Potent drug delivery enhancement of betulinic acid and NVX-207 into equine skin in vitro - a comparison between a novel oxygen flow-assisted transdermal application device and microemulsion gels.

BACKGROUND: Gray horses are predisposed to equine malignant melanoma (EMM) with advancing age. Depending on the tumor's location and size, they can cause severe problems (e.g., defaecation, urination, feeding). A feasible therapy for EMM has not yet been established and surgical excision can be difficult depending on the location of the melanoma. Thus, an effective and safe therapy is needed. Naturally occurring betulinic acid (BA), a pentacyclic triterpene and its synthetic derivate, NVX-207 (3-acetyl-betulinic acid-2-amino-3-hydroxy-2-hydroxymethyl-propanoate) are known for their cytotoxic properties against melanomas and other tumors and have already shown good safety and tolerability in vivo. In this study, BA and NVX-207 were tested for their permeation potential into equine skin in vitro in Franz-type diffusion cell (FDC) experiments after incubation of 5 min, 30 min and 24 h, aiming to use these formulations for prospective in vivo studies as a treatment for early melanoma stages. Potent permeation was defined as reaching or exceeding the half maximal inhibitory concentrations (IC50) of BA or NVX-207 for equine melanoma cells in equine skin samples. The active ingredients were either dissolved in a microemulsion (ME) or in a microemulsion gel (MEG). All of the formulations were transdermally applied but the oil-in-water microemulsion was administered with a novel oxygen flow-assisted (OFA) applicator (DERMADROP TDA). RESULTS: All tested formulations exceeded the IC50 values for equine melanoma cells for BA and NVX-207 in equine skin samples, independently of the incubation time NVX-207 applied with the OFA applicator showed a significant time-dependent accumulation and depot-effect in the skin after 30 min and 24 h (P < 0.05). CONCLUSIONS: All tested substances showed promising results. Additionally, OFA administration showed a significant accumulation of NVX-207 after 30 min and 24 h of incubation. Further in vivo trials with OFA application are recommended.

Drug release profile of a novel exenatide long-term drug delivery system (OKV-119) administered to cats.

Beneficial weight-loss properties of glucagon-like peptide-1 receptor agonists (GLP-1RA) in obese people, with corresponding improvements in cardiometabolic risk factors, are well established. OKV-119 is an investigational drug delivery system that is being developed for the long-term delivery of the GLP-1RA exenatide to feline patients. The purpose of this study was to evaluate the drug release characteristics of subcutaneous OKV-119 implants configured to release exenatide for 84 days. Following a 7-day acclimation period, five purpose-bred cats were implanted with OKV-119 protypes and observed for a 112-day study period. Food intake, weekly plasma exenatide concentrations and body weight were measured. Exenatide plasma concentrations were detected at the first measured timepoint (Day 7) and maintained above baseline for over 84 Days. Over the first 28 days, reduced caloric intake and a reduction in body weight were observed in four of five cats. In these cats, a body weight reduction of at least 5% was maintained throughout the 112-day study period. This study demonstrates that a single OKV-119 implant can deliver the GLP-1RA exenatide for a months long duration. Results suggest that exposure to exenatide plasma concentrations ranging from 1.5 ng/ml to 4 ng/ml are sufficient for inducing weight loss in cats.

Floating Ricobendazole Delivery Systems: A 3D Printing Method by Co-Extrusion of Sodium Alginate and Calcium Chloride.

At present, the use of benzimidazole drugs in veterinary medicine is strongly limited by both pharmacokinetics and formulative issues. In this research, the possibility of applying an innovative semi-solid extrusion 3D printing process in a co-axial configuration was speculated, with the aim of producing a new gastro-retentive dosage form loaded with ricobendazole. To obtain the drug delivery system (DDS), the ionotropic gelation of alginate in combination with a divalent cation during the extrusion was exploited. Two feeds were optimized in accordance with the printing requirements and the drug chemical properties: the crosslinking ink, i.e., a water ethanol mixture containing CaCl2 at two different ratios 0.05 M and 0.1 M, hydroxyethyl cellulose 2% w/v, Tween 85 0.1% v/v and Ricobendazole 5% w/v; and alginate ink, i.e., a sodium alginate solution at 6% w/v. The characterization of the dried DDS obtained from the extrusion of gels containing different amounts of calcium chloride showed a limited effect on the ink extrudability of the crosslinking agent, which on the contrary strongly influenced the final properties of the DDS, with a difference in the polymeric matrix toughness and resulting effects on floating time and drug release.

In vitro evaluation of novel (nanoparticle) oral delivery systems allow selection of gut immunomodulatory formulations.

Oral delivery is the most convenient way to vaccinate cultured fish, however it is still problematic, primarily due to a lack of a commercially valid vaccine vehicle to protect the antigen against gastric degradation and ensure its uptake from the intestine. With the goal of advancing the potential to vaccinate orally, this study evaluates a novel silicon nanoparticle-based vehicle (VacSaf carrier). Aeromonas salmonicida antigens were formulated with the VacSaf carrier using different preparation methods to generate dry powder and liquid formulations. Twelve formulations were first subjected to an in vitro evaluation where the A. salmonicida bacterin conjugated to VacSaf carriers were found superior at inducing pro-inflammatory cytokine expression in primary leucocyte cultures and the macrophage/monocyte cell line RTS-11 compared with A. salmonicida bacterin alone. This was especially apparent after exposure to acid conditions to mimic stomach processing. One formulation (FD1) was taken forward to oral delivery using two doses and two administration schedules (5 days vs 10 days, the latter 5 days on, 5 days off, 5 days on), and the transcript changes of immune genes in the intestine (pyloric caeca, midgut and hindgut) and spleen were evaluated by qPCR and serum IgM was measured by ELISA. The VacSaf carrier alone was shown to be safe for use in vivo, in that no side-effects were seen, but it did induce expression of some cytokines, and may have value as an oral adjuvant candidate. The FD1 bacterin formulation was effective at inducing a range of cytokines associated with innate and adaptive immunity, mainly in the pyloric caeca, compared to A. salmonicida bacterin alone (which had almost no effect), and confirms the immune competence of this gut region following appropriate oral vaccination. These results reveal that in vitro screening of formulations for oral delivery has value and can be used to assess the most promising formulations to test further.

Pharmacokinetics and diuretic effect of furosemide after single intravenous, oral tablet, and newly developed oral disintegrating film administration in healthy beagle dogs.

BACKGROUND: Furosemide, a diuretic that acts on the loop of Henle, is commonly used to treat congestive heart failure in veterinary medicine. Some owners have difficulty in administering oral tablet medication to animal patients, which leads to noncompliance, especially during long-term administration. Oral disintegrating film (ODF) has the advantages of easy administration via a non-invasive route, rapid dissolution, and low suffocating risk. The objective of this study was to research the pharmacokinetic (PK) profiles and diuretic effect of furosemide after intravenous (IV), orally uncoated tablet (OUT), and newly developed ODF administration in healthy beagle dogs. In this study, a furosemide-loaded ODF (FS-ODF) formulation was developed and five beagle dogs were administered a single dose (2 mg/kg) of furosemide via each route using a cross-over design. RESULTS: The most suitable film-forming agent was sodium alginate; thus, this was used to develop an ODF for easy drug administration. No significant differences were detected in the PK profiles between OUT and FS-ODF. In the blood profiles, the concentration of total protein was significantly increased compared to the baseline (0 h), whereas no significant difference was detected in the concentration of creatinine and hematocrit compared to the baseline. FS-ODF resulted in a similar hourly urinary output to OUT during the initial 2 h after administration. The urine specific gravity was significantly decreased compared to the baseline in each group. The peak times of urine electrolyte (sodium and chloride) excretion per hour were 1 h (IV), 2 h (OUT), and 2 h (FS-ODF). CONCLUSIONS: These results suggest that the PK/PD of furosemide after administration of newly developed FS-ODF are similar to those of OUT in healthy dogs. Therefore, the ODF formulation has the benefits of ease and convenience, which would be helpful to owners of companion animals, such as small dogs (< 10 kg), for the management of congestive heart failure.

Melatonin and CIDR improved the follicular and luteal haemodynamics, uterine and ovarian arteries vascular perfusion, ovarian hormones and nitric oxide in cyclic cows.

This study hypothesizes that melatonin with exogenous progesterone (CIDR) can improve follicular, luteal, ovarian and uterine haemodynamic of heat-stressed cows. Holstein cows (N = 12) studied for two spontaneous oestrous cycles during winter then divided equally during summer into the CIDR group received CIDR for 7 days and the melatonin group (Mel) received three injections of melatonin (75 mg/head) at the CIDR insertion, removal and ovulation days. Blood samples were collected to assay oestradiol (E2), progesterone (P4) and nitric oxide (NO). On day 0 (Ovulation), Mel had more small follicles (p < .05), higher ipsilateral and contralateral ovarian arteries (Ov.A.) peak systolic velocity (PSV), higher ipsilateral uterine artery (Ut.A.) PSV (p = .031) and blood flow volume (BFV), also Mel elevated contralateral Ut.A. PSV and BFV (p < .0001) but lowered contra Ut.A. pulsatility index (PI, p < .0001), E2 (p < .01) and NO (p < .0001). Mel increased the corpus luteum diameter (CL, p < .001), coloured area (p < .007) and P4 (p < .0001) on day 5 and reduced them (p < .05; p < .01) on Day 14. On day 10, Mel obtained CL diameter (p < .03) and coloured area (p < .002) of spontaneous that was higher than CIDR and decreased P4 (p < .003). Mel increased CL diameter, area and coloured area and decreased them thereafter. Mel increased the ipsilateral ovarian and uterine arteries PSV and BFV before ovulation and until day 8. Mel increased P4 and decreased NO until days 6 and 14. In conclusion, the improvement in follicular, luteal, ovarian and uterine haemodynamic and the decrease of NO production proved our hypothesis Melatonin doses higher than 75 mg/head is recommended to improve the heat-stressed cow's fertility.

Role of nanotechnology in animal production and veterinary medicine.

Nanotechnology is the discipline and technology of small and specific things that are < 100 nm in size. Because of their extremely miniscule size, any changes in their chemical and physical structure may show higher reactivity and solubility than larger particles. Nanotechnology plays a vital role in every field of life. It is considered one of the most bleeding edge field of scientific research. It has already several applications in a myriad of disciplines while its application in the field of animal production and veterinary medicine is still experimental in nature. But, in recent years, the role of nanotechnology in the aforementioned fields of scientific inquiry has shown great progress. These days, nanotechnology has been employed to revolutionize drug delivery systems and diagnose atypical diseases. Applications of nanoparticle technology in the field of animal reproduction and development of efficacious vaccines have been at the forefront of scientific endeavors. Additionally, their impacts on meat and milk quality are also being judiciously inquired in recent decades. Veterinary nanotechnology has great potential to improve diagnosis and treatment, and provide new tools to this field. This review focuses on some noteworthy applications of nanoparticles in the field of animal production and their future perspectives.

In ovo delivery of various biological supplements, vaccines and drugs in poultry: current knowledge.

The technique of delivering various nutrients, supplements, immunostimulants, vaccines, and drugs via the in ovo route is gaining wide attention among researchers worldwide for boosting production performance, immunity and safeguarding the health of poultry. It involves direct administration of the nutrients and biologics into poultry eggs during the incubation period and before the chicks hatch out. In ovo delivery of nutrients has been found to be more effective than post-hatch administration in poultry production. The supplementation of feed additives, nutrients, hormones, probiotics, prebiotics, or their combination via in ovo techniques has shown diverse advantages for poultry products, such as improved growth performance and feed conversion efficiency, optimum development of the gastrointestinal tract, enhancing carcass yield, decreased embryo mortality, and enhanced immunity of poultry. In ovo delivery of vaccination has yielded a better response against various poultry pathogens than vaccination after hatch. So, this review has aimed to provide an insight on in ovo technology and its potential applications in poultry production to deliver different nutrients, supplements, beneficial microbes, vaccines, and drugs directly into the developing embryo to achieve an improvement in post-hatch growth, immunity, and health of poultry. © 2019 Society of Chemical Industry.

A novel CAV derived cell-penetrating peptide efficiently delivers exogenous molecules through caveolae-mediated endocytosis.

Cell-penetrating peptide (CPP) is a promising cargo for delivering bioactive molecules. In this study, the N terminus of VP1 from chicken anemia virus, designated as CVP1, was found to carry enriched arginine residues with α-helix. By confocal imaging, flow cytometry and MTT assay, we identified CVP1 as a novel, safe and efficient CPP. CVP1-FITC peptide could entry different types of cells tested with dose dependence, but without cytotoxic effects. Compared with TAT-FITC peptide, the CVP1-FITC peptide showed much higher cell-penetrating activity. Moreover, CVP1 could successfully deliver ß-glycosidase, poly (I:C) and plasmid into HCT116 cells. Inhibitors and temperature sensitivity analysis further indicated that the cell-penetrating activity of CVP1 was based on ATP-dependent and caveolae-mediated endocytosis. All these data demonstrate that CVP1 has efficient cell-penetrating activity and great potential for developing a novel delivery vector.

Novel amoxicillin nanoparticles formulated as sustained release delivery system for poultry use.

Amoxicillin is used in the treatment and prevention of a wide range of diseases in poultry breeding. However, its short half-life and low bioavailability restrict its clinical application in these species. Entrapment of drugs into polymeric nanoparticles (nps) presents a means to improve gastrointestinal absorption and oral bioavailability of drugs. This study was aimed to overcome limitation of amoxicillin use in poultry breeding. Amoxicillin was loaded into sodium alginate-polyvinyl alcohol (NaAlg-PVA) blend nps, and characterization of the prepared nps was performed. For pharmacokinetic study, commercial male broilers were used and comparative pharmacokinetics of free and nanoparticle form of amoxicillin were investigated. Twenty-one broilers were divided into three groups. All groups received 10 mg/kg drug. Blood samples were collected, and drug plasma concentrations were determined by HPLC. The results demonstrated that the particle size, zeta potential, encapsulation efficiency, and loading capacity of the nps were 513.96 ± 19.46 nm, -45.36 ± 1.35 mV, 43.66 ± 3.30, and 12.06 ± 0.83%, respectively. In vitro drug release exhibited a biphasic pattern with an initial burst release of 18% within 2 hr followed by a sustained release over 22 hr. The pharmacokinetic results showed that amoxicillin nps have higher bioavailability and longer plasma half-life (p < .01) than free amoxicillin. These results indicate that amoxicillin nano formulation is suitable for oral administration in broilers.

Single-walled carbon nanotubes as delivery vehicles enhance the immunoprotective effects of a recombinant vaccine against Aeromonas hydrophila.

To reduce the economic losses caused by diseases in aquaculture industry, more efficient and economic prophylactic measures should be urgently investigated. In this research, the effects of a novel functionalized single-walled carbon nanotubes (SWCNTs) applied as a delivery vehicle for recombinant Aeromonas hydrophila vaccine administration via bath or injection in juvenile grass carp were studied. The results showed that SWCNT as a vector for the recombinant protein aerA, augmented the production of specific antibodies, apparently stimulated the induction of immune-related genes, and induced higher level of survival rate compared with free aerA subunit vaccine. Furthermore, we compared the routes of bath and intramuscular injection immunization by SWCNTs-aerA vaccine, and found that similar antibody levels induced by SWCNTs-aerA were observed in both immunization routes. Meanwhile, a similar relative percentage survival (approximately 80%) was found in both a 40 mg/L bath immunization group, and a 20 µg injection group. The results indicate that functionalized SWCNTs could be a promising delivery vehicle to potentiate the immune response of recombinant vaccines, and might be used to vaccinate juvenile fish by bath administration method.

Vaginal histological changes after using intravaginal sponges for oestrous synchronization in anoestrous ewes.

To characterize the histological and cytological vaginal changes generated by the use of intravaginal sponge (IS) applied in oestrous synchronization treatments in ewes during mid-non-breeding season. Thirty-five multiparous ewes were allocated to three experimental groups according to the moment in which the samples were taken: (i) ewes treated with IS containing 60 mg of medroxyprogesterone acetate for 14 days, sampled the day of IS removal (group ISR; n = 10), (ii) or after sponge removal at time of oestrus or 72 h after removal (group AR; n = 14) and (iii) ewes without sponge treatment that were sampled at the day of IS removal of the other groups (group CG; n = 11). Vaginal biopsies and cytological samples were taken from the anterior vaginal fornix area. The vagina of the CG group had a stratified squamous epithelium with a moderate degree of cellular infiltration with lymphocytes and plasma cells in the lamina propia. Treated ewes (ISR and AR) had epithelial hyperplasia and hypertrophy. ISR ewes had haemorrhage and perivascular infiltrate and an increased number of epithelial cells, neutrophils, macrophages and erythrocytes at IS removal. The use of IS generated histological and cytological alterations in the vaginal wall when used for oestrous synchronization in anoestrous ewes.

Preliminary investigation on the design of biodegradable microparticles for ivermectin delivery: set up of formulation parameters.

The aim was to design sterile biodegradable microparticulate drug delivery systems based on poly(dl-lactide) (PLA) and poly(ε-caprolactone) (PCL) and containing ivermectin (IVM), an antiparasitic drug, for subcutaneous administration in dogs. The drug delivery system should: (i) ensure a full 12-month protection upon single dose administration; (ii) be safe with particular attention regarding IVM dosage and its release, in order to prevent over dosage side effects. This preliminary work involves: polymer selection, evaluation of the effects of γ-irradiation on the polymers and IVM, investigation and set up of suitable microparticle preparation process and parameters, IVM-loaded microparticles in vitro release evaluation. Results of gel permeation chromatography analysis on the irradiated polymers and IVM mixtures showed that combination of IVM with the antioxidant α-tocopherol (TCP) reduces the damage extent induced by irradiation treatment, independently on the polymer type. Solvent evaporation process was successfully used for the preparation of PLA microparticles and appropriately modified; it was recognized as suitable for the preparation of PCL microparticles. Good process yields were achieved ranging from 76.08% to 94.72%; encapsulation efficiency was between 85.76% and 91.25%, independently from the polymer used. The type of polymer and the consequent preparation process parameters affected microparticle size that was bigger for PCL microparticles (480-800 µm) and solvent residual that was >500 ppm for PLA microparticles. In vitro release test showed significantly faster IVM release rates from PCL microparticles, with respect to PLA microparticles, suggesting that a combination of the polymers could be used to obtain the suitable drug release rate.

Plant-based solutions for veterinary immunotherapeutics and prophylactics.

An alarming increase in emergence of antibiotic resistance among pathogens worldwide has become a serious threat to our ability to treat infectious diseases according to the World Health Organization. Extensive use of antibiotics by livestock producers promotes the spread of new resistant strains, some of zoonotic concern, which increases food-borne illness in humans and causes significant economic burden on healthcare systems. Furthermore, consumer preferences for meat/poultry/fish produced without the use of antibiotics shape today's market demand. So, it is viewed as inevitable by the One Health Initiative that humans need to reduce the use of antibiotics and turn to alternative, improved means to control disease: vaccination and prophylactics. Besides the intense research focused on novel therapeutic molecules, both these strategies rely heavily on the availability of cost-effective, efficient and scalable production platforms which will allow large-volume manufacturing for vaccines, antibodies and other biopharmaceuticals. Within this context, plant-based platforms for production of recombinant therapeutic proteins offer significant advantages over conventional expression systems, including lack of animal pathogens, low production costs, fast turnaround and response times and rapid, nearly-unlimited scalability. Also, because dried leaves and seeds can be stored at room temperature for lengthy periods without loss of recombinant proteins, plant expression systems have the potential to offer lucrative benefits from the development of edible vaccines and prophylactics, as these would not require "cold chain" storage and transportation, and could be administered in mass volumes with minimal processing. Several biotechnology companies currently have developed and adopted plant-based platforms for commercial production of recombinant protein therapeutics. In this manuscript, we outline the challenges in the process of livestock immunization as well as the current plant biotechnology developments aimed to address these challenges.

Nano-emulsified black soldier fly oil concerning performance traits, health, and immunity of broilers.

In the antibiotic-free era, traditional antibiotics have been suggested as alternatives to antibiotic-based growth promoters. Among the various methods, self-nano-emulsifying drug delivery systems (SNEDDS) are increasingly utilized to improve the bioavailability of oils containing essential substances. In this study, we evaluated the effects of black soldier fly oil (BSFO) SNEDDS in chicken drinking water on growth performance, small intestine histomorphology, and poultry health status. We divided 225 male Indian River strain broiler chickens into five treatment groups, each consisting of 5 replicates. The chickens were reared from to 0 to 35 d of age in a controlled environmental housing system. The BSFO SNEDDS was administered via drinking water. Treatments included ordinary drinking water (P1), bacitracin (P2), and 10 mL/L (P3), 20 mL/L (P4), and 30 mL/L (P5) BSFO SNEDDS. The observed parameters included growth performance, carcass yield, blood hematology, intestinal histomorphology, digestive microflora, and immunoglobulin (Ig) levels. Data were analyzed using analysis of variance (ANOVA) and Duncan's test. The results indicated that administering BSFO SNEDDS via drinking water improved feed conversion (P < 0.05), enhanced the performance index (P < 0.05), increased carcass percentage (P < 0.001), and increased the weight of the carcass parts. Additionally, it increased villus height (P < 0.01), lowered jejunal pH (P < 0.001), reduced pathogenic bacteria in the jejunum, and decreased the leukocyte count. BSFO SNEDDS in drinking water is expected to be used as a traditional antibiotic with the potential to replace synthetic antibiotic growth promoters in broiler chickens.

Effects of treatment with human chorionic gonadotrophin or intravaginal progesterone-releasing device after AI on circulating progesterone concentrations in lactating dairy cows.

Adequate circulating progesterone (P4) is important for pregnancy. Lactating dairy cattle have lower circulating P4, particularly when smaller follicles are ovulated during timed AI protocols. The aim of the present study was to determine the supplementation strategy that resulted in P4 concentrations in lactating dairy cattle similar to those in heifers. Lactating Holstein cows (n=61) were synchronised using the Double-Ovsynch method and, on Day 5, were randomly assigned to receive no treatment (control), controlled internal drug release (CIDR), human chorionic gonadotrophin (hCG; 3300 IU) or CIDR+hCG. Heifers after normal oestrus were followed as controls (n=10). Profiles of circulating P4 concentrations were compared using repeated-measures ANOVA. Heifers had greater P4 concentrations than control cows at all times after Day 5 (P<0.0001). Cows receiving CIDR had lower P4 concentrations than heifers (P=0.0037) on Days 8-16. Treatment with hCG generally caused ovulation and resulted in circulating P4 concentrations greater than those in control lactating cows by 3 days after treatment (Day 8 after AI), but the treatment×time interaction (P=0.01) showed that cows treated with hCG generally had lower P4 concentrations than heifers. Supplementation with CIDR+hCG resulted in P4 concentration profiles similar to those in heifers. Thus, the use of CIDR and the production of an accessory corpus luteum with hCG elevates P4 concentrations in lactating cows to those seen in heifers. This information may be useful for designing future trials into P4 supplementation and fertility.

A five-day progesterone plus eCG-based fixed-time AI protocol improves fertility over spontaneous estrus in high-producing dairy cows under heat stress.

This study compared the efficiency of a five-day or standard (nine-day) progesterone-based regimen combined with equine chorionic gonadotrophin (eCG) in a fixed-time AI (FTAI) protocol for dairy cows. The data examined were derived from 3577 inseminations conducted in three dairy herds. Animals with no estrus signs detected over 21 days were randomly assigned to a PRID-9 or PRID-5 group. Cows in each group received a progesterone intravaginal device (PRID) for 9 or 5 days, respectively, PGF(2α) and eCG on PRID removal, and GnRH 48 h later. Fixed-time AI was performed 12 h after the GnRH dose. Cows artificially inseminated following spontaneous estrus during the study period were considered as controls. Based on the odds ratio, the likelihoods of animals in PRID-9 in the warm (conception rate [CR] of 22.3%) and cool (32% CR) periods, and control animals in the warm period (26.6% CR) becoming pregnant were reduced (by factors of 0.6, 0.3 and 0.4, respectively) compared with the control animals in the cool period (CR of 43.7%). The risk of a twin pregnancy was higher (51.4%) for cystic PRID-9 cows (by a factor of 3.6) and lower (9.9%) for cyclic PRID-5 animals (by a factor of 0.4) compared with the PRID-9 cyclic cows. These findings indicate that the proposed protocol achieves similar results during the cool or warm season to those obtained when AI is conducted at spontaneous estrus during the cool season. In addition, PRID-5 reduced twin pregnancy compared with PRID-9.

Alteration of the endometrial EGF profile as a potential mechanism connecting the alterations in the ovarian steroid hormone profile to embryonic loss in repeat breeders and high-producing cows.

Poor reproductive efficiency is a worldwide problem that has affected the dairy industry during the last several decades. In an attempt to explain the changes in reproductive physiology caused by high milk production, a model of elevated steroid metabolism in lactating dairy cows has been proposed. A slow increase in levels and low peak levels of estradiol (E2) and progesterone (P4) characterize endocrine changes in high producing cows. Similar changes have been reported in the repeat breeder cows. The abnormal changes in E2 and P4 concentrations of these cows may cause an improper uterine environment due to disturbed expression of growth factors and cytokines in the endometrium. This review focuses on the alteration in epidermal growth factor (EGF) profile in the endometrium during the estrous cycle. The normal cow has two peaks of EGF concentrations on days 2-4 and 13-14. Low concentrations of EGF on these days distinguished both high-producing and repeat breeder cows from normal cows. Alteration of the EGF profile could be found in 70 and 40% of the repeat breeder and high-producing cows, respectively. Treatment with a high dose of estradiol benzoate and an intravaginal progesterone-releasing device restored the normal EGF profile in about 70% of the affected cows. The cows having a normal EGF profile after treatment showed a higher pregnancy rate than the cows with the altered profile. Further studies to understand the etiology of the alteration in the EGF profile are needed to develop another treatment option and preventive management for this problem.

Construction of an attenuated Salmonella delivery system harboring genes encoding various virulence factors of avian pathogenic Escherichia coli and its potential as a candidate vaccine for chicken colibacillosis.

An attenuated Salmonella (deltalon, deltacpxR, and deltaasdA16) delivery system containing the genes encoding P-fimbriae (papa and papG), aerobactin receptor (iutA), and CS31A surface antigen (clpG) of avian pathogenic Escherichia coli (APEC) was constructed, and its potential as a vaccine candidate against APEC infection in chickens was evaluated. The birds were divided into three groups designated group A (nonvaccinated control), group B (given a single immunization), and group C (administered prime and boost immunizations). Prime and booster vaccinations with the constructions were administered to 1-day-old and 14-day-old birds, respectively. Immune responses were measured postimmunization, and the birds were challenged via an intra-air sac route with a virulent APEC strain at the second, third, and fourth weeks of age. Group B birds were partially protected against the challenge and showed increased levels of plasma immunoglobulin (Ig)G, mucosal IgA antibodies, and lymphocyte proliferation. Group C birds showed greater protection against the challenge, with significantly stronger immune responses compared with the birds in the other groups. Overall, our data suggest that the Salmonella delivery system with recombinant constructs is capable of inducing robust immune responses and induces effective protection against colibacillosis caused by APEC.