Effect of calcium stearoyl-2 lactylate and lipase supplementation on growth performance, gut health, and nutrient digestibility of broiler chickens.

OBJECTIVE: To evaluate calcium stearoyl-2 lactylate (CSL) performance as an exogenous emulsifier together with lipase for broiler diets. METHODS: In total, 252 one-day-old Ross 308 broiler chickens were allocated in a completely randomized design to give 6 replications per treatment with 7 birds in each cage. There were six dietary treatments representing a 2×3 factorial arrangement consisted of two energy levels (standard energy [positive control, PC] and -100 kcal/kg of the requirement level [negative control, NC]) and three dietary treatments (without additives [CON], CON+CSL [CSL], and CON+CSL+lipase [CSL-Lipase]). Corn and soybean meal-based experimental diets containing vegetable oil were formulated. Growth performance, blood parameters, visceral organ weights, ileal morphology, nutrient digestibility, and cytokine gene expression were measured. RESULTS: Birds fed a diet including CSL increased (p<0.05) lipase level in blood compared to birds fed a diet including CSL-Lipase on day 21. Similarly, higher (p<0.05) liver weight was observed in birds fed a diet including either CSL or CSL-Lipase on day 21. Birds fed NC diet with CSL improved (p<0.05) nutrient digestibility compared to the NC diet on day 21. However, birds fed a diet supplemented with CSL or CSL-Lipase did not affect (p>0.05) the weight gain, feed efficiency, ileal morphology, and cytokine concentrations during the experiment period, regardless of dietary energy levels. CONCLUSION: Our results indicated that CSL has a role in improving nutrient digestibility in young birds when supplemented to a corn-soybean meal based broiler diet.

Physiological impact on layer chickens fed corn distiller's dried grains with solubles naturally contaminated with deoxynivalenol.

OBJECTIVE: An experiment was conducted to investigate the response of laying hens fed corn distiller's dried grains with solubles (DDGS) that are naturally contaminated with deoxynivalenol (DON). METHODS: One hundred and sixty 52-week-old Lohmann Brown Lite hens were randomly allotted to five dietary treatments with 8 replicates per treatment. The dietary treatments were formulated to provide a range of corn DDGS contaminated with DON from 0% to 20% (i.e., 5% scale of increment). All laying hens were subjected to the same management practices in a controlled environment. Body weight, feed intake and egg production were measured biweekly for the entire 8-week experiment. The egg quality was measured biweekly for 8 weeks. On weeks 4 and 8, visceral organ weights, blood metabolites, intestinal morphology, and blood cytokine concentrations were measured. RESULTS: The inclusion of corn DDGS contaminated with DON in the diet did not alter (p> 0.05) the body weight, feed intake, hen-day egg production, egg mass and feed efficiency of the laying hens. No difference was found (p>0.05) in the egg quality of hens that were fed the dietary treatments. Furthermore, hens that were fed a diet containing corn DDGS contaminated with DON showed no change (p>0.05) in the visceral organ weights, the blood metabolites, and the cytokine concentrations. The crypt depth increased (p<0.05) as the amount of corn DDGS contaminated with DON increased. Proportionately, the villus height to crypt depth ratio of the laying hens decreased (p<0.05) with the increasing level of corn DDGS contaminated with DON in the diet. CONCLUSION: The inclusion of corn DDGS contaminated with DON up to 20% in layer diets did not cause changes in egg production performance and egg quality, which indicates that DON is less toxic at the concentration of 1.00 mg DON/kg.

Autophagy and ubiquitin-proteasome system contribute to sperm mitophagy after mammalian fertilization.

Maternal inheritance of mitochondria and mtDNA is a universal principle in human and animal development, guided by selective ubiquitin-dependent degradation of the sperm-borne mitochondria after fertilization. However, it is not clear how the 26S proteasome, the ubiquitin-dependent protease that is only capable of degrading one protein molecule at a time, can dispose of a whole sperm mitochondrial sheath. We hypothesized that the canonical ubiquitin-like autophagy receptors [sequestosome 1 (SQSTM1), microtubule-associated protein 1 light chain 3 (LC3), gamma-aminobutyric acid receptor-associated protein (GABARAP)] and the nontraditional mitophagy pathways involving ubiquitin-proteasome system and the ubiquitin-binding protein dislocase, valosin-containing protein (VCP), may act in concert during mammalian sperm mitophagy. We found that the SQSTM1, but not GABARAP or LC3, associated with sperm mitochondria after fertilization in pig and rhesus monkey zygotes. Three sperm mitochondrial proteins copurified with the recombinant, ubiquitin-associated domain of SQSTM1. The accumulation of GABARAP-containing protein aggregates was observed in the vicinity of sperm mitochondrial sheaths in the zygotes and increased in the embryos treated with proteasomal inhibitor MG132, in which intact sperm mitochondrial sheaths were observed. Pharmacological inhibition of VCP significantly delayed the process of sperm mitophagy and completely prevented it when combined with microinjection of autophagy-targeting antibodies specific to SQSTM1 and/or GABARAP. Sperm mitophagy in higher mammals thus relies on a combined action of SQSTM1-dependent autophagy and VCP-mediated dislocation and presentation of ubiquitinated sperm mitochondrial proteins to the 26S proteasome, explaining how the whole sperm mitochondria are degraded inside the fertilized mammalian oocytes by a protein recycling system involved in degradation of single protein molecules.

A hot-water extract of Sanguisorba officinalis ameliorates endotoxin-induced septic shock by inhibiting inflammasome activation.

The inflammasome is a multiprotein signaling complex that mediates inflammatory innate immune responses through caspase 1 activation and subsequent IL-1ß secretion. However, because its aberrant activation often leads to inflammatory diseases, targeting the inflammasome holds promise for the treatment of inflammation-related diseases. In this study, it was found that a hot-water extract of Sanguisorba officinalis (HSO) suppresses inflammasome activation triggered by adenosine 5'-triphosphate, nigericin, microbial pathogens, and double stranded DNA in bone marrow-derived macrophages. HSO was found to significantly suppress IL-1ß production in a dose-dependent manner; this effect correlated well with small amounts of caspase 1 and little ASC pyroptosome formation in HSO-treated cells. The anti-inflammatory activity of HSO was further confirmed in a mouse model of endotoxin-induced septic shock. Oral administration of HSO reduced IL-1ß titers in the serum and peritoneal cavity, increasing the survival rate. Taken together, our results suggest that HSO is an inhibits inflammasome activation through nucleotide-binding domain and leucine-rich repeat pyrin domain 3, nucleotide-binding domain and leucine-rich repeat caspase recruitment domain 4 and absent in melanoma 2 pathways, and may be useful for treatment of inflammasome-mediated diseases.

A hot water extract of Aralia cordata activates bone marrow-derived macrophages via a myeloid differentiation protein 88-dependent pathway and protects mice from bacterial infection.

In traditional Asian medicine, Aralia cordata (AC) is a known as a pain reliever and anti-inflammatory drug. Although several of its biological activities have been reported, the immunomodulatory effects of a hot water extract of AC (HAC) have not yet been described. The aim of this study was to investigate whether HAC modulates the activation of macrophages, which play important roles in innate immune responses against microbial pathogens, and if so, to determine the molecular mechanisms by which HAC mediates this process. It was found that HAC activates bone marrow-derived macrophages (BMDM) and increases amounts of nitric oxide and proinflammatory cytokines in a dose-dependent manner. In addition, HAC was found to induce phosphorylation of NF-κB and mitogen-activated protein kinases (MAPKs), including c-Jun N-terminal kinases, extracellular signal-regulated kinases and p38. Interestingly, these effects were absent in BMDM prepared from myeloid differentiation protein 88-knockout mice. Polysaccharides from HAC exerted stronger immunostimulatory effects than HAC itself. Furthermore, orally administered HAC clearly enhanced clearance of the intracellular pathogen Listeria monocytogenes by boosting innate immune responses. These results demonstrate that HAC exerts immunostimulatory effects through the TLR/MyD88 and NF-κB/MAPK signal transduction pathways.

Transgenic pig carrying green fluorescent proteasomes.

Among its many functions, the ubiquitin-proteasome system regulates substrate-specific proteolysis during the cell cycle, apoptosis, and fertilization and in pathologies such as Alzheimer's disease, cancer, and liver cirrhosis. Proteasomes are present in human and boar spermatozoa, but little is known about the interactions of proteasomal subunits with other sperm proteins or structures. We have created a transgenic boar with green fluorescent protein (GFP) tagged 20S proteasomal core subunit α-type 1 (PSMA1-GFP), hypothesizing that the PSMA1-GFP fusion protein will be incorporated into functional sperm proteasomes. Using direct epifluorescence imaging and indirect immunofluorescence detection, we have confirmed the presence of PSMA1-GFP in the sperm acrosome. Western blotting revealed a protein band corresponding to the predicted mass of PSMA1-GFP fusion protein (57 kDa) in transgenic spermatozoa. Transgenic boar fertility was confirmed by in vitro fertilization, resulting in transgenic blastocysts, and by mating, resulting in healthy transgenic offspring. Immunoprecipitation and proteomic analysis revealed that PSMA1-GFP copurifies with several acrosomal membrane-associated proteins (e.g., lactadherin/milk fat globule E8 and spermadhesin alanine-tryptophan-asparagine). The interaction of MFGE8 with PSMA1-GFP was confirmed through cross-immunoprecipitation. The identified proteasome-interacting proteins may regulate sperm proteasomal activity during fertilization or may be the substrates of proteasomal proteolysis during fertilization. Proteomic analysis also confirmed the interaction/coimmunoprecipitation of PSMA1-GFP with 13/14 proteasomal core subunits. These results demonstrate that the PSMA1-GFP was incorporated in the assembled sperm proteasomes. This mammal carrying green fluorescent proteasomes will be useful for studies of fertilization and wherever the ubiquitin-proteasome system plays a role in cellular function or pathology.

Protein deubiquitination during oocyte maturation influences sperm function during fertilisation, antipolyspermy defense and embryo development.

Ubiquitination is a covalent post-translational modification of proteins by the chaperone protein ubiquitin. Upon docking to the 26S proteasome, ubiquitin is released from the substrate protein by deubiquitinating enzymes (DUBs). We hypothesised that specific inhibitors of two closely related oocyte DUBs, namely inhibitors of the ubiquitin C-terminal hydrolases (UCH) UCHL1 (L1 inhibitor) and UCHL3 (L3 inhibitor), would alter porcine oocyte maturation and influence sperm function and embryo development. Aberrant cortical granule (CG) migration and meiotic spindle defects were observed in oocytes matured with the L1 or L3 inhibitor. Embryo development was delayed or blocked in oocytes matured with the general DUB inhibitor PR-619. Aggresomes, the cellular stress-inducible aggregates of ubiquitinated proteins, formed in oocytes matured with L1 inhibitor or PR-619, a likely consequence of impaired protein turnover. Proteomic analysis identified the major vault protein (MVP) as the most prominent protein accumulated in oocytes matured with PR-619, suggesting that the inhibition of deubiquitination altered the turnover of MVP. The mitophagy/autophagy of sperm-contributed mitochondria inside the fertilised oocytes was hindered by DUB inhibitors. It is concluded that DUB inhibitors alter porcine oocyte maturation, fertilisation and preimplantation embryo development. By regulating the turnover of oocyte proteins and mono-ubiquitin regeneration, the DUBs may promote the acquisition of developmental competence during oocyte maturation.

Relative Expression of Low Molecular Weight Protein, Tyrosine Phosphatase (Wzb Gene) of Herbaspirillum sp. GW103 Toward Arsenic Stress and Molecular Modeling.

This study investigated the expression rate and molecular modeling of Wzb gene, a low molecular weight protein tyrosine phosphatase, under As stress in Herbaspirillum sp. GW103. Expression of Wzb gene was quantified at transcriptional level through real-time quantitative PCR. The results showed up- and down-regulations of Wzb gene in the presence of As (50 and 100 mg/L). The maximum Wzb transcript expression was 1.2-fold after 72 h exposure to 50 mg/L of As. However, the minimum expression was 0.1-fold after 48 h exposure to 100 mg/L of As. The Wzb protein sequence was retrieved from NCBI sequence database and was used for in silico analysis. 3D structure of Wzb gene was predicted by comparative modeling using modeler 9v9. Further, the model was validated for its quality by Ramachandran plot, ERRAT, Verify 3D, and SAVES server which revealed structure and quality of the Wzb gene model.

Identification and characterization of RING-finger ubiquitin ligase UBR7 in mammalian spermatozoa.

The ubiquitin-proteasome system (UPS) controls intracellular protein turnover in a substrate-specific manner via E3-type ubiquitin ligases. Mammalian fertilization and particularly sperm penetration through the oocyte vitelline coat, the zona pellucida (ZP), is regulated by UPS. We use an extrinsic substrate of the proteasome-dependent ubiquitin-fusion degradation pathway, the mutant ubiquitin UBB(+1), to provide evidence that an E3-type ligase activity exists in sperm-acrosomal fractions. Protein electrophoresis gels from such de novo ubiquitination experiments contained a unique protein band identified by tandem mass spectrometry as being similar to ubiquitin ligase UBR7 (alternative name: C14ORF130). Corresponding mRNA was amplified from boar testis and several variants of the UBR7 protein were detected in boar, mouse and human sperm extracts by Western blotting. Genomic analysis indicated a high degree of evolutionary conservation, remarkably constant purifying selection and conserved testis expression of the UBR7 gene. By immunofluorescence, UBR7 was localized to the spermatid acrosomal cap and sperm acrosome, in addition to hotspots of proteasomal activity in spermatids, such as the cytoplasmic lobe, caudal manchette, nucleus and centrosome. During fertilization, UBR7 remained with the ZP-bound acrosomal shroud following acrosomal exocytosis. Thus, UBR7 is present in the acrosomal cap of round spermatids and within the acrosomal matrix of mature boar spermatozoa. These data provide the first evidence of ubiquitin ligase activity in mammalian spermatozoa and indicate UBR7 involvement in spermiogenesis.

Feeding dietary non-starch polysaccharides supplemented with xylanase could improve the performance of broilers.

The impact of dietary non-starch polysaccharides (NSP) on performance and carcass traits of broilers fed wheat-bran substituted into corn-soybean meal-based diets supplemented with xylanase was investigated. A total of 280 (7-day-old) Ross 308 broilers were randomly allotted to one of five dietary treatments with 8 replicates, 7 chicks per pen. Treatments were; i) CON: Control diet, ii) CON-X (CON + 3,000 U/kg xylanase), iii) L-X: low NSP (2% wheat bran in CON + 3,000 U/kg xylanase), iv) M-X: medium NSP (4% wheat bran in CON + 3,000 U/kg xylanase), v) H-X: higher NSP (8% wheat bran in CON+ 3,000 U/kg xylanase). Birds fed the H-X diet increased (p < 0.05) daily gains, and average daily feed intake and had marginally improved body weights (p = 0.074) on day 35. Relatively, the H-X diet tended to increase the average daily gains (p = 0.053; p = 0.073) of birds during the grower phase (d 24-35) and the entire experimental period (d 8-35), respectively. Moreover, there were no significant differences among treatments in the feed conversion ratio of birds throughout the entire experiment period. Birds fed diets CON-X, L-X, and M-X had improved (p < 0.05) the ileal digestibility of energy on d 24 and 35 compared to those fed the H-X diet. Furthermore, birds fed diet CON-X improved (p < 0.05) N digestibility on d 24. Improved carcass moisture content and lowered crude fat of leg meat (p < 0.05) were noted in birds fed the diet M-X and H-X on d 35, respectively. The intestinal viscosity was reduced (p < 0.05) in xylanase-supplemented treatments CON-X, L-X, M-X, and H-X diets when compared to CON. Our results suggest that supplementing 3,000 U/kg xylanase in a higher NSP (8% wheat bran substituted level) diet could improve the intestinal viscosity and growth performance of broilers.

Effect of different bedding depths of rice hulls on growth performance and carcass traits of White Pekin ducks.

Duck meat is recognized as a healthier poultry product that contains higher amounts of unsaturated and essential fatty acids, iron, and excellent amounts of protein. It has been found to possess the ability to reduce low-density lipoprotein cholesterol and subsequently, blood pressure in the human body; and improve the immunity system. The current study investigated the appropriate bedding depths of rice hulls as a preferred bedding material by evaluating the growth performance and carcass traits of White Pekin ducks raised for 42 days. A total of 288 one-day-old White Pekin ducklings were randomly allotted to floor cages with one of four bedding depths at 4 cm, 8 cm, 12 cm, and 16 cm. Ducklings were fed standard duck starter (days 1-21) and finisher (days 22-42) diets. The birds were stocked at a rate of 6 birds/m2 with 6 replicates per treatment. Growth performance evaluation for the body weight, average daily gain, and average daily feed intake were measured to calculate the weekly feed conversion ratio. Breast, leg, and carcass yield were assessed as carcass traits. The muscle color and proximate composition were also analyzed for meat quality. Footpad dermatitis was also evaluated on day 42. Ducks reared on 16 cm bedding depth over the 42 days recorded higher (p < 0.05) body weight, average daily, average daily feed intake, and improved feed conversion ratios compared to other groups. The crude fat in breast meat also lowered (p < 0.05) in ducks reared at 16 cm (1.02%) when compared to ducks raised at 4 cm bedding depth (2.11%). Our results showed improved redness (p < 0.05) when the depth of bedding materials was elevated. Except for the breast meat fat, the dissimilar bedding depths did not affect (p < 0.05) the breast and leg meat composition, footpad dermatitis, and mortality for the current study. In conclusion, this study indicated that the bedding depths would directly or indirectly affect the growth performance and meat color of White Pekin ducks; and the bedding depth of rice hulls at 16 cm improved the growth performance of White Pekin ducks for 42 days.

Inactivation of mammalian spermatozoa on the exposure of TiO2 nanorods deposited with noble metals.

Titanium dioxide (TiO2) nanorods (NRs) are well-known semiconducting and catalytic material that has been widely applied, but their toxicities have also attracted recent interest. In this study, we investigated and compared the toxic effects of TiO2 NRs and TiO2 NRs loaded with Ag or Au NPs on boar spermatozoa. As a result, sperm incubated with Ag-TiO2 NRs showed lower motility than sperm incubated with controls (with or without TiO2 NRs) or Au-TiO2 NRs. In addition, sperm viability and acrosomal integrity were defective in the presence of Ag-TiO2 NRs, and the generation of intracellular reactive oxygen species (ROS) increased significantly when spermatozoa were incubated with 20 µg/ml Ag-TiO2 NRs. We discussed in depth the charge transfer mechanism between enzymatic NADPH and Ag-TiO2 NRs in the context of ROS generation in spermatozoa. The effects we observed reflected the fertilization competence of sperm incubated with Ag-TiO2 NRs; specifically sperm penetration and embryonic development rates by in vitro fertilization were reduced by Ag-TiO2 NRs. To summarize, our findings indicate that exposure to Ag-TiO2 NRs could affect male fertilization fecundity and caution that care be exercised when using these NRs.

Change in intestinal alkaline phosphatase activity is a hallmark of antibiotic-induced intestinal dysbiosis.

OBJECTIVE: Intestinal alkaline phosphatase (IAP) maintains intestinal homeostasis by detoxifying bacterial endotoxins and regulating gut microbiota, and lipid absorption. Antibiotics administered to animals can cause gut dysbiosis and barrier disruption affecting animal health. Therefore, the present study sought to investigate the role of IAP in the intestinal environment in dysbiosis. METHODS: Young male mice aged 9 weeks were administered a high dose of antibiotics to induce dysbiosis. They were then sacrificed after 4 weeks to collect the serum and intestinal organs. The IAP activity in the ileum and the level of cytokines in the serum samples were measured. Quantitative real-time polymerase chain reaction analysis of RNA from the intestinal samples was performed using primers for tight junction proteins (TJPs) and proinflammatory cytokines. The relative intensity of IAP and toll-like receptor 4 (TLR4) in intestinal samples was evaluated by western blotting. RESULTS: The IAP activity was significantly lower in the ileum samples of the dysbiosisinduced group compared to the control. The interleukin-1 beta, interleukin-6, and tumor necrosis factor-alpha concentrations were significantly higher in the ileum samples of the dysbiosis-induced group. The RNA expression levels of TJP2, claudin-3, and claudin-11 showed significantly lower values in the intestinal samples from the dysbiosis-induced mice. Results from western blotting revealed that the intensity of IAP expression was significantly lower in the ileum samples of the dysbiosis-induced group, while the intensity of TLR4 expression was significantly higher compared to that of the control group without dysbiosis. CONCLUSION: The IAP activity and relative mRNA expression of the TJPs decreased, while the levels of proinflammatory cytokines increased, which can affect intestinal integrity and the function of the intestinal epithelial cells. This suggests that IAP is involved in mediating the intestinal environment in dysbiosis induced by antibiotics and is an enzyme that can potentially be used to maintain the intestinal environment in animal health care.

The bisphenol S contamination level observed in human follicular fluid affects the development of porcine oocytes.

Bisphenol S (BPS), the main replacement for bisphenol A (BPA), is thought to be toxic, but limited information is available on the effects of Bisphenol S on ovarian follicles. In our study, we demonstrated the presence of Bisphenol S in the follicular fluid of women at a concentration of 22.4 nM. The effect of such concentrations of Bisphenol S on oocyte maturation and subsequent embryo development is still unknown. Therefore, we focused on the effect of Bisphenol S on in vitro oocyte maturation, fertilization, and embryo development. As a model, we used porcine oocytes, which show many physiological similarities to human oocytes. Oocytes were exposed to Bisphenol S concentrations similar to those detected in female patients in the ART clinic. We found a decreased ability of oocytes to successfully complete meiotic maturation. Mature oocytes showed an increased frequency of meiotic spindle abnormalities and chromosome misalignment. Alarming associations of oocyte Bisphenol S exposure with the occurrence of aneuploidy and changes in the distribution of mitochondria and mitochondrial proteins were demonstrated for the first time. However, the number and quality of blastocysts derived from oocytes that successfully completed meiotic maturation under the influence of Bisphenol S was not affected.

Combinatorial Effect of Dietary Oregano Extracts and 3,4,5-Trihydroxy Benzoic Acid on Growth Performance and Elimination of Coccidiosis in Broiler Chickens.

We aimed to compare the combinatorial effect of 3,4,5-trihydroxybenzoic acid (THB) and oregano extracts (OE) with THB alone on the growth performance and elimination of deleterious effects in coccidiosis-infected broilers. A total of 210 one-day-old broilers were randomly assigned to one of five dietary treatments, with six replicates each, for 35 days. Dietary treatments were: 1) non-challenged, non-treated (NC); 2) challenged, non-treated (PC); 3) PC+ Salinomycin (0.05 g/kg; AB); 4) PC+THB (0.1 g/kg; THB); and 5) PC+THB+OE (0.1 g/kg; COM). On day 14, all groups except for NC were challenged with a 10-fold dose of Livacox® T anticoccidial vaccine to induce mild coccidiosis. All treatments significantly improved (P<0.05) body weight, average daily gain, and average daily feed intake, compared to PC, on days 21, 28, and 35. However, all treatments significantly reduced (P<0.05) the feed conversion ratio of PC by more than 14.60% on day 35, 11.76% during growing period, and 10.36% through the entire period. Broilers receiving anticoccidial treatments had 54.23% and 51.86% lower lesion scores (P<0.05) at 4 and 7 days post-infection, respectively, compared to PC. Additionally, the villus height of COM was significantly longer (P < 0.05) than that of THB. Although the molecular action of COM remains unclear, OE addition to THB reduced the shedding of oocysts better than THB alone (P<0.05, 9-11 days post-infection). Most importantly, COM effectively minimized the mortality of challenged birds from as high as 11.90% (PC) to 0%, a level similar to NC and AB, while THB maintained a mortality of 2.38%. In conclusion, the anticoccidial effect of THB can be enhanced by the addition of OE for better animal performance and the elimination of deleterious effects from coccidiosis-infected broilers for 35 days.

Artemisia vulgaris extract causes precocious acrosome reaction and viability loss but low rate of membrane damage in mouse spermatozoa.

Several herbs including Artemisia are known to possess conceptive property. In the present study, mouse spermatozoa were incubated with ethanol extract of Artemisia vulgaris leaves. The effect of extract on acrosome exocytosis was studied by labeling spermatozoa with fluorescein isothiocyanate (FITC) peanut agglutinin and by staining with Coomassie blue. Viability and membrane integrity were studied by Trypan-blue staining and hypo-osmotic swelling test. Artemisia extract at very low concentration caused precocious acrosome reaction and loss of sperm viability. Acrosome reaction increased remarkably from 22.63% to 88.42% with increasing extract concentration from 0 to 2,000 µg/mL. However, the viability loss of spermatozoa was increased from 11.71% in control to 63.73% in samples treated, evaluated by Trypan-blue staining method. Membrane damage caused by the extract, evaluated by hypo-osmotic swelling test was even low, ranging from 2.27% to only 24.23%. These results indicate that Artemisia extract might block fertilization by causing precocious acrosome exocytosis in spermatozoa. A direct contraceptive effect was tested by injecting the plant extract into the vagina of female mice and then allowing them to mate with normal males. The treated female mice delivered significantly fewer litters in comparison to the control.

Mammalian Cell-Free System Recapitulates the Early Events of Post-Fertilization Sperm Mitophagy.

Propagation of paternal sperm-contributed mitochondrial genes, resulting in heteroplasmy, is seldom observed in mammals due to post-fertilization degradation of sperm mitochondria, referred to as sperm mitophagy. Whole organelle sperm mitochondrion degradation is thought to be mediated by the interplay between the ubiquitin-proteasome system (UPS) and the autophagic pathway (Song et al., Proc. Natl. Acad. Sci. USA, 2016). Both porcine and primate post-fertilization sperm mitophagy rely on the ubiquitin-binding autophagy receptor, sequestosome 1 (SQSTM1), and the proteasome-interacting ubiquitinated protein dislocase, valosin-containing protein (VCP). Consequently, we anticipated that sperm mitophagy could be reconstituted in a cell-free system consisting of permeabilized mammalian spermatozoa co-incubated with porcine oocyte extracts. We found that SQSTM1 was detected in the midpiece/mitochondrial sheath of the sperm tail after, but not before, co-incubation with oocyte extracts. VCP was prominent in the sperm mitochondrial sheath both before and after the extract co-incubation and was also detected in the acrosome and postacrosomal sheath and the subacrosomal layer of the spermatozoa co-incubated with extraction buffer as control. Such patterns are consistent with our previous observation of SQSTM1 and VCP associating with sperm mitochondria inside the porcine zygote. In addition, it was observed that sperm head expansion mimicked the early stages of paternal pronucleus development in a zygote during prolonged sperm-oocyte extract co-incubation. Treatment with anti-SQSTM1 antibody during extract co-incubation prevented ooplasmic SQSTM1 binding to sperm mitochondria. Even in an interspecific cellular environment encompassing bull spermatozoa and porcine oocyte extract, ooplasmic SQSTM1 was recruited to heterospecific sperm mitochondria. Complementary with the binding of SQSTM1 and VCP to sperm mitochondria, two sperm-borne pro-mitophagy proteins, parkin co-regulated gene product (PACRG) and spermatogenesis associated 18 (SPATA18), underwent localization changes after extract coincubation, which were consistent with their degradation observed inside fertilized porcine oocytes. These results demonstrate that the early developmental events of post-fertilization sperm mitophagy observed in porcine zygote can be reconstituted in a cell-free system, which could become a useful tool for identifying additional molecules that regulate mitochondrial inheritance in mammals.

Broiler responses to dietary 3,4,5-trihydroxybenzoic acid and oregano extracts under Eimeria challenge conditions.

This study was conducted to evaluate the efficacy of a combination 3,4,5-trihydroxybenzoic acid (THB) and oregano extracts (i.e., Carvacrol and Thymol) at intake/dietary different levels on growth performance, intestinal health indicators, immune responses and fecal oocyst shedding in broiler chickens under Eimeria challenged condition. A total of 336 one-day-old broilers were randomly assigned to one of six dietary treatments with seven replications per treatment. Dietary treatments were: i) Non-challenged bird without any dietary treatment (NCNT), ii) Challenged bird without any dietary treatment (CNT), iii) Challenged birds fed a THB diet (0.1 g/kg, THB), iv) Challenged birds fed a combination of THB and oregano extracts diet (0.1 g/kg, COM 100), and a gradual increase of combination of THB and oregano extracts likely v) 0.15 g/kg (COM 150), and 0.2 g/kg (COM 200). On day 14, all groups except for NCNT have orally challenged with a 10-fold dose of Livacox® T anticoccidial vaccine to trigger coccidiosis. The results indicated that Eimeria-challenged broilers fed COM 100 and COM 200 diets increased (p < 0.05) body weight than CNT diet on day 35. Furthermore, birds fed COM 100 and COM 200 diets increased (p < 0.05) average daily gain compared to those fed CNT diets for the entire experimental period. There is no significant (p > 0.05) in average daily feed intake, feed efficiency between NCNT and birds fed with combined THB and oregano extracts for the entire experimental period. A combination of THB and oregano extract regardless of concentration levels or THB alone reduced (p < 0.05) lesion score in ileum compared to the CNT diet for 7 days post-infection (dpi). Birds fed COM 100 diet had lower (p < 0.05) intestinal lesion scores in jejunum and caeca on 7 dpi compared to those were in the CNT diet. No (p > 0.05) difference was observed in the oocysts per gram of feces count, intestinal morphology, carcass traits and blood cytokine concentration among the infected treatments. Collectively, we conclude that birds fed with a combination of THB and oregano extracts regardless of the ratios that were used demonstrated better recovery of health after the coccidial challenge than using only THB alone.

Carbohydrate-mediated binding and induction of acrosomal exocytosis in a boar sperm-somatic cell adhesion model.

The molecular basis underlying the binding of spermatozoa to their homologous eggs and the subsequent induction of acrosomal exocytosis remain a major unresolved issue in mammalian fertilization. Novel cell adhesion systems are now being explored to advance this research. Triantennary and tetraantennary N-glycans have previously been implicated as the major carbohydrate sequences that mediate the initial binding of spermatozoa to the specialized egg coat (zona pellucida) in the murine and porcine models. Mouse spermatozoa also undergo binding to rabbit erythrocytes (rRBCs), presumably via the interaction of their lectin-like egg-binding proteins with branched polylactosamine sequences present on these somatic cells. Experiments presented in this study confirm that boar spermatozoa also bind to rRBCs. However, unlike mouse spermatozoa, boar spermatozoa also undergo acrosomal exocytosis within 30 min after binding to rRBCs. Both binding and induction of acrosomal exocytosis in this system did not require the participation of terminal Galalpha1-3Gal sequences that are found on rRBCs. Pronase glycopeptides derived from rRBCs inhibited the binding of boar sperm to porcine oocytes by 91% at a final concentration of 0.3 mg/ml under standard IVF conditions. Binding in this porcine cell adhesion model was also completely blocked at this concentration of glycopeptide. Thus, adhesion results from the interaction of the egg-binding protein expressed on the surface of boar spermatozoa with the glycans presented on rRBCs. This cell adhesion model will be useful for investigating the molecular basis of gamete binding and the induction of acrosomal exocytosis in the pig.

Interference with the 19S proteasomal regulatory complex subunit PSMD4 on the sperm surface inhibits sperm-zona pellucida penetration during porcine fertilization.

Proteolysis of ubiquitinated sperm and oocyte proteins by the 26S proteasome is necessary for the success of mammalian fertilization, including but not limited to acrosomal exocytosis and sperm-zona pellucida (ZP) penetration. The present study examined the role of PSMD4, an essential non-ATPase subunit of the proteasomal 19S regulatory complex responsible for proteasome-substrate recognition, in sperm-ZP penetration during porcine fertilization in vitro (IVF). Porcine sperm-ZP penetration, but not sperm-ZP binding, was blocked in the presence of a monoclonal anti-PSMD4 antibody during IVF. Inclusion in the fertilization medium of mutant ubiquitins (Ub+1 and Ub5+1), which are refractory to processing by the 19S regulatory complex and associated with Alzheimer's disease, also inhibited fertilization. This observation suggested that subunit PSMD4 is exposed on the sperm acrosomal surface, a notion that was further supported by the binding of non-cell permeant, biotinylated proteasomal inhibitor ZL3VS to the sperm acrosome. Immunofluorescence localized PSMD4 in the sperm acrosome. Immunoprecipitation and proteomic analysis revealed that PSMD4 co-precipitated with porcine sperm-associated acrosin inhibitor (AI). Ubiquitinated species of AI were isolated from boar sperm extracts by affinity purification of ubiquitinated proteins using the recombinant UBA domain of p62 protein. Some proteasomes appeared to be anchored to the sperm head inner acrosomal membrane, as documented by co-fractionation studies. In conclusion, the 19S regulatory complex subunit PSMD4 is involved in the sperm-ZP penetration during fertilization. The recognition of substrates on the ZP by the 19S proteasomal regulatory complex is essential for the success of porcine/mammalian fertilization in vitro.