ADAMTS Proteases: Importance in Animal Reproduction.

Many reproductive physiological processes, such as folliculogenesis, ovulation, implantation, and fertilization, require the synthesis, remodeling, and degradation of the extracellular matrix (ECM). The ADAMTS (A Disintegrin and Metalloproteinase with Thrombospondin Motifs) family genes code for key metalloproteinases in the remodeling process of different ECM. Several genes of this family encode for proteins with important functions in reproductive processes; in particular, ADAMTS1, 4, 5 and 9 are genes that are differentially expressed in cell types and the physiological stages of reproductive tissues. ADAMTS enzymes degrade proteoglycans in the ECM of the follicles so that the oocytes can be released and regulate follicle development during folliculogenesis, favoring the action of essential growth factors, such as FGF-2, FGF-7 and GDF-9. The transcriptional regulation of ADAMTS1 and 9 in preovulatory follicles occurs because of the gonadotropin surge in preovulatory follicles, via the progesterone/progesterone receptor complex. In addition, in the case of ADAMTS1, pathways involving protein kinase A (PKA), extracellular signal regulated protein kinase (ERK1/2) and the epidermal growth factor receptor (EGFR) might contribute to ECM regulation. Different Omic studies indicate the importance of genes of the ADAMTS family from a reproductive aspect. ADAMTS genes could serve as biomarkers for genetic improvement and contribute to enhance fertility and animal reproduction; however, more research related to these genes, the synthesis of proteins encoded by these genes, and regulation in farm animals is needed.

Properties of Oaxaca Cheese Elaborated with Ultrasound-Treated Raw Milk: Physicochemical and Microbiological Parameters.

The effect of ultrasound-treated fresh raw milk upon yield, physicochemical and microbiological quality of Oaxaca cheese was evaluated under a factorial design. The ultrasound frequencies tested were 25 and 45 kHz, during 15 or 30 min. The cheeses made with the ultrasonicated milk (30 min, high-intensity ultrasound, HIU) had greater luminosity without significant changes in hue or chroma, as compared to the controls with no HIU. The yield improved significantly (by up to 2.8 kg/100 L of milk), as the ultrasound treatment time increased. Such cheese yield is attributable to the higher protein content, which was up to 1.5% higher, after sonication. Long-treatment time (30 min) at 25 kHz significantly lowered mesophilic bacteria counts down to limits allowed by current regulations and favors the growth of lactic acid bacteria (LAB) while lowering mold and yeast counts. The absence of E. coli and Salmonella spp. and the decrease in S. aureus counts in Oaxaca cheese were attributed to the mixing of the paste with hot water, inherent to the traditional elaboration process, and to the antagonistic effect of the ultrasound-triggered increased LAB on pathogenic bacteria. Since the artisanal elaboration of Oaxaca cheese does not comply with the current Mexican regulations regarding mesophiles, ultrasound could be a suitable technology to protect its genuine elaboration process with raw milk.

Dynamics of Gastrointestinal Activity and Ruminal Absorption of the Methane-Inhibitor, Nitroethane, in Cattle.

Nitroethane is a potent methane-inhibitor for ruminants but little is known regarding simultaneous effects of repeated administration on pre- and post-gastric methane-producing activity and potential absorption and systemic accumulation of nitroethane in ruminants. Intraruminal administration of 120 mg nitroethane/kg body weight per day to Holstein cows (n = 2) over a 4-day period transiently reduced (P < 0.05) methane-producing activity of rumen fluid as much as 3.6-fold while concomitantly increasing (P < 0.05) methane-producing activity of feces by as much as 8.8-fold when compared to pre-treatment measurements. These observations suggest a bacteriostatic effect of nitroethane on ruminal methanogen populations resulting in increased passage of viable methanogens to the lower bovine gut. Ruminal VFA concentrations were also transiently affected by nitroethane administration (P < 0.05) reflecting adaptive changes in the rumen microbial populations. Mean (± SD) nitroethane concentrations in plasma of feedlot steers (n = 6/treatment) administered 80 or 160 mg nitroethane/kg body weight per day over a 7-day period were 0.12 ± 0.1 and 0.41 ± 0.1 µmol/mL 8 h after the initial administration indicating rapid absorption of nitroethane, with concentrations peaking 1 day after initiation of the 80 or 160 mg nitroethane/kg body weight per day treatments (0.38 ± 0.1 and 1.14 ± 0.1 µmol/mL, respectively). Plasma nitroethane concentrations declined thereafter to 0.25 ± 0.1 and 0.78 ± 0.3 and to 0.18 ± 0.1 and 0.44 ± 0.3 µmol/mL on days 2 and 7 for the 80 or 160 mg nitroethane/kg body weight per day treatment groups, respectively, indicating decreased absorption due to increased ruminal nitroethane degradation or to more rapid excretion of the compound.

A variant associated to IGF-1 mRNA and protein expression in sheep.

The insulin-like growth factor-1 (IGF-1), a key hormone in muscle development was investigated for single-nucleotide polymorphisms (SNPs) upstream of the IGF-1 gene and their effects upon its cognate mRNA and hormone levels in sheep. A 70 d feeding trial was conducted with 22 F1 (Dorper × Pelibuey) lambs, individually allocated and fed a diet with a forage-to-concentrate ratio of 36:64 and 17% crude protein. Sequence analyses of 265 bp upstream the IGF-1 gene revealed the variant NC_040254.1:g.[184028491G > C;184028493G > A]. These SNPs generate alleles A and B, with frequencies of 0.66 and 0.34 in F1 lambs and of 0.73 and 0.27 in 81 pure Dorper lambs, respectively. Females were grouped by genotype AA, AB and BB (n = 3). IGF-1 hormone concentrations at 14, 42 and 70 d were higher (p < 0.05) in AA lambs compared to AB + BB lambs. The IGF-1 mRNA level was 2.6-fold higher in AA animals (n = 5, p < 0.05) than in AB + BB lambs (n = 7). A DNA binding site for the Inhibitor of Growth family member 4 (ING4) was found in allele B but not in allele A, which could explain the lower mRNA and hormone expression levels for AB + BB animals. The variant reported here appears to function as an eQTL with a negative effect on the level of IGF-1.

Astragallus mollissimus plant extract: a strategy to reduce ruminal methanogenesis.

Ruminal methanogenesis is considered an inefficient process as it can result in the loss of 4 to 12% of the total energy consumed by the ruminant. Recent studies have shown that compounds such as nitroethane, 2-nitroethanol, 2-nitro-1-propanol, and 3-nitro-1-propionic acid are capable of inhibiting methane production during in vitro studies. However, all of these nitrocompounds came from a synthetic origin, which could limit their use. In contrast, some plants of the Astragallus genus produce a natural nitrocompound, although its anti-methanogenic effect has not been evaluated. To determine the anti-methanogenic effect, in vitro cultures of freshly collected mixed populations of ruminal microbes were supplemented with A. mollissimus extracts (MISER). Cultures supplemented with 2-nitroethanol, ethyl 2-nitroacetate, or nitroethane were used as positive controls whereas distilled water was added to the untreated control tubes. After a 24 h incubation period, the methane production was reduced by more than 98% for the samples treated with A. mollissimus extract (P < 0.05) compared to the untreated controls (10.2 ± 0.1 mmol mL-1 incubated liquid). Cultures supplemented with MISER produced a greater (P < 0.05) amount of total VFA, compared to the rest of treated and untreated cultures. Considering that there are significant differences between MISER treatment, positive controls and untreated cultures (P < 0.05) regarding the amounts of total gas, gas composition (CH4 and H2), and the amount of VFA produced, it is concluded that Astragallus mollissimus poses an alternative strategy to reduce ruminal methanogenesis. To further explore such alternative, it is necessary to determine if the metabolization byproducts are safe and/or useful for the animal.

In vitro assessment of two novel Cellulases from Trabulsiella odontotermitis for agricultural waste utilization.

BACKGROUND: The production of agricultural wastes still growing as a consequence of the population growing. However, the majority of these residues are under-utilized due their chemical composition, which is mainly composed by cellulose. Actually, the search of cellulases with high efficiency to degrade this carbohydrate remains as the challenge. In the present experiment, two genes encoding an endoglucanase (EC 3.2.1.4) and ß-glucosidase (EC 3.2.1.21) were overexpressed in Escherichia coli and their recombinant enzymes (egl-FZYE and cel-FZYE, respectively) characterized. Those genes were found in Trabulsiella odontermitis which was isolated from the gut of termite Heterotermes sp. Additionally, the capability to release sugars from agricultural wastes was evaluated in both enzymes, alone and in combination. RESULTS: The results have shown that optimal pH was 6.0 and 6.5, reaching an activity of 1051.65 ± 47.78 and 607.80 ± 10.19 U/mg at 39 °C, for egl-FZYE and cel-FZYE, respectively. The Km and Vmax for egl-FZYE using CMC as substrate were 11.25 mg/mL and 3921.57 U/mg, respectively, whereas using Avicel were 15.39 mg/mL and 2314.81 U/mg, respectively. The Km and Vmax for cel-FZYE using Avicel as substrate were 11.49 mg/mL and 2105.26 U/mg, respectively, whereas using CMC the enzyme did not had activity. Both enzymes had effect on agricultural wastes, and their effect was improved when they were combined reaching an activity of 955.1 ± 116.1, 4016.8 ± 332 and 1124.2 ± 241 U/mg on corn stover, sorghum stover and pine sawdust, respectively. CONCLUSIONS: Both enzymes were capable of degrading agricultural wastes, and their effectiveness was improved up to 60% of glucose released when combined. In summary, the results of the study demonstrate that the recombinant enzymes exhibit characteristics that indicate their value as potential feed additives and that the enzymes could be used to enhance the degradation of cellulose in the poor-quality forage generally used in ruminant feedstuffs.

Influence of sodium chlorate, ferulic acid, and essential oils on Escherichia coli and porcine fecal microbiota.

The influence of sodium chlorate (SC), ferulic acid (FA), and essential oils (EO) was examined on the survivability of two porcine diarrhetic enterotoxigenic Escherichia coli (ETEC) strains (F18 and K88) and populations of porcine fecal bacteria. Fecal bacterial populations were examined by denaturing gradient gel electrophoresis (DGGE) and identification by 16S gene sequencing. The treatments were control (no additives), 10 mM SC, 2.5 mg FA /mL, a 1.5% vol/vol solution of an EO mixture as well as mixtures of EO + SC, EO + FA, and FA + SC at each of the aforementioned concentrations. EO were a commercial blend of oregano oil and cinnamon oil with water and citric acid. Freshly collected porcine feces in half-strength Mueller Hinton broth was inoculated with E. coli F18 (Trial 1) or E. coli K88 (Trial 2). The fecal-E. coli suspensions were transferred to crimp top tubes preloaded with the treatment compounds. Quantitative enumeration was at 0, 6, and 24 h. All treatments reduced (P < 0.05) the counts of E. coli F18 at 6 and 24 h. With the exception of similarity coefficient (%SC), all the other treatments reduced (P < 0.05) the K88 counts at 24 h. The most effective treatments to reduce the F18 and K88 CFU numbers were those containing EO. Results of DGGE revealed that Dice percentage similarity coefficients (%SC) of bacterial profiles among treatment groups varied from 81.3% to 100%SC. The results of gene sequencing showed that, except for SC at 24 h, all the other treatments reduced the counts of the family Enterobacteriaceae, while Lactobacillaceae and Ruminococcaceae increased and Clostridiaceae decreased in all treatments. In conclusion, all treatments were effective in reducing the ETEC, but EO mixture was the most effective. The porcine microbial communities may be influenced by the studied treatments.

Cloning, expression, purification and biochemical characterization of recombinant metallothionein from the white shrimp Litopenaeus vannamei.

Metallothioneins (MTs) are cysteine rich proteins with antioxidant capacity that participate in the homeostasis and detoxification of metals and other cellular processes, and help to counteract the oxidative stress produced by Reactive Oxygen Species (ROS). The production of ROS increases during several stress conditions, including metal intoxication and hypoxia (oxygen deficiency). During hypoxia the expression of the MT gene is induced in the shrimp Litopenaeus vannamei; however, the MT protein coded by this gene has not been purified nor characterized. In this work, the coding sequence of L. vannamei MT was cloned and overexpressed in Escherichia coli as a fusion protein, containing an intein and a chitin binding domain (CBD). The MT was purified by chitin affinity chromatography and its antioxidant capacity and ability to bind cadmium (Cd) and copper (Cu) were evaluated. This MT has an antioxidant capacity of 27.23 µM equivalent to Trolox in a 100 µg/mL solution. Addition of CdCl2 to the culture media augments 273-fold the Cd content, while addition of CuCl2 increases Cu content 569-fold in the purified MT. Thus, the shrimp MT gene codes for a functional protein that has antioxidant capacity and binds Cu and Cd.

Molecular cloning and modeling of the Tp53-induced glycolysis and apoptotic regulator (TIGAR) from the Pacific white shrimp Litopenaeus vannamei and its expression in response to hypoxia.

Hypoxia is a common stressor for aquaculture species. The Pacific white shrimp Litopenaeus vannamei survives low dissolved oxygen (DO) conditions by adjusting its energy metabolism. In vertebrates, the transcription factor p53 regulates glucose metabolism under stress through diverse target genes like the Tp53-induced glycolysis and apoptotic regulator (TIGAR), a protein similar to fructose-2,6-bisphosphatase that has a pro-survival role in cells participating in the defense against oxidative damage. Until now, TIGAR has been not reported in any invertebrate species, including crustaceans. In this work, we report the molecular cloning of the white shrimp TIGAR. The cDNA sequence is 765 bp encoding a 254 amino acid protein. Bioinformatics analyses predicted that although the overall sequence identities of L. vannamei TIGAR and vertebrate proteins are not very high (33.61%-35.34%), they have a remarkable predicted structural similarity with full conservation of catalytic residues, secondary and three-dimensional structures. Gene expression analysis by RT-qPCR revealed that the mRNA abundance of TIGAR in white shrimp is tissue-specific under normal oxygen conditions, with higher expression in gills than hepatopancreas and muscle. Also, gene expression in gills and hepatopancreas is modified by environmental hypoxia, suggesting that TIGAR participates in the cellular tolerance of L. vannamei to this stressor.

Cell cycle regulation and apoptosis mediated by p53 in response to hypoxia in hepatopancreas of the white shrimp Litopenaeus vannamei.

Although hypoxic aquatic environments cause negative effects on shrimp, these animals can withstand somewhat hypoxia, but the cellular mechanisms underlying this capacity are still poorly understood. In humans, mild hypoxia causes the induction of many proteins to allow cell survival. In contrast, apoptosis is induced during severe hypoxia leading to cell death. p53 is a key transcription factor that determines cells fate towards cell cycle arrest or induction of apoptosis in humans. The aim of this work was to study the role of p53 in cell cycle regulation and apoptosis in response to hypoxia in hepatopancreas of the white shrimp Litopenaeus vannamei. p53 was silenced by RNAi and afterwards the shrimp were exposed to hypoxia. Cdk-2 was used as indicator of cell cycle progression while caspase-3 expression and caspase activity were analyzed as indicators of apoptosis. p53 levels in hepatopancreas were significantly higher at 48 h after hypoxic treatment. Increased expression levels of Cdk-2 were found in p53-silenced shrimp after 24 and 48 h in the normoxic treatments as well as 48 h after hypoxia, indicating a possible role of p53 in cell cycle regulation. In response to hypoxia, unsilenced shrimp showed an increase in caspase-3 expression levels, however an increase was also observed in caspase activity at 24 h of normoxic conditions in p53-silenced shrimps. Taken together these results indicate the involvement of p53 in regulation of cell cycle and apoptosis in the white shrimp in response to hypoxia.

Hypoxia drives apoptosis independently of p53 and metallothionein transcript levels in hemocytes of the whiteleg shrimp Litopenaeus vannamei.

The cellular mechanisms used by the shrimp Litopenaeus vannamei to respond to hypoxia have been studied from the energetic metabolism and antioxidant angles. We herein investigated the participation of p53 and metallothionein (MT) in the apoptotic process in response to hypoxia in shrimp hemocytes. The Lvp53 or LvMT genes were efficiently silenced by injection of double stranded RNA for p53 or MT. The effects of silencing on apoptosis were measured as caspase-3 activity and flow cytometry in hemocytes after 24 and 48 h of hypoxia (1.5 mg DO L(-1)). Hemocytes from unsilenced animals had significantly higher apoptosis levels upon both times of hypoxia. The apoptotic levels were diminished but not suppressed in dsp53-silenced but not dsMT-silenced hemocytes after 24 h of hypoxia, indicating a contribution of Lvp53 to apoptosis. Apoptosis in normoxia was significantly higher in dsp53-and dsMT-silenced animals compared to the unsilenced controls, pointing to a possible cytoprotective role of LvMT and Lvp53 during the basal apoptotic program in normoxia. Overall, these results indicate that hypoxia augments apoptosis in shrimp hemocytes and high mRNA levels of Lvp53 and LvMT are not necessary for this response.

A novel functional glucose transporter in the white shrimp Litopenaeus vannamei -LvGLUT2- is up-regulated during hypoxia in hepatopancreas.

In hypoxia conditions, the white shrimp Litopenaeus vannamei shifts its energetic metabolism from aerobic to anaerobic, requiring more glucose uptake into the cells by GLUT proteins. We here report a novel glucose transporter in shrimp. The Lvglut2 cDNA is 2473 bp-long containing an ORF of 1458 bp encoding 486 amino acid residues. The deduced protein has the features of a facilitative sugar transporter. The Lvglut2 gene product tagged with GFP was expressed in the cell membrane of Xenopus oocytes. In the same expression system, untagged LvGLUT2 resulted to be a bidirectional glucose transporter that functions moving glucose down its concentration gradient in and out of the cell. Lvglut2 mRNA is expressed in hepatopancreas while in muscle and gills it was not detected. Hypoxia up-regulates the expression of Lvglut2 transcripts in hepatopancreas. These results provide a better understanding of facilitative glucose transporters and gene regulation during hypoxia in crustaceans.

Expression of fructose 1,6-bisphosphatase and phosphofructokinase is induced in hepatopancreas of the white shrimp Litopenaeus vannamei by hypoxia.

Marine organisms are exposed to hypoxia in natural ecosystems and during farming. In these circumstances marine shrimp survive and synthesize ATP by anaerobic metabolism. Phosphofructokinase (PFK) and fructose 1,6-bisphosphatase (FBP) are key enzymes in carbohydrate metabolism. Here we report the cDNA of FBP from the shrimp Litopenaeus vannamei hepatopancreas and expression of PFK and FBP under normoxia and hypoxia. Hypoxia induces PFK and FBP expression in hepatopancreas but not in gills and muscle. Induction in hepatopancreas of the glycolytic and gluconeogenic key enzymes, PFK and FBP, suggests that PFK could be a key factor for increasing anaerobic rate, while FBP is probably involved in the activation of gluconeogenesis or the pentose-phosphates pathway during hypoxia in the highly active metabolism of hepatopancreas.

The metallothionein gene from the white shrimp Litopenaeus vannamei: characterization and expression in response to hypoxia.

Aquatic animals encounter variation in oxygen tension that leads to the accumulation of reactive oxygen species (ROS) that can harm the organisms. Under these circumstances some organisms have evolved to tolerate hypoxia. In mammals, metallothioneins (MTs) protect against hypoxia-generated ROS. Here we report the MT gene from the shrimp Litopenaeus vannamei (LvMT). LvMT is differentially expressed in hemocytes, intestine, gills, pleopods, heart, hepatopancreas and muscle, with the highest levels in hepatopancreas and heart. LvMT mRNA increases during hypoxia in hepatopancreas and gills after 3 h at 1.5 mg L(-1) dissolved oxygen (DO). This gene structure resembles the homologs from invertebrates and vertebrates possessing three exons, two introns and response elements for metal response transcription factor 1 (MTF-1), hypoxia-inducible factor 1 (HIF-1) and p53 in the promoter region. During hypoxia, HIF-1/MTF-1 might participate inducing MT to contribute towards the tolerance to ROS toxicity. MT importance in aquatic organisms may include also ROS-detoxifying processes.