oocytes or for nuclear matured oocytes (i.e. MII stage). To evaluate the level of reactive oxygen species (ROS), matured-denuded oocytes were labelled using 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA) and the intracellular fluorescence of DCF was immediately measured by fluorescence microscopy (3 replicates, 425 COC). Heat shock increased ROS level relative to the control (68.8 ± 5.32 v. 33.9 ± 4.08 pixels intensity, respectively; P < 0.05) and melatonin tended to alleviate the effect of heat stress to some extent (56.0 ± 4.78; P < 0.06). In summary, heat shock impairs the process of nuclear maturation, expressed by reduced proportion of MII-stage oocytes at the end of maturation. Melatonin partly overcomes this effect, perhaps through its antioxidant activity.
Cyclic-di-GMP regulation promotes survival of a slow-replicating subpopulation of intracellular Salmonella Typhimurium
. Proceedings of the National Academy of Sciences 2019
, 6335. Publisher's VersionAbstract
Cyclic di-GMP is a bacterial second messenger that transmits extracellular signals to the intracellular environment via sensor cyclic-di-GMP−metabolizing enzymes. Here a fluorescent biosensor is used to accurately measure cyclic-di-GMP concentrations in thousands of individual intracellular Salmonella Typhimurium. Furthermore, three enzymes that reduce cyclic-di-GMP concentrations were identified and shown to be essential for reduction of cyclic di-GMP, intracellular survival, and full virulence for mice. This was due to cyclic-di-GMP−mediated overproduction of cellulose that specifically affected a population of slowly replicating bacteria. These results further our knowledge of mechanisms of virulence and persistence of this important pathogen.Salmonella Typhimurium can invade and survive within macrophages where the bacterium encounters a range of host environmental conditions. Like many bacteria, S. Typhimurium rapidly responds to changing environments by the use of second messengers such as cyclic di-GMP (c-di-GMP). Here, we generate a fluorescent biosensor to measure c-di-GMP concentrations in thousands of individual bacteria during macrophage infection and to define the sensor enzymes important to c-di-GMP regulation. Three sensor phosphodiesterases were identified as critical to maintaining low c-di-GMP concentrations generated after initial phagocytosis by macrophages. Maintenance of low c-di-GMP concentrations by these phosphodiesterases was required to promote survival within macrophages and virulence for mice. Attenuation of S. Typhimurium virulence was due to overproduction of c-di-GMP−regulated cellulose, as deletion of the cellulose synthase machinery restored virulence to a strain lacking enzymatic activity of the three phosphodiesterases. We further identified that the cellulose-mediated reduction in survival was constrained to a slow-replicating persister population of S. Typhimurium induced within the macrophage intracellular environment. As utilization of glucose has been shown to be required for S. Typhimurium macrophage survival, one possible hypothesis is that this persister population requires the glucose redirected to the synthesis of cellulose to maintain a slow-replicating, metabolically active state.
Chapter 16 - Corpus Luteum Formation
. In The Ovary (Third Edition)
; The Ovary (Third Edition); Academic Press, 2019; pp. 255 - 267. Publisher's VersionAbstract
The ovulatory surge of LH triggers extensive structural, cellular, and molecular changes in the periovulatory follicle, leading to ovulation and corpus luteum (CL) formation. In recent years, it has become apparent that hypoxia plays an important complementary role in CL formation. The hypoxic microenvironment in this fast-growing tissue is a major contributor to its ability to survive by inducing an intricate vascular network. The intense angiogenic process during CL formation results in an extremely high rate of blood flow in the mature gland, which facilitates nutrient intake and hormonal (progesterone) discharge. Vascular endothelial growth Factor A (VEGFA), fibroblast growth Factor 2 (FGF2), and prostaglandin endoperoxide 2 (PGE2) are implicated in luteal angiogenesis. The neovascularization is accompanied by extensive extracellular matrix (ECM) remodeling. Cellular responses to hypoxia are mediated by hypoxia-inducible factor 1A (HIF1), an oxygen-regulated transcriptional activator. Two prominent HIF1-dependent luteal genes are discussed: VEGFA and endothelin-2; these factors affect ovulation, angiogenesis, and CL formation. MicroRNA-210 (miR-210), known as hypoxiamiR, is a critical element of HIF1A-dependent responses in granulosa-lutein cells. These HIF1A/miR-210pathways are similarly used by the two main signals driving CL formation, namely, hypoxia and LH.
Establishment of specific enzyme-linked immunosorbent assay (ELISA) for measuring Fsh and Lh levels in medaka (Oryzias latipes), using recombinant gonadotropins
. MethodsX 2019
1473 - 1479. Publisher's VersionAbstract
The paucity of information on understanding the regulatory mechanisms that are involved in the control of piscine Fsh and Lh synthesis, secretion, and function, prompted the present work. Part of the problem is related to the molecular heterogeneity and the unavailability of Fsh and Lh assays for quantifying gonadotropins, in particular assays regarding the measurement of Fsh, and such assays are available today for only a few teleost species. The present study reports the development and validation of competitive ELISAs for quantitative determination of medaka Fsh and Lh by first producing medaka recombinant (md) gonadotropins mdFshβ, mdLhβ, mdFshβα, and mdLhβα by Pichia pastoris, generating specific antibodies against their respective β subunits, and their use within the development of ELISAs. The advantages of this protocol include: •The reproducibility of the ELISA demonstrated was relatively high, as shown by reasonably low intra- (Fsh 2.7%, Lh 3%) and interassay CVs (Fsh 5.3%, Lh 5.7%).•The high degree of parallelism between serial dilutions of the recombinant and native pituitary-derived Fsh and Lh, may be a sign of similar structures and immunologically similarity.•Two new competitive ELISAs for the quantification of medaka Fsh and Lh were established for the first time.
Melatonin receptors in Atlantic salmon stimulate cAMP levels in heterologous cell lines and show season-dependent daily variations in pituitary expression levels
. Journal of Pineal Research 2019
, e12590. Publisher's VersionAbstract
Abstract The hormone melatonin connects environmental cues, such as photoperiod and temperature, with a number of physiological and behavioural processes, including seasonal reproduction, through binding to their cognate receptors. This study reports the structural, functional and physiological characterization of five high-affinity melatonin receptors (Mtnr1aaα, Mtnr1aa?, Mtnr1ab, Mtnr1al, Mtnr1b) in Atlantic salmon. Phylogenetic analysis clustered salmon melatonin receptors into three monophyletic groups, Mtnr1A, Mtnr1Al and Mtnr1B, but no functional representative of the Mtnr1C group. Contrary to previous studies in vertebrates, pharmacological characterization of four receptors in COS-7, CHO and SH-SY5Y cell lines (Mtnr1Aaα, Mtnr1Aa?, Mtnr1Ab, Mtnr1B) showed induction of intracellular cAMP levels following 2-iodomelatonin or melatonin exposure. No consistent response was measured after N-acetyl-serotonin or serotonin exposure. Melatonin receptor genes were expressed at all levels of the hypothalamo-pituitary-gonad axis, with three genes (mtnr1aa?, mtnr1ab and mtnr1b) detected in the pituitary. Pituitary receptors displayed daily fluctuations in mRNA levels during spring, prior to the onset of gonadal maturation, but not in autumn, strongly implying a direct involvement of melatonin in seasonal processes regulated by the pituitary. To the best of our knowledge, this is the first report of cAMP induction mediated via melatonin receptors in a teleost species.
Data on Western blot and ELISA analysis of medaka (Oryzias latipes) follicle-stimulating hormone (Fsh) and luteinizing hormone (Lh) using recombinant proteins expressed with Pichia pastoris
. Data in Brief 2019
, 1057 - 1063. Publisher's VersionAbstract
The gonadotropins follicle-stimulating hormone (Fsh) and luteinizing hormone (Lh) play essential roles in vertebrate reproduction. This article presents data on molecular weight validation of recombinant medaka (Oryzias latipes) (md) gonadotropins Fshβ (mdFshβ), Lhβ (mdLhβ), Fshβα (mdFshβα), and Lhβα (mdLhβα) generated by Pichia pastoris, as well as data on a validation of produced antibodies against Fshβ and Lhβ by Western blot analysis. Furthermore, the article includes data on Fsh and Lh protein levels in male medaka pituitaries using recombinant mdFshβα and mdLhβα within enzyme-linked immunosorbent assays (ELISAs), in which protein amounts were analyzed related to body weight and age of the fish. This dataset is associated with the research article entitled “Medaka Follicle-stimulating hormone (Fsh) and Luteinizing hormone (Lh): Developmental profiles of pituitary protein and gene expression” (Burow et al., in press).
Medaka follicle-stimulating hormone (Fsh) and luteinizing hormone (Lh): Developmental profiles of pituitary protein and gene expression levels
. General and Comparative Endocrinology 2019
, 93 - 108. Publisher's VersionAbstract
The two gonadotropins follicle-stimulating hormone (Fsh) and luteinizing hormone (Lh) are of particular importance within the hypothalamic-pituitary-gonadal (HPG) axis of vertebrates. In the current study, we demonstrate the production and validation of Japanese medaka (Oryzias latipes) recombinant (md) gonadotropins Fshβ (mdFshβ), Lhβ (mdLhβ), Fshβα (mdFshβα), and Lhβα (mdLhβα) by Pichia pastoris, the generation of specific rabbit antibodies against their respective β subunits, and their use within the development and validation of competitive enzyme-linked immunosorbent assays (ELISAs) for quantification of medaka Fsh and Lh. mdFsh and mdLh were produced as single-chain polypeptides by linking the α subunit with mdFshβ or mdLhβ mature protein coding sequences to produce a “tethered” polypeptide with the β-chain at the N-terminal and the α-chain at the C-terminal. The specificity of the antibodies raised against mdFshβ and mdLhβ was determined by immunofluorescence (IF) for Fshβ and Lhβ on medaka pituitary tissue, while comparison with fluorescence in situ hybridization (FISH) for fshb and lhb mRNA was used for validation. Competitive ELISAs were developed using antibodies against mdFshβ or mdLhβ, and the tethered proteins mdFshβα or mdLhβα for standard curves. The standard curve for the Fsh ELISA ranged from 97.6 pg/ml to 50 ng/ml, and for the Lh ELISA from 12.21 pg/ml to 6.25 ng/ml. The sensitivity of the assays for Fsh and Lh was 44.7 and 70.8 pg/ml, respectively. A profile of pituitary protein levels of medaka Fsh and Lh comparing juveniles with adults showed significant increase of protein amount from juvenile group (body length from 12 mm to 16.5 mm) to adult group (body length from 21 mm to 26.5 mm) for both hormones in male medaka. Comparing these data to a developmental profile of pituitary mRNA expression of medaka fshb and lhb, the mRNA expression of lhb also increased during male maturation and a linear regression analysis revealed a significant increase of lhb expression with increased body length that proposes a linear model. However, fshb mRNA expression did not change significantly during male development and therefore was not correlated with body length. In summary, we have developed and validated homologous ELISA assays for medaka Fsh and Lh based on proteins produced in P. pastoris, assays that will be used to study the functions and regulations of Fsh and Lh in more detail.
Early posthatch thermal stress causes long-term adverse effects on pectoralis muscle development in broilers
. Poultry Science 2019
. Publisher's VersionAbstract
Broiler chicks in the immediate posthatch handling period are exposed to thermal stress, with potentially harmful consequences for muscle growth and structure (e.g., less protein and more fat deposition). We addressed the effects of broiler chicks' exposure to various ambient temperatures during the first 13 D posthatch on their performance, as well as on muscle development and structure, up to day 35. Body weight and pectoralis muscle growth were lower throughout the entire period in the high-heat-exposed chicks (39°C, Hot) and to a lesser extent in the mild-heat-exposed chicks (35°C, Hot Mild) than in the Control chicks that were raised under a commercial protocol. In the cold-exposed chicks (29oC, Cold), BW and pectoralis muscle absolute growth were similar to the Control group throughout the entire period. The lower body and muscle growth in the Hot and Hot Mild groups were reflected in a lower number of myonuclei expressing proliferating cell nuclear cell in pectoralis major muscle cross sections sampled on day 8, in the distribution of myofibers as the experiment progressed, and in mean myofiber diameter on day 35, whereas in the Cold group, these numbers exceeded that of the Control group. However, TUNEL assay revealed similar cell survival in all groups. Hematoxylin–eosin and Oil red O staining revealed the highest fat deposition in the pectoralis muscle derived from the Hot group, whereas lower fat deposition was observed in the Control Cold group. These results were corroborated by immunostaining for CCAAT/enhancer binding protein β in the pectoralis muscle, the levels of which were significantly higher in the Hot and Hot Mild groups on day 35 than in the Control group. Similar results were observed with Sirius red staining for collagen content in the pectoralis muscle. Together, the results imply long-term effects of chronic heat stress vs. cold stress in the early posthatch period on the broiler's body and muscle growth in general and myodegeneration of the pectoralis muscle in particular.
Intra-muscular and oral vaccination using a Koi Herpesvirus ORF25 DNA vaccine does not confer protection in common carp (Cyprinus carpio L.)
. Fish & Shellfish Immunology 2019
, 90 - 98. Publisher's VersionAbstract
Koi Herpes Virus (KHV or Cyprinid Herpesvirus 3, CyHV-3) is among the most threatening pathogens affecting common carp production as well as the highly valuable ornamental koi carp. To date, no effective commercial vaccine is available for worldwide use. A previous study reported that three intramuscular injections with an ORF25-based DNA vaccine, led to the generation of neutralizing antibodies and conferred significant protection against an intraperitoneal challenge with KHV. In the present study, we set out to optimize an ORF25-based DNA vaccination protocol that required fewer injections and would confer protection upon a challenge that better resembled the natural route of infection. To this end, ORF25 was cloned in pcDNA3 either as a soluble protein or as a full-length transmembrane GFP-fusion protein. We tested our ORF25-based DNA vaccines in multiple vaccination trials using different doses, vaccination routes (i.m. injection and oral gavage) and challenge methods (bath and cohabitation). Furthermore, we analysed local and systemic responses to the i.m. injected DNA vaccine through histological and RT-qPCR analysis. We observed a strong protection when fish received three injections of either of the two DNA vaccines. However, this protection was observed only after bath challenge and not after cohabitation challenge. Furthermore, protection was insufficient when fish received one injection only, or received the plasmid orally. The importance of choosing a challenge model that best reflects the natural route of infection and the possibility to include additional antigens in future DNA vaccination strategies against KHV will be discussed.
Multiple interacting QTLs affect disease challenge survival in common carp (Cyprinus carpio)
. Heredity 2019
. Publisher's VersionAbstract
With the steady growth of the human population, food security becomes a prime challenge. Aquaculture is the fastest growing sector providing proteins from an animal source, but outbreaks of infectious diseases repeatedly hamper the production and further development of this sector. Breeding of disease-resistant strains is a desired sustainable solution to this problem. Cyprinid herpes virus-3 (CyHV-3) is a dsDNA virus damaging production of common carp, an important food and ornamental fish. Previously, we have demonstrated successful introgression of CyHV-3 resistance from a feral strain to commercial strains. Here, we used genotyping by sequencing to identify two novel quantitative trait loci (QTLs) for disease survival that map to different linkage groups than two other QTLs that we previously identified. Effects of these four QTLs were validated and further studied in 14 families with various levels of disease resistance. CyHV-3 survival was found to be a quantitative trait conditioned by mild additive QTL effects and by intricate dominant allelic and epistatic QTL–QTL interactions. Both rare feral alleles and alleles common to feral and cultured strains contributed to survival. This and other advantages of feral alleles introgression were demonstrated. These QTLs, which affected survival of individuals within families, had no significant effect on variation in cumulative family % survival, suggesting that more between family variation remains to be explored. Unraveling the underlying genetics of survival is important for enhancing the breeding of resistant strains and our knowledge of disease resistance mechanisms.
Milk Lipids: A Complex Nutrient Delivery System
. Nestlé Nutr Inst Workshop Ser
, 2019, 90
, 217 - 225. Publisher's VersionAbstract
The evolution of lactation and the composition, structures, and functions of milk's biopolymers illustrates that the Darwinian pressure on lactation selected for gene products with considerable structural complexity and diverse functions within the digestive system. For example, complex sugar polymers - oligosaccharides - possess unique properties in guiding the growth of intestinal bacteria that are not possible by feeding their simple sugars. The proteins of milk are diverse with some exhibiting enzymatic activities towards other milk components rendering those components both more digestible but also releasing biologically active products. Thus, research into milk's biopolymers has been most enlightening when milk was investigated for the formation and disassembly of its structures and for the functions within the infant. To date however, the most complex structure in mammalian milk, the fat globule, has not been effectively examined beyond its simple composition. The globules of milk are heterogeneous in size, composition, and function. With new research tools, scientists are beginning to understand the mechanisms that control the assembly of globules in the mammary gland and the disassembly within the infant.
Changes in lipid droplets morphometric features in mammary epithelial cells upon exposure to non-esterified free fatty acids compared with VLDL
. PLOS ONE 2019
, e0209565 -. Publisher's VersionAbstract
The effects of the macrostructure of long chain fatty acids on the lipid metabolism and biosynthesis of lipid droplets (LD) was studied in mammary epithelial cells (MEC). MEC were exposed to similar compositions and concentrations of fatty acids in the form of either triglycerides (Tg), as part of the very-low-density lipids (VLDL) isolated from lactating cow plasma, or as non-esterified- free fatty acids (FFA). Exposing MEC to FFA resulted in two distinct processes; each independently could increase LD size: an elevation in Tg production and alterations in phospholipid (PL) composition. In particular, the lower PC/PE ratio in the FFA treatment indicated membrane destabilization, which was concomitant with the biosynthesis of larger LD. In addition, 6 fold increase in the cellular concentration of the exogenously added linoleic acid (C18:2) was found in MEC treated with FFA, implying that long chain fatty acids administrated as FFA have higher availability to MEC, enabling greater PL synthesis, more material for the LD envelope, thereby enhancing LD formation. Availability of long chain fatty acids administrated as VLDL-Tg, is dependent on LPL which its activity can be inhibited by the hydrolysis products. Therefore, we used increasing concentrations of albumin, to reduce the allosteric inhibition on LPL by the hydrolysis products. Indeed, a combined treatment of VLDL and albumin, increased LD size and number, similar to the phenotype found in the FFA treatment. These results reveal the role played by the macrostructure of long chain fatty acids in the regulation of LD size in MEC which determine the size of the secreted MFG.
Symposium review: Milk fat globule size: Practical implications and metabolic regulation
. Journal of Dairy Science 2019
, 2783 - 2795. Publisher's VersionAbstract
ABSTRACTMilk fat globule (MFG) size ranges over 3 orders of magnitude, from less than 200 nm to over 15 µm. The significance of MFG size derives from its tight association with its lipidome and proteome. More specifically, small MFG have relatively higher content of membrane compared with large globules, and this membrane exerts diverse positive health effects, as reported in human and animal studies. In addition, MFG size has industrial significance, as it affects the physicochemical and sensory characteristics of dairy products. Studies on the size regulation of MFG are scarce, mainly because various confounders indirectly affect MFG size. Because MFG size is determined before and during its secretion from mammary epithelial cells, studies on the size regulation of its precursors, the intracellular lipid droplets (LD), have been used as a proxy for understanding the mechanisms controlling MFG size. In this review, we provide evidence for 2 distinct mechanisms regulating LD size in mammary epithelial cells: co-regulation of fat content and triglyceride-synthesis capacity of the cells, and fusion between LD. The latter is controlled by the membrane's polar lipid composition and involves mitochondrial enzymes. Accordingly, this review also discusses MFG size regulation in the in vivo metabolic context, as MFG morphometric features are often modulated under conditions that involve animals' altered energy status.
Thrombospondin-1 at the crossroads of corpus luteum fate decisions
. Reproduction 2018
The multimodular matricellular protein thrombospondin-1 (THBS1) was among the first identified endogenous antiangiogenic molecules. Recent studies have shown THBS1-mediated suppression of angiogenesis and other critical activities for corpus luteum (CL) regression. THBS1 is specifically induced by prostaglandin F2alpha in mature CL undergoing regression, whereas luteinizing signals such as luteinizing hormone and insulin reduced its expression. THBS1 interacts both synergistically and antagonistically with other essential luteal factors, such as fibroblast growth factor 2, transforming growth factor beta1, and serpin family E member 1, to promote vascular instability, apoptosis, and matrix remodeling during luteal regression. Expression of THBS1 is also downregulated by pregnancy recognition signals to maintain the CL during early pregnancy. This dynamic pattern of luteal expression, the extensive interactivity with other luteal factors, and strong antiangiogenic and proapoptotic activities indicate that THBS1 is a major determinant of CL fate.
Innate immune functions of avian intestinal epithelial cells: Response to bacterial stimuli and localization of responding cells in the developing avian digestive tract
. PLoS One 2018
Intestinal epithelial cells are multi-tasked cells that participate in digestion and absorption as well as in protection of the digestive tract. While information on the physiology and immune functions of intestinal epithelial cells in mammals is abundant, little is known of their immune function in birds and other species. Our main objectives were to study the development of anti-bacterial innate immune functions in the rapidly developing gut of the pre- and post-hatch chick and to determine the functional diversity of epithelial cells. After establishing primary intestinal epithelial cell cultures, we demonstrated their capacity to uptake and process bacteria. The response to bacterial products, LPS and LTA, induced expression of pro-inflammatory cytokine genes (IL-6, IL-18) as well as the expression of the acute phase proteins avidin, lysozyme and the secretory component derived from the polymeric immunoglobulin receptor. These proteins were then localized in gut sections, and the goblet cell was shown to store avidin, lysozyme as well as secretory component. Lysozyme staining was also located in a novel rod-shaped intestinal cell, situated at different loci along the villus, thus deviating from the classical Paneth cell in the mammal, that is restricted to crypts. Thus, in the chicken, the intestinal epithelium, and particularly goblet cells, are committed to innate immune protection. The unique role of the goblet cell in chicken intestinal immunity, as well as the unique distribution of lysozyme-positive cells highlight alternative solutions of gut protection in the bird.
Plasma Lipoprotein Particle Subclasses in Preterm Infants
. Am J Perinatol 2018
OBJECTIVE: A pilot study to determine lipoprotein classes and subclasses in premature infants and examine associations with nutritional intake, gestational age (GA), and morbidity.
STUDY DESIGN: Plasma lipoprotein particle concentrations were analyzed in a cohort of 15 premature infants in the first 5 days of life and again at 2 weeks. Breast milk samples were analyzed for fatty acid content. Associations between lipoprotein particle subclasses and GA, breast milk intake, milk fatty acid intake, and chronic lung disease (CLD) were determined.
RESULTS: At 2 weeks of age, more premature infants had higher concentrations of total very low-density lipoprotein and lower concentrations of total high-density lipoprotein (HDL) and large HDL particles (similar to profiles seen in adults and children with infectious disease, cardiometabolic disease, and diabetes). Lower total HDL, large HDL, and medium HDL and a higher small HDL:total HDL ratio at 2 weeks were each associated with CLD with GA a likely confounder. Intake of human milk C18 and C20 fatty acids was inversely correlated with plasma total LDL concentration at 2 weeks of age.
CONCLUSION: Dyslipidemia was common in extremely premature infants and was associated with CLD and with lower intake of specific long chain fatty acids.
The Effect of Iodophor Post-Milking Teat Disinfection on Iodine Content in Goat Milk
. Israel Journal of Veterinary Medicine 2018
, 14 - 22.Abstract
Iodine intake is important for thyroid function and human health. Goat milk can be an important source of iodine for human nutrition. However, data regarding the effect of iodophor post-milking teat disinfection on iodine content in goat milk is lacking. Our aim was to assess the iodine concentrations in raw milk of dairy goats and to investigate the effect of post-milking teat-dipping iodophor practice on iodine content in goat milk. Two groups of dairy goats (n=6 in each) were treated with different post-milking teat-dipping disinfection: iodine-free solution (iodine-free group) and iodine-based solution (4,000 μg/L) (iodophors group). Treatments were carried out for 19 experimental days, following a 14-day pre-experimental period, in which only iodine-free sanitizer was used for both groups. The results showed that Iodine concentrations in milk of all goats were 49 ± 23, 49, 17-86 μg/100g (mean ± SD, median, range) at days-3,-2,-1 and 45 ± 26, 42, 14-96 μg/100g (mean ± SD, median, range) at days 17, 18, 19 of treatment. Iodine concentration increased by 7 μg/100g (mean) in the iodophors group while iodine concentration decreased by 15 μg/100g by day 17-19 of treatment in the iodine free group. It was concluded that relatively high iodine concentrations were found in raw milk of dairy goats whose teats were dipped post-milking in disinfectants with or without iodine. Post-milking teat-dipping iodophor practice may increase iodine content in goat milk within an average period of 20 days. This information can help in controlling iodine content in goat milk and iodine intake in the public.
Fluorimetric Techniques for the Assessment of Sperm Membranes
. JoVE 2018
, e58622. Publisher's VersionAbstract
Standard spermiograms describing sperm quality are mostly based on the physiological and visual parameters, such as ejaculate volume and concentration, motility and progressive motility, and sperm morphology and viability. However, none of these assessments is good enough to predict the semen quality. Given that maintenance of sperm viability and fertilization potential depends on membrane integrity and intracellular functionality, evaluation of these parameters might enable a better prediction of sperm fertilization competence. Here, we describe three feasible methods to evaluate sperm quality using specific fluorescent probes combined with fluorescence microscopy or flow cytometry analyses. Analyses assessed plasma membrane integrity using 4',6-diamidino-2-phenylindole (DAPI) and propidium iodide (PI), acrosomal membrane integrity using fluorescein isothiocyanate-conjugated Pisum sativum agglutinin (FITC-PSA) and mitochondrial membrane integrity using 5,5',6,6'-tetra-chloro-1,1',3,3'-tetraethylbenzimidazolyl carbocyanine iodide (JC-1). Combinations of these methods are also presented. For instance, use of annexin V combined with PI fluorochromes enables assessing apoptosis and calculating the proportion of apoptotic sperm (apoptotic index). We believe that these methodologies, which are based on examining spermatozoon membranes, are very useful for the evaluation of sperm quality.