Thrombospondin-1 (THBS1) affects corpus luteum (CL) regression. Highly induced during luteolysis, it acts as a natural anti-angiogenic, proapoptotic compound. THBS1 expression is regulated in bovine luteal endothelial cells (LECs) by fibroblast growth factor-2 (FGF2) and transforming growth factor-beta1 (TGFB1) acting in an opposite manner. Here we sought to identify specific microRNAs (miRNAs) targeting THBS1 and investigate their possible involvement in FGF2 and TGFB1-mediated THBS1 expression. Several miRNAs predicted to target THBS1 mRNA (miR-1, miR-18a, miR-144, miR-194, and miR-221) were experimentally tested. Of these, miR-221 was shown to efficiently target THBS1 expression and function in LECs. We found that this miRNA is highly expressed in luteal cells and in mid-cycle CL. Consistent with the inhibition of THBS1 function, miR-221 also reduced Serpin Family E Member 1 [SERPINE1] in LECs and promoted angiogenic characteristics of LECs. Plasminogen activator inhibitor-1 (PAI-1), the gene product of SERPINE1, inhibited cell adhesion, suggesting that PAI-1, like THBS1, has anti-angiogenic properties. Importantly, FGF2, which negatively regulates THBS1, elevates miR-221. Conversely, TGFB1 that stimulates THBS1, significantly reduces miR-221. Furthermore, FGF2 enhances the suppression of THBS1 caused by miR-221 mimic, and prevents the increase in THBS1 induced by miR-221 inhibitor. In contrast, TGFB1 reverses the inhibitory effect of miR-221 mimic on THBS1, and enhances the upregulation of THBS1 induced by miR-221 inhibitor. These data support the contention that FGF2 and TGFB1 modulate THBS1 via miR-221. These in vitro data propose that dynamic regulation of miR-221 throughout the cycle, affecting THBS1 and SERPINE1, can modulate vascular function in the CL. © The Author(s) 2017. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved.

In ruminants, uterine pulses of prostaglandin (PG) F 2α characterize luteolysis, while increased PGE 2 /PGE 1 distinguish early pregnancy. This study evaluated intrauterine (IU) infusions of PGF 2α and PGE 1 pulses on corpus luteum (CL) function and gene expression. Cows on day 10 of estrous cycle received 4 IU infusions (every 6 h; n = 5/treatment) of saline, PGE 1 (2 mg PGE 1), PGF 2α (0.25 mg PGF 2α), or PGE 1 + PGF 2α. A luteal biopsy was collected at 30 min after third infusion for determination of gene expression by RNA-Seq. As expected, IU pulses of PGF 2α decreased (P < 0.01) P4 luteal volume. However, there were no differences in circulating P4 or luteal volume between saline, PGE 1, and PGE 1 + PGF 2α, indicating inhibition of PGF 2α -induced luteolysis by IU pulses of PGE 1. After third pulse of PGF 2α, luteal expression of 955 genes were altered (false discovery rate [FDR] < 0.01), representing both typical and novel luteolytic transcriptomic changes. Surprisingly, after third pulse of PGE 1 or PGE 1 + PGF 2α, there were no significant changes in luteal gene expression (FDR > 0.10) compared to saline cows. Increased circulating concentrations of the metabolite of PGF 2α (PGFM; after PGF 2α and PGE 1 + PGF 2α) and the metabolite PGE (PGEM; after PGE 1 and PGE 1 + PGF 2α) demonstrated that PGF 2α and PGE 1 are entering bloodstream after IU infusions. Thus, IU pulses of PGF 2α and PGE 1 allow determination of changes in luteal gene expression that could be relevant to understanding luteolysis and pregnancy. Unexpectedly, by third pulse of PGE 1, there is complete blockade of either PGF 2α transport to the CL or PGF 2α action by PGE 1 resulting in complete inhibition of transcriptomic changes following IU PGF 2α pulses. © The Author(s) 2017. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail:.

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. © 2018 Mesilati-Stahy, Argov-Argaman. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

This paper reports the use of a novel automatic vitrification device (Sarah, Fertilesafe, Israel) for cryopreservation of oocytes and embryos.

ABSTRACT This study aimed to identify individual characteristics differing among high-efficiency (HEf; upper 20

Tilapia lake virus (TiLV) is an emerging pathogen of Tilapiines associated with high mortalities of wild and farmed tilapia posing great threat to the fishery industry worldwide. The virus has been reported in Israel, Ecuador, Colombia, Thailand, Egypt, Taiwan, India and Malaysia. In this study, a reverse transcription polymerase chain reaction (RT-PCR) assay was developed and used to detect TiLV genome in Nile tilapia from Lake Victoria. Nile tilapia samples were collected from the Tanzanian (108 fish) and Ugandan (83 fish) parts of Lake Victoria in 2015 and 2016, respectively. Samples were screened for TiLV by using RT-PCR and the PCR products were sequenced. The findings show that out of the 191 fish examined, 28 had PCR products showing the presence of TiLV genome. The TiLV nucleic acids were detected in the spleen (10.99%, N=191), head kidney (7.69%, N=65), heart (3.45%, N=29) and liver (0.71%, N=140) samples while no PCR amplification was detected in the brain by the developed RT-PCR method. Generally, the findings show that the lymphoid organs, mainly comprising of the head kidney and spleen had the highest number of samples with positive nucleic acids for TiLV followed by heart samples. On the contrary, the liver and brain that have previously been shown to be target organs during acute infection either did not have or had the lowest level of TiLV nucleic acids detected in the present study. All the 28 sequences retrieved had an average length of 768 bp. A blast analysis on NCBI showed that all sequences obtained were homologous to TiLV segment-2 sequences obtained from previous outbreaks in Israel and Thailand. To our knowledge, this is the first detection of TiLV subclinical infections in Nile tilapia in Lake Victoria, a none-outbreak area.

Intensive pond cultivation of Gracilaria may achieve maximal yields by enrichment of seawater with ammonia and CO2. In the present study, we examined the use of organic waste material as a source of ammonia and inorganic carbon. A biofiltration system was constructed, consisting of several fermentation tanks and a fluidized bed reactor for the required bioconversion of organic wastes. Gracilaria conferta was cultured in a 30m2 pond from which seawater was circulated through the biofiltration system for a period of nine months. As compared to the filtered seawater used for water supply, the outlets of the fermentation tanks and the fluidized bed reactor showed significant increases in dissolved inorganic carbon and ammonia concentrations and significant decreases in oxygen concentrations and redox potentials. The most efficient organic waste compound tested had the highest C/N ratio. No significant differences were found between the Gracilaria yields of the control and biofiltration pond systems. These results might contribute a fundamental improvement in the economy of Gracilaria pond cultivation by water recycling through such a biofiltration system. Statement of relevance The system might contribute a fundamental improvement in the economy of Gracilaria pond cultivation.

The accumulation of geosmin and 2-methylisoborneol in culture water and fish is a common problem encountered in recirculating aquaculture systems (RAS). In these systems, like in other aquaculture systems, abatement of this problem is usually conducted by purging the fish with clean water prior to their marketing. In indoor RAS, mainly heterotrophic prokaryotes underlie the production of geosmin and MIB. While production of these off flavor compounds has been linked with the organic-rich parts of these systems, no further information on factors that promote the growth of the geosmin and MIB-producing microorganisms under these conditions is currently available. Thus far, geosmin and MIB removal from RAS has mainly been conducted by ozonation, albeit with limited success. Biodegradation of geosmin and MIB might serve as an additional method for removal of these compounds from RAS. Geosmin and MIB degradation has been detected in the latter systems, yet factors which promote the activity of geosmin and MIB degraders in these systems remain largely unknown. In the present review, the current knowledge on geosmin and MIB production and removal in RAS is presented. Emphasis is placed on the biodegradation of these off-flavor compounds, a process which has received little attention thus far.

ABSTRACT Previous studies demonstrated that in-ovo photostimulation with monochromatic green light increased the somatotropic axis expression in broilers embryos. The objective of the current study was to detect the critical period for in-ovo GL photostimulation, in order to find the optimal targeted photostimulation period during the incubation process. Three hundred thirty-six fertile broiler eggs were divided into 4 groups. The first group was incubated under dark conditions as a negative control. The second incubated under intermittent monochromatic green light using light-emitting diode (LED) lamps with an intensity of 0.1 W\m2 at shell level from d 0 of the incubation as a positive control. The third group incubated under intermittent monochromatic green light from d 10 of the incubation. The last group incubated under intermittent monochromatic green light from d 15 of the incubation. In-ovo green light photostimulation from embryonic d 0 (ED0) increased plasma growth hormone (GH), as well as hypothalamic growth hormone releasing hormone (GHRH) and liver growth hormone receptor (GHR) and insulin-like growth factor-1 (IGF-1) mRNA levels. In-ovo green light photostimulation from ED10 increased the GH plasma levels compared to the negative control group, without affecting somatotropic axis mRNA genes expressions of GHRH, GHR, and IGF-1. In-ovo green light photostimulation from ED15 caused an increase in both the plasma GH levels and the somatotropic axis mRNA genes expressions of GHRH, GHR, and IGF-1, compared to the negative control group. These results suggest that the critical period of somatotropic axis acceleration by GL photostimulation start at 15 d of incubation.

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.

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.

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.

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.

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.

The oocyte achieves its developmental competence through the lengthy process of folliculogenesis. It can therefore potentially be exposed to various stressors while enclosed in the follicle. Oocyte maturation relies mainly on maternal sources. These include nuclear, cytoplasmic, and molecular maturation, which involve DNA and RNA organization. Maternal transcripts are dominant through the first embryonic cleavages, up until embryonic genome activation. Thus, it is suggested that any perturbations during oocyte storage, in particular of the maternal transcripts, might lead to genetic and/or epigenetic changes, which might be further expressed in the developing embryo. The review discusses the effects of three representative stressors?environmental heat stress, endocrine-disrupting compounds (phthalates), and inflammatory stress (mastitis)?shown to be involved in reduced fertility. The review highlights the carryover response from the oocyte to the developing embryo; it includes intracellular and molecular disruptive mechanisms with an emphasis on maternal transcripts. The review provides insights into the oocyte?s cellular and molecular responses with an emphasis on the effects of various stressors on the maternal (nuclear and mitochondrial) transcripts and the association with embryonic development. A comparison between stressors might clarify, at least in part, a few open questions. For instance, (a) whether stress-induced alterations share the same mechanism and if so (b) whether this mechanism involves alterations of maternal transcripts; (c) whether stress-induced alterations in the maternal transcript are further expressed at the developing blastocyst stage, that is, after embryonic genome activation.

ABSTRACTStress can affect reproductive performance of lactating cows by targeting the ovarian pool of follicles and their enclosed oocytes. Among the documented stressors are heat stress (i.e., high temperature-humidity index) as well as environmental and food toxins. Oocytes collected during the hot season are of lower quality than those collected in the winter, expressed by reduced oocyte maturation and developmental competence. A similar pattern has been reported for oocytes exposed to endocrine-disrupting chemicals. Whereas the underlying mechanism might differ among stressors, accumulating evidence suggests that stress-induced impairment of oocyte developmental competence involves alterations in mitochondrial functioning. Within the oocyte, mitochondria are involved in ATP generation, calcium homeostasis, regulation of cytoplasmic reduction–oxidation, signal transduction, and apoptosis. Summer heat stress is strongly associated with alterations in mitochondrial distribution and alterations in mitochondria membrane potential. Heat stress impairs the expression of mitochondrion-associated genes, in particular those related to mitochondrial DNA transcription and replication and encoding oxidative phosphorylation complexes for ATP production. Reduction of ATP levels below the required threshold is suggested to compromise the progression of oocyte maturation and, subsequently, embryonic development. Another mechanism associated with mitochondrial function is the increase in reactive oxygen species (ROS), which has been documented in oocytes exposed to heat stress or environmental toxicants. Oxidative phosphorylation in mitochondria is the major source of ROS. Under physiological conditions, ROS are essential for nuclear maturation; however, disequilibrium between ROS production and antioxidative capacity might lead to DNA damage and apoptosis. The current review provides new insights into the oocyte's cellular and molecular responses to stress with an emphasis on the mitochondria. It discusses some strategies to mitigate the effects of stress on the mitochondria, such as incorporation of coenzyme Q10—a key component of the mitochondrial respiratory chain—administration of antioxidants, and injection of healthy mitochondria. Exploring the oocyte's cellular and molecular responses, in particular that of the mitochondria, might lead to the development of new strategies to mitigate the effects of various stressors on fertility.

Aflatoxins are poisonous byproducts of the soilborne fungus Aspergillus, involved in the decomposition of plant materials. Aflatoxins can be found in various food products, such as maize, sorghum, millet, rice and wheat. AFB1 is the most toxic of these, classified as a carcinogen and mutagen for both humans and animals. AFB1 has been detected in human cord blood and placenta; however, its toxic effect on sperm is less known. The current study examines sperm responses associated with AFB1 exposure. These included acrosome integrity and function, mitochondrial polarity, DNA fragmentation, fertilization competence and early embryonic development. Spermatozoa were obtained from bull ejaculate and epididymis and capacitated in vitro for 4h with 0, 0.1, 1, 10 and 100μM AFB1. Following capacitation, acrosome reaction (AR) was induced by Ca2+ ionophore. The integrity and functionality of sperm were examined simultaneously by florescent staining. A Halosperm DNA fragmentation kit was used to evaluate DNA integrity. An in-vitro culture system was used to evaluate fertilization competence and blastocyst formation rate, using bovine oocytes. Findings indicate dose-responsive variation among compartments to AFB1 exposure. Sperm viability, expressed by integrity of the plasma membrane, was lower in sperm isolated from ejaculate or epididymis after culturing with AFB1. Exposure to AFB1 reduced the proportion of sperm from the epididymis tail undergoing acrosome reaction induced by Ca2+ ionophore. AFB1 impaired mitochondrial membrane potential (ΔYm) in sperm isolated from ejaculate and the epididymis tail. Exposing ejaculated sperm to AFB1 increased the proportion of sperm with fragmented DNA and reduced the proportion of embryos that cleaved to the 2- to 4-cell stage, 42h postfertilization, however, the proportion of embryos that developed to blastocysts, 7days postfertilization, did not differ among groups. The findings explore the harmful effects of AFB1 on sperm viability, ΔΨm and DNA integrity associated with fertility competence. We postulate that AFB1-induced fragmentation in paternal DNA might have a carryover effect on the quality of developing embryos. Further evaluation for the quality of blastocysts derived from sperm exposed to AFB1 is warranted.

The mesotocinergic (MTergic) and dopaminergic (DAergic) systems have been documented to play pivotal roles in maternal behaviors in native Thai chickens. In native Thai chickens, plasma prolactin (PRL) concentrations are associated with maternal behaviors, which are also controlled by the DAergic system. However, the role of MT in conjunction with the roles of DA and PRL on the neuroendocrine regulation of the transition from incubating to rearing behavior has never been studied. Therefore, the aim of this study was to investigate the association of MT, DA, and PRL during the transition from incubating to rearing behavior in native Thai hens. Using an immunohistochemistry technique, the numbers of MT-immunoreactive (-ir) and tyrosine hydroxylase-ir (TH-ir, a DA marker) neurons were compared between incubating hens (INC; n = 6) and hens for which the incubated eggs were replaced with 3 newly hatched chicks for 3 days after 6, 10, and 14 days of incubation (REC; n = 6). Plasma PRL concentrations were determined by enzyme-linked immunosorbent assay. The results revealed that the numbers of MT-ir neurons within the nucleus supraopticus, pars ventralis (SOv), nucleus preopticus medialis (POM), and nucleus paraventricularis magnocellularis (PVN) increased in the REC hens when compared with those of the INC hens at 3 different time points (at days 9, 13, and 17). On the other hand, the number of TH-ir neurons in the nucleus intramedialis (nI) decreased in the REC13 and REC17 hens when compared with those of the INC hens. However, the number of TH-ir neurons in the nucleus mamillaris lateralis (ML) only decreased in the REC13 hens when compared with the INC13 hens. The decrease in the numbers of TH-ir neurons within the nI and ML is associated with the decrease in the levels of plasma PRL. This study suggests that the presence of either eggs or chicks is the key factor regulating the MTergic system within the SOv, POM, and PVN and the DAergic system within the nI and ML during the transition from incubating to rearing behavior in native Thai chickens. The results further indicate that these two systems play pivotal roles in the transition from incubating to rearing behavior in this equatorial species.