Feline infectious peritonitis (FIP) is an infectious disease characterized by non-specific laboratory changes and clinical signs. Clinical symptoms include anorexia, jaundice, fever, and weight loss. Moreover, some lesions are found in the digestive and respiratory systems. FIP, whose virulence varies, cannot be distinguished using several diagnostic methods. Moreover, feline coronaviruses (FCoVs) can be classified into two serotypes based on differences in their amino acid sequences, spike (S) protein sequences, and antibody (Ab) neutralization. There are two pathotypes, namely those caused by FCoV, which are often referred to as feline enteric coronavirus and FIP virus (FIPV). Furthermore, FIPV infection can be caused by sub-neutralizing levels of anti-FIPV S Abs. Therefore, a supporting diagnosis is needed to confirm FIP because there are no specific symptoms. 

Buffalo is the principal dairy animal and plays a major role in the economic growth of the dairy industry, contributing nearly 50% of the country’s milk production. The Buffalo core body temperature is typically 38.5^°C, but it can rise to 41.5^°C in the summer, causing heat stress, which leads to the generation of reactive oxygen species or oxidative stress and affects the reproductive physiology of animals. Curcumin acts as an antioxidant, improves cellular development, and combats the effect of heat stress on in vitro-produced embryos. This study aimed to examine the impact of curcumin on developmental competence and the expression of important genes under normal and heat-stressed conditions during in vitro embryo production in buffalo.  Group-1: All embryo production steps (i.e., in vitro maturation [IVM], in vitro fertilization [IVF], and in vitro culture [IVC]) were conducted at 38.5^°C. The presumed zygotes were cultured in media supplemented with different concentrations of curcumin, that is, 0 μM, 5 μM, and 10 μM of curcumin. Group-2: All embryo production steps (i.e., IVM, IVF, and IVC) were carried out at 38.5^°C. The presumed zygotes were cultured in media supplemented with different concentrations of curcumin, that is, 0 μM, 5 μM, and 10 μM of curcumin, but the early cleaved embryos were exposed to heat stress (39.5^°C) for 2 h after 48 h of IVF and then cultured at 38.5^°C for embryo production.  Blastocyst production was 16.63 ± 1.49%, 21.46 ± 0.67%, and 6.50 ± 1.17% at control, 5 μM and 10 μM of curcumin at 38.5^°C, respectively, whereas at 39.5^°C, it was 8.59 ± 1.20%, 15.21 ± 1.31%, and 3.03 ± 1.20% at control, 5 μM and 10 μM curcumin, respectively. The blastocyst rate was found to be significantly higher (p < 0.05) at 5 μM curcumin compared with the control or 10 μM at 38.5^°C and 39.5^°C. The antioxidant, antiapoptotic, and pluripotency-related genes exhibited higher (p < 0.05) expression in the presence of 5 μM curcumin compared to 10 μM or control at both temperatures.  Curcumin supplementation in embryo culture media effectively enhances embryo production in vitro and mitigates the adverse effects of heat stress. 

Goats are valuable livestock because they can generate meat and milk for human consumption. Goat husbandry is becoming more intensive due to the growing demand for goat products, which may impact animal welfare and natural behavior. This study aimed to investigate the impact of natural rubber (para rubber)-based scratchers as an environmental enrichment on scratching behavior, cortisol levels, and semen quality in stable bucks (male goats/goats).  Nine male goats were used in this study. Scratching behavior and cortisol levels were used as welfare indicators, whereas semen quality was evaluated as an indicator of reproductive potential. These indicators were analyzed before and after scratcher installation.  After installing the scratchers, the goats showed a significant increase in scratching behavior and a notable decrease in cortisol levels (p < 0.001). Notably, the goats exhibited a marked preference for scratching against the scratcher (p < 0.001) compared to the stable. They significantly preferred using their heads for scratching (p < 0.001) instead of other body parts. In addition, goats preferred to scratch on the softest rubber scratchers at specific installation locations (p < 0.001). Although there was a slight improvement in semen quality, there was no statistically significant difference (p > 0.05).  A natural rubber-based scratcher can increase scratching behavior and reduce cortisol levels, indicating its potential to improve the welfare of farm goats. Selecting an appropriate hardness and preferred location is essential to ensure that the scratcher effectively encourages animals to use it. 

Skin antisepsis plays a crucial role in pre-operative skin preparation, with chlorhexidine gluconate and alcohol being historically the preferred choice. However, concerns have risen regarding the development of bacterial resistance to chlorhexidine. Polyhexamethylene biguanide (PHMB) combined with Tris-ethylenediaminetetraacetic acid (Tris-EDTA) has recently emerged as a skin and wound antiseptic. This study aimed to compare the antibacterial efficacy and local safety of 2% chlorhexidine gluconate with 70% alcohol (CG+Alc) and 0.3% PHMB with 6% Tris and 1.86% EDTA (PHMB+Tris-EDTA) for pre-operative skin preparation in dogs.  Twenty-four adult dogs underwent aseptic preparation on both sides of their ventral abdomens, with one side receiving CG+Alc and the other side receiving PHMB+Tris-EDTA, assigned randomly. Skin swab samples were collected pre-antisepsis and at 3-, 10-, and 60-min post-antisepsis to quantify bacterial colony-forming units (CFUs). Local skin reactions (erythema and edema) were evaluated after hair clipping, pre-antisepsis, and at 3-, 10-, 30-, and 60-min post-antisepsis.  There was no significant difference in bacterial CFU counts between the two antiseptic groups pre-antiseptic. Both solutions significantly reduced CFU counts (p < 0.05) at all post-antisepsis sampling times compared with pre-antisepsis. However, dogs treated with PHMB+Tris-EDTA showed a significantly higher incidence of edema at 10 min (p = 0.02) and 30 min (p = 0.003) and a higher incidence of erythema at 10 min (p = 0.043) post-antisepsis compared with CG+Alc. No skin reactions were observed in either group at 60 min post-antisepsis.  CG+Alc and PHMB+Tris-EDTA reduced bacterial counts in pre-operative skin preparation in dogs. However, acute transient skin reactions were observed more frequently following the application of PHMB+Tris-EDTA. 

The global prevalence of gastrointestinal (GI) and blood parasite infections in horses is a significant concern due to their substantial impact on morbidity, mortality, and economic losses in the horse industry. In Thailand, limited research has been conducted on these parasites in horse populations, and data from southern Thailand are lacking. Consequently, this study aimed to estimate the prevalence of GI and blood parasites in horses in Nakhon Si Thammarat province, Thailand.  In total, 79 fecal and blood samples were collected from horses across 11 farms in Nakhon Si Thammarat province. The fecal examination was conducted using simple flotation, formalin-ethyl acetate sedimentation, and a modified McMaster technique. Conventional polymerase chain reaction (PCR) was used to identify blood and strongyle parasites. The influence of sex, age, and body condition score on the prevalence of GI parasites was also analyzed.  Six GI parasites were detected: four nematodes (Oxyuris equi, Parascaris equorum, strongyles, and Strongyloides westeri), one trematode (Gastrodiscus aegyptiacus), and one protozoan (Eimeria leuckarti). The overall prevalence of GI parasites was 74.7%, with single strongyle infections accounting for the highest proportion at 50.6%, followed by co-infections of strongyles and G. aegyptiacus at 10.1%. All 11 pooled strongyle samples were positive for cyathostomins and Strongylus vulgaris using conventional PCR with specific primers. Sex was significantly associated with the overall prevalence of GI parasites, whereas both sex and age were significant risk factors for infection by strongyle parasites. Theileria equi was the only blood parasite species detected in the surveyed horses, with a prevalence of 1.3% (n = 1/79).  This study is the first to estimate the prevalence of GI and blood parasites in horses from Nakhon Si Thammarat province, Thailand. These findings highlight the importance of implementing control measures against GI parasites and are pivotal for developing effective infection prevention strategies. 

Foot and mouth disease (FMD) is highly contagious in cloven-hoofed animals, and it causes outbreaks in Indonesia and several countries worldwide. This disease is caused by the FMD virus (FMDV), which belongs to the genus Aphthovirus and family Picornaviridae. In 1990, the World Organization for Animal Health Office International des Epizooties recognized Indonesia as an FMD-free country. A new FMDV outbreak in Indonesia was reported in April 2022 and confirmed in May 2022, resulting in economic losses to the beef cattle sector. This study aimed to determine the genotype and amino acid content of viral proteins (VP1) gene.  Samples were obtained from vesicle swabs from the mouth and feet of cattle in Banyuwangi Regency, Lamongan Regency, and Surabaya City, East Java, Indonesia. Samples were identified using one-step reverse transcriptase-polymerase chain reaction with a pair of specific primers encoding the VP1O serotype with a target of 1165 bp.  Sequencing revealed that the FMDV subtype belonged to the O/ME-SA/Ind2001e. Phylogenetic analysis showed that our isolate was 100% amino acid-identical to the Indonesian outbreak isolates from 2022 and 95% identical to isolates from Southeast Asia. The amino acid substitutions found in the G-H Loop of the VP1were S134C, D138E, T140A, and A156T. Only the K135Q mutation was detected in Lamongan.  The spread of the subtype O/ME-SA/Ind2001e in South-east Asia caused an outbreak in Indonesia due to less stringent animal traffic control measures. Surveillance studies and whole-genome sequence analyses are important for monitoring FMDV genetics in Indonesia. 

Reproductive disorders in swine herds pose significant challenges to pig breeding due to both infectious and non-infectious factors. In large-scale pig farming, coinfections are increasingly common, affecting sow health and herd productivity. This study aimed to determine occurrence and coinfection patterns of Mycoplasma suis and porcine circovirus type 3 in Vietnamese pig farms and to evaluate their association with reproductive disorders and clinical signs in affected herds.  We collected 291 samples from 15 farms, composed of whole blood and various tissues from fetuses and weak-born piglets. Molecular biological testing was conducted to detect key pathogens of interest. Consistently, porcine circovirus type 3 (PCV3) and porcine Hemoplasma were detected and sequenced for the whole genome and partial 16S rRNA, respectively. The genetic diversity of PCV3 and Mycoplasma suis was analyzed.  Various clinical signs, including abortion, stillborn, mummified, and weak-born piglets, and dermatitis, were recorded. M. suis was detected in 252/291 (86.59%) samples from all 15 surveyed farms, with an occurrence of 100%. PCV3 was detected in 35.05% (102/291) samples and 73.3% (11/15) of farms. PCV3 and M. suis coinfections were observed in 29.21% of the positive samples. It should be noted that most PCV3 Ct-values were above 30, indicating the existence of PCV3 in the herd but with insufficient data to confirm its pathogenic potential. The complete genomes of 10 PCV3 strains identified in this study exhibited high sequence homology, with >97% nucleotide identity. In addition, the eight partial 16S rRNA porcine Hemoplasma sequences shared absolute identity with M. suis isolates from pigs in China and Germany.  This report on the occurrence of M. suis and PCV3 in pigs from farms with reproductive failure provides important insights into the expanding global distribution of these pathogens. Our findings warrant further investigations of the pathogenic potential and economic implications of M. suis and PCV3 in pigs with reproductive failure in Vietnam. 

Ascaridia galli, a nematode that frequently infects the digestive tract of chickens, is a significant concern for poultry health. In response, the use of medicinal plant-derived anthelmintics was proposed as a potential solution. This study observed the in vitro effectiveness of a single, graded dose of the ethanol extract of Andrographis paniculata, Phyllanthus niruri L., Curcuma xanthorrhiza Roxb., and Curcuma aeruginosa Roxb. on the movement activity of adult A. galli every hour for 6 h, followed by an analysis of worm cuticle damage in A. galli.  A randomized block design was used. Adult A. galli were collected from the intestinal lumen of fresh free-range chickens. Each petri dish contained two A. galli for each treatment with three replications. Each plant extract (A. paniculata, P. niruri L., C. xanthorrhiza Roxb., and C. aeruginosa Roxb.) was evaluated with three distinct doses, which were 250 μg/mL, 500 μg/mL, and 1000 μg/mL; 0.9% sodium chloride solution was used as a negative control, and 500 μg/mL Albendazole solution was used as a positive control. The active compound content of A. paniculata, P. niruri L., C. xanthorrhiza Roxb., and C. aeruginosa Roxb. extracts were analyzed using ultra-performance liquid chromatography-mass spectrometry. The movement activity of A. galli was determined by the percentage score value from the 1st to the 6th h in each treatment group, followed by analysis of damage to the A. galli cuticle layer using a nano-microscope and histopathological images.  Analysis of variance demonstrated that at doses of 250 μg/mL and 500 μg/mL, the ethanol extracts of A. paniculata, P. niruri L., C. xanthorrhiza Roxb., and C. aeruginosa Roxb. did not have a significant effect on the effectiveness of A. galli’s motility (>0.005). However, at a dose of 1000 μg/mL, the ethanol extract of A. paniculata, P. niruri L., C. xanthorrhiza Roxb., and C. aeruginosa Roxb. reduced the motility of A. galli. Importantly, the motility of A. galli in the dose of 1000 μg/mL A. paniculata and P. niruri L. extract groups was very weak and significantly different (p < 0.001) compared to the negative control group. The content of the active compound Andrographolide in the ethanol extract of A. paniculata and the active compound 5-Methoxybenzimidazole in the extract of P. niruri L. are strongly suspected to play an important role in damaging and shedding the cuticle layer of A. galli.  All herbal extracts have anthelmintic activity at a concentration of 1000 μg/mL. Extracts of A. paniculata, P. niruri L., C. xanthorrhiza Roxb., and C. aeruginosa Roxb. have activities that can damage and dissolve the cuticle layer of A. galli, resulting in the weakening of the motility of A. galli. 

To address the problems associated with the availability and prices of conventional feeds, researchers have started to include alternative feeds to reduce the cost of diets and increase profitability. This study examined the influences of olive cake (OC), either alone or in combination with Saccharomyces cerevisiae (SC), in the diet of black kids.  Thirty kids were distributed into three treatments: A free OC diet (control [CON]), OC (20% OC), and OCSC (20% OC with 1 g SC head/d). While penned individually, the kids were fed daily and allowed to acclimate to their diets and pens for 7 days. After acclimation, the kids were fed the prescribed diets for 63 days. Five kids were chosen from each group on day 50 of the feeding period and moved to the metabolic cages to evaluate nutrient digestibility and N balance. At the end of the study, all kids were slaughtered to evaluate carcass characteristics and meat quality. Feeding the OCSC diet increased (p = 0.035) DM intake for kids compared with the OC and CON. The CP intake was higher (p = 0.021) in the OCSC group than in the CON group. Growth performance parameters were higher (p ≤ 0.031) in the OCSC group than in the CON diet. Feed conversion efficiency was improved (p = 0.052) by incorporating OCSC compared with the CON diet. Consequently, economic return was enhanced (p = 0.003) by feeding the OC and OCSC groups. Kids fed the OCSC had a higher cold carcass weight (p = 0.054) than those fed the CON diet. The OCSC group also showed increased (p ≤ 0.027) loin cut and intermuscular fat weights compared with the OC and CON groups. All measured serum metabolites were similar in CON and groups incorporating OCs or OCSCs.  The results showed that adding 20% OC combined with SC to the diet of growing kids improved some growth and carcass characteristic parameters without affecting their health status. Notably, using OC along with SC could be an option for feed cost reduction in kids’ diets. 

Rodents are carriers or reservoirs of various bacteria, protozoa, viruses, and ectoparasites. Given the proximity of various rodent species and humans, there is a potential for the transmission of pathogens. Data on ecto- and endo-parasite prevalence in rodent populations in Gabon are limited. To fill this gap, we conducted a study in Libreville to investigate the occurrence of ecto- and endo-parasites in rodents.  We captured and euthanized 68 rodents belonging to the genus Praomys and examined their ecto- and endo-parasite fauna, dissected their gastrointestinal tract for helminths, and prepared blood smears to examine blood-borne pathogens.  Our analyses identified three pathogen taxa: helminths (Protospirura spp., Trichuris spp., and Taenia spp.), protozoa (Babesia spp.), bacteria (Anaplasma spp.), and arthropods (Laelaps and Haemaphysalis). Overall, 91.2% of the rodents were infected with at least one pathogen and ectoparasite, with helminth and ectoparasite occurrence rate of 63.2% and ectoparasite occurrence at 44.1%. Protozoan infections (Babesia spp.) were found in 10.3% of the rodents, whereas bacteria (Anaplasma spp.) had an occurrence rate of 39.7%.  Native rodents in Libreville harbor various infectious agents, ecto- and endo-parasites. These findings highlight the potential health risks associated with Praomys rodents for the transmission of various diseases to human population in Gabon and emphasize the need for investigation of rodents for their role as disease carriers. 

The positive effects of Lactobacillus farciminis and Lactobacillus rhamnosus on growth and feed consumption indicators have been described; however, the underlying mechanisms remain unclear. This study aimed to determine whether the addition of L. farciminis CNCM-I-3699 (2.1010 GU/g) and L. rhamnosus CNCM-I-3698 (2.1010 GU/g) to the feed of Ross 308 specific-pathogen-free (SPF) broiler chickens (at a dose of 4 g/10 kg feed) affects live weight gain, the feed conversion ratio (FCR), and duodenal development in SPF broiler chickens.  In total, 780 SPF broiler chicks were randomly divided into two groups (three replicates per group) immediately after hatching: The control group (n = 390) and the probiotic group (n = 390). Live body weight (g) and FCR were measured on days 1, 7, 14, 21, 28, and 35 of the study. Histological examinations (hematoxylin and eosin staining) of the duodenum were performed, and the villus height (VH), villus width, crypt depth (CD), muscle layer thickness, and VH: CD ratio were measured. In addition, immunohistochemical examinations were performed to determine the number of proliferating cell nuclear antigen (PCNA)-positive cells.  Feeding a probiotic mixture containing L. farciminis and L. rhamnosus to SPF broiler chickens for 35 days increased the duodenal absorption area and muscle layer thickness. In addition, it accelerated the histological development of the duodenum, as evidenced by the significantly higher number of PCNA-positive cells within the crypts. Although SPF broiler chickens in the ProL group exhibited greater live weight gain and lower FCR throughout the study, these differences were not statistically significant. These results suggest that L. farciminis and L. rhamnosus can serve as additives to SPF broiler chicken feed to promote growth and development. 

In animal husbandry, antibiotics are frequently used as growth promoters, as well as for illness prevention and treatment. They are considered important toxic and allergenic contaminants of food and a serious risk factor for the spread of antibiotic resistance. National and international regulatory authorities have established limits on the permissible residue of antibiotics in food. Immunochromatographic test strips are the most efficient tools for the simple and rapid control of antibiotics for food safety. In these tests, each antibody specific to a particular antibiotic is labeled with a marker, which complicates the manufacturing technology and increases the cost of the test. This study aimed to develop a multiplex immunochromatographic assay (ICA) to determine streptomycin (STR), tetracycline (TC), and chloramphenicol (CAP) residues in milk by indirect labeling of antibiotic-specific antibodies.  Test strips were composed using 15 μm pore size CNPC nitrocellulose membranes, GFB-R4 separation, and AP045 adsorption pads. The applied reactants include TC and STR conjugates with bovine serum albumin, and CAP-soybean trypsin inhibitor conjugate; anti-TC, anti-STR, and anti-CAP mouse monoclonal antibodies; goat anti-mouse immunoglobulin G (GAMI) conjugated with gold nanoparticles (GNPs) and staphylococcal protein A. Milk samples were collected from cows and goats that had not been injected with any antibiotics. STR and TC/CAP at concentrations of 0.27–600 ng/mL and 0.04–30 ng/mL were added to skim milk, respectively. Milk samples were tested by ICA and calibration curves were constructed to determine the sensitivity of the assay for each antibiotic used.  A multiplex ICA of three antibiotic residues in milk was achieved through labeling of immune complexes using a single reagent, GNPs-GAMI. The visual limits of detection (LOD) were 600 ng/mL, 10 ng/mL, and 30 ng/mL for STR, TC, and CAP in cow and goat milk, respectively. Instrumental LODs gave higher sensitivity when analyzed goat milk to STR, TC, and CAP (1.2, 0.05, and 1.3 ng/mL) than cows’ milk (7.27, 0.96, and 2.07 ng/mL, respectively).  The developed approach for manufacturing multiplex ICA tests for the detection of antibiotic residues in milk does not involve labeling specific antibodies and is implemented using only GNP conjugates with anti-species antibodies. 

Coat color is a phenotypic trait that is affected by many functional genes. In addition, coat color is an important characteristic of breeds in livestock. This study aimed to determine functional genes for coat color patterns in Sumatran native cattle in Indonesia using a genome-wide association study method.  A bovine single nucleotide polymorphism (SNP) 50K BeadChip was used for the investigation. A total of 46 Sumatran native cattle of three colors as follows: Brown (36 animals), white (9 animals), and black (1 animal), were used as experimental animals.  Results showed that the SNP markers ARS-BFGL-NGS-75486 (p = 2.46 × 10^-7) and BTB-01992588 (p = 1.06 × 10^-5) were selected as two genetic markers for coat color variation in animals under study, which were located at the cytoplasmic FMR1-interacting protein 2 (CYFIP2) gene at BTA7 and small G protein signaling modulator 1(SGSM1) genes at BTA17, respectively. The polymorphic informative content values of both SNP markers were 0.33 (ARS-BFGL-NGS-75486) and 0.13 (BTB-01992588). In this study, a genetic marker for coat color patterns in Sumatran native cattle was obtained based on the haplotypes of both SNP markers.  It can be concluded that CYFIP2 and SGSM1 are two coloration genes that affect the phenotype characteristics of Sumatran native cattle. 

One of the worst zoonotic illnesses, avian influenza (AI), or commonly referred to as bird flu, is caused by viruses belonging to the genus Influenza viruses, which are members of the Orthomyxoviridae family. The harmful effects of AI illness can affect both human and animal health and cause financial losses. Globally, the AI virus lacks political purpose and is not limited by geographical limits. It has been isolated from poultry, wild birds, and captive birds in Asia, North America, Europe, Australia, and South America. Their virulence is divided into highly pathogenic AI (HPAI) and low pathogenic AI (LPAI). The AI virus can also be diagnosed in a laboratory setting using molecular tests like real-time polymerase chain reaction or serological tests like the hemagglutinin inhibition test, agar gel immunodiffusion, antigen detection enzyme-linked immunosorbent assay, and other immunoassays. The type of AI virus and host species determines the clinical manifestations, severity, and fatality rates of AI. Human infection with AI viruses typically results from direct transmission from infected birds to humans. AI outbreaks in domestic and wild birds are uncommon; however, an infection can pose a significant threat to public, veterinary, and medical health. Successful vaccination reduces the probability of AI H5N1 virus infection in meat and other poultry products and prevents systemic infection in chickens. This review will provide information that can be used as a reference for recognizing the dangers of AI and for preventing and controlling the disease, considering its potential to become a serious pandemic outbreak. 

Fowl adenovirus (FAdV) is the etiological agent of inclusion body hepatitis (IBH) and hepatitis-hydropericardium syndrome (HHS) in poultry. It is also detected in chickens with runting and stunting syndrome (RSS). FAdV has been detected worldwide, and genotypes 8a, 8b, and 11 have been identified in chickens with enteric problems in Brazil. Nevertheless, none of them have been isolated; therefore, these viruses propagate; thus, the viral behavior and pathogenicity are unknown in Brazil. This study aimed to isolate FAdV from the enteric content of chickens affected by RSS.  Enteric content samples from chickens affected with RSS and a positive polymerase chain reaction (PCR) test for FAdV were inoculated into specific pathogen-free chicken embryonated eggs (CEEs) through the yolk and chorioallantoic membrane routes on 8 and 10 days of age, respectively and the eggs were incubated for five days for viral isolation.  The embryos exhibited dwarfism, beak atrophy, and pale claws. In addition, some embryos displayed edema and gelatin-like characteristics. The liver exhibited hepatomegaly and multiple necrotic foci, resembling the appearance of nutmeg. In addition, the kidneys appeared enlarged and pale. After 8 days of incubation, the hearts of the inoculated embryos showed hydropericardium. Microscopic evaluation revealed the presence of hepatitis, which was characterized by the presence of intranuclear inclusion bodies and cellular necrosis. Viral FAdV particles were observed in kidney cells using electron microscopy. Viral DNA was detected in the embryos in all three passages, and viral gene copies were also measured in some organs, with high FAdV gene copies detected in the spleen and bursa beyond the liver.  The molecular characterization of FAdV revealed that the isolated strain belonged to genotype 8a of FAdV. Here, FAdV-8a from chickens infected with RSSs produced IBH/HHS in CEE, and FAdV-8a detected in RSS outbreaks, in addition to producing IBH/HHS in chicken embryos, could be a possible viral agent that causes IBH/HHS in chickens. 

Bacterial lipopolysaccharide (LPS)-induced neuroinflammation can be the most dependable animal model for studying neurodegeneration mechanisms driven by systemic inflammation-induced neuroinflammation. Hence, this study aimed to standardize the LPS model of neuroinflammation by comparing the effect of relatively low-dose LPS administered for different durations on the induction of neurodegeneration in Wistar rats.  Six groups of six adult Wistar rats per group were used in the study. Group 1 was the control group, and the other five were administered single weekly dose of LPS (170 μg/kg) for increasing durations, ranging from 4 weeks to 8 weeks. The study endpoints included behavioral parameters, neuronal assay results, and the expression of microglia and astrocytes in the frontal cortex, dentate gyrus, and hippocampus.  We observed a significant reduction in the number of neurons and an increase in glial cells at 5 weeks of exposure, along with a decline in memory. Thereafter, these changes were gradual until 7 weeks of exposure. However, at 8 weeks of exposure, there was no further statistically significant worsening compared with the group exposed for 7 weeks.  To effectively induce neuroinflammation and cause neuronal damage, a minimum of five weekly LPS administrations at a dose of 170 μg/kg is required. Moreover, our results recommend a maximum of 7 weeks of LPS exposure to create a chronic inflammatory model of neuroinflammation. 

Mycobacterium avium subspecies paratuberculosis (MAP) causes Johne’s disease (paratuberculosis), which is a chronic wasting disease. No previous study has been found to investigate the coinfection of gastrointestinal parasites with paratuberculosis. This study aimed to investigate the relationship between paratuberculosis and gastrointestinal parasitism in naturally infected local Awassi (AS) and imported Tsurcana (TS) sheep.  A total of 207 sheep (118 AS and 89 TS) were randomly selected from local butcher shops and slaughterhouses. Fecal and tissue samples from the ileum and mesenteric lymph nodes were collected. Fecal samples were screened for the presence of parasitic ova and oocysts. Hematoxylin-and-eosin-stained and Ziehl–Neelsen-stained tissues were examined for evidence of inflammation, acid-fast bacteria, and parasitic structures. Risk factor analysis was performed using multivariate logistic regression analysis.  Mucosal thickening and corrugation of the ileum were found in AS sheep (74/113 [65.5%], 10/113 [8.8%], respectively) and TS sheep (21/88 [23.9%], 8/88 [9.1%], respectively). Histopathologically, diffuse multibacillary/histiocytic form was detected only in the AS sheep breed. AS sheep had higher levels of acid–fast bacteria in the ileum (70/113 [61.9%]) compared with TS sheep (34/88 [38.6%]). In addition, parasitic structure and severe eosinophilic inflammation were detected in AS (10/113 [8.8%], 108/113 [95.6%], respectively) and TS sheep (2/88 [2.3%], 70/88 [79.5%], respectively). Parasitic infections were detected in fecal samples from 15/118 (12.7%) AS sheep and 11/89 (12.4%) TS sheep. Coinfection between gastrointestinal parasites and paratuberculosis was detected histologically in 8/118 (7.1%) and 2/88 (2.3%) AS and TS sheep, respectively.  Risk factor analysis indicated that the ileum from the AS sheep breed was 2.164 times more likely to have acid–fast bacteria and 2.874 times more likely to have eosinophilic infiltrations than the TS sheep breed. Results of this preliminary study may indicate the role of the sheep in the pathogenesis and shedding of MAP. 

Tick-borne pathogens pose a significant problem in canines, other animals, and humans worldwide. This study aimed to estimate the prevalence of Borrelia burgdorferi, Ehrlichia canis, and Coxiella burnetii in dogs and associated ticks in Egypt.  Blood samples from 110 tick-infested dogs and 550 whole ticks (divided into 110 pools) were collected and tested for the targeted pathogens using polymerase chain reaction (PCR).  Of the 110 dog blood samples, B. burgdorferi DNA was detected in three samples, E. canis in six samples, and C. burnetii in one kenneled dog. Among the 110 tick pools, B. burgdorferi was detected in four pools, E. canis in 12 pools, and C. burnetii in three pools from kenneled dogs. The overall prevalence of the three agents in dog and tick samples were 3.18%, 8.18%, and 1.81%, respectively. Simultaneous positive PCR reactions in both dogs and their associated tick pools were observed in four cases. B. burgdorferi and E. canis were simultaneously detected in two dogs and two tick pools, whereas C. burnetii was detected in one dog but not in any tick pools. The three agents were simultaneously detected in one dog, but none were found in the corresponding tick pools. A mixed infection of C. burnetii and B. burgdorferi was observed in one dog and one tick pool.  Molecular diagnosis is the most reliable method for detecting B. burgdorferi, E. canis, and C. burnetii in dogs and associated ticks. E. canis showed the highest prevalence in both dog and tick samples followed by B. burgdorferi while C. burnetti showed the lowest prevalence. The potential transmission of these diseases from companion dogs to humans through ticks presents a significant challenge for the One Health concept. 

Potassium peroxymonosulfate (PPMS) is a broad-spectrum disinfectant that oxidizes viral protein capsids. The effectiveness of PPMS in killing viruses depends on several factors, including its concentration, contact time, and present of organic materials. This study evaluated the efficacy of PPMS in an aqueous phase. It also applied PPMS to artificially avian influenza virus (AIV)-contaminated carrier surfaces and clothes and compared its effectiveness with that of sodium dichloroisocyanurate (NaDCC) and quaternary ammonium compounds (QAC).  Four PPMS concentrations (1×, 0.5×, 0.25×, and 0.125×), were evaluated for their virucidal efficacy against Newcastle disease virus (NDV) and AIV in an aqueous phase. The evaluation included testing in the absence and presence of organic materials under different exposure times, such as 5 s, 30 s, 1 min, 3 min, 5 min, 10 min, and 15 min. AIV inactivation was assessed on contaminated carrier surfaces, such as stainless steel, rubber, plastic, and artificially contaminated clothes.  In aqueous phase, concentrations of 1×, 0.5×, 0.25×, and 0.125× inactivated NDV in the absence of organic materials within 5 s, 5 s, 5 min, and 15 min at concentrations of 1×, 0.5×, 0.25×, and 0.125×, respectively. In the presence of organic material contamination, NDV could be inactivated within 30 s for 1×, 1 min for 0.5×, and 10 min for 0.25×; however, 0.125× PPMS did not achieve inactivation within 15 min. PPMS concentrations of 1×, 0.5×, 0.25×, and 0.125× inactivated AIV within 5 s, 5 s, 5 s, and 30 s, respectively, in both the absence and presence of organic materials. PPMS at a concentration of 1× could inactivate AIV on all carriers within 30 s. PPMS at 0.5× and 0.25× concentrations could inactivate AIV within 30 s on rubber and plastic; inactivation occurred within 1 min on stainless steel. However, 0.125× PPMS and 1× QAC could not achieve inactivation within 3 min on all carriers. Finally, PPMS concentrations of 1×, 0.5×, 0.25×, and 0.125× inactivated AIV on rayon sheets within 5 s, 30 s, 5 min, and 15 min, respectively. However, the recommended NaDCC concentration achieved inactivation within 10 min, whereas QAC did not achieve inactivation within 15 min.  PPMS can inactivate enveloped viruses such as NDV and AIV. Furthermore, PPMS is superior to NaDCC and QAC for inactivating viruses on various carrier surfaces and artificially contaminated clothes. However, the virucidal efficacy of PPMS depends on the optimal concentration, organic material conditions, and exposure/contact timing. Therefore, PPMS is a promising alternative disinfectant crucial for enhancing biosecurity and controlling viruses that contaminate animal farms, slaughterhouses, and hospitals. 

Coxiella burnetii is a highly contagious zoonotic bacterial micro-organism. This study aimed to estimate the prevalence of C. burnetii in dogs using serological and molecular methods. Furthermore, a sequencing analysis of C. burnetii dog isolates was conducted.  A total of 172 dogs, including 93 pet dogs, 21 police dogs, 38 guardian dogs, and 20 stray dogs, were selected. Venous blood was drained from the dogs and examined serologically by indirect enzyme-linked immunosorbent assay (ELISA) and molecularly by polymerase chain reaction (PCR) for C. burnetii. A sequencing analysis of C. burnetii dog isolates was conducted.  The overall prevalence of C. burnetii was 16.86%, accounting for 55% in stray dogs, 9.68% in pet dogs, 19.05% in police dogs, and 13.16% in guardian dogs. Strong positive sera were observed in stray dogs (4.84 ± 0.29), whereas weak sera were observed in pet dogs (3.22 ± 0.18). PCR analysis revealed 6.4% positive dogs, accounting for 1.08%, 4.76%, 2.63%, and 40% in pet, police, guardian, and stray dogs, respectively. Phylogenetic tree analysis of local C. burnetii isolates revealed a total rate of similarity and mutations/changes between 95.47% and 100% and 0.059%, respectively. Subsequently, the local isolates were significantly similar to Chinese hedgehog, Iraqi camel, and Colombian human C. burnetii National Center for Biotechnology Information-GenBank isolates.  This is the first study on prevalence of C. burnetii in dogs in Iraq. To prevent transmission of C. burnetii to humans, the role of dogs or other domestic and wild animals as sources of infection must be investigated extensively. In addition, the prevalence of C. burnetii in other Iraqi regions should be surveyed using the most sensitive and specific diagnostic assays, such as ELISA and PCR. 

Leptospirosis is an infectious zoonotic disease that significantly affects animal health, particularly the reproduction of ruminants. However, some aspects of epidemiology and clinical characteristics have not been clarified. This study aimed to estimate the prevalence and identify risk factors of leptospirosis in female bovines at reproductive age in the Ecuadorian Amazon rainforest.  A total of 213 bovines were studied in the Amazon province of Zamora Chinchipe, in which a microscopic agglutination test was used to diagnose a panel of eight serovars of Leptospira borgpetersenii (Sejroe) and Leptospira interrogans, Australis, Bataviae, Canicola, Tarassovi, Icterohaemorrhagiae, Wolffi, and Hardjo. An epidemiological survey was conducted to identify risk factors by animal and herd and clinical symptoms associated with Leptospira spp. infection; and blood samples were collected to determine the differences between seropositive and seronegative animals regarding hematocrit, hemoglobin (Hb), mean corpuscular Hb concentration, total red blood cell count, total platelet count, leukocytes, total proteins, creatinine, and ureic nitrogen.  The prevalence of bovine leptospirosis was 12.21% (26/213), with positive reactions in the Australis, Sejroe, Bataviae, Canicola, and Tarassovi serovars. No variables were considered risk factors, nor clinical signs associated with the infection, nor were there differences in the hematological parameters between the seropositive and seronegative animals.  These findings indicate the persistence of Leptospira on cattle farms in the Ecuadorian Amazon and highlight the interaction between domestic and wild species. It is crucial to implement control measures and improvements in management practices under the One Health approach to reduce accidental infections from contact with wildlife; the awareness of farmers is essential for effective prevention. 

Zoonotic diseases caused by various blood parasites are important public health concerns that impact animals and humans worldwide. The traditional method of microscopic examination for parasite diagnosis is labor-intensive, time-consuming, and prone to variability among observers, necessitating highly skilled and experienced personnel. Therefore, an innovative approach is required to enhance the conventional method. This study aimed to develop a self-supervised learning (SSL) approach to identify zoonotic blood parasites from microscopic images, with an initial focus on parasite species classification.  We acquired a public dataset featuring microscopic images of Giemsa-stained thin blood films of trypanosomes and other blood parasites, including Babesia, Leishmania, Plasmodium, Toxoplasma, and Trichomonad, as well as images of both white and red blood cells. The input data were subjected to SSL model training using the Bootstrap Your Own Latent (BYOL) algorithm with Residual Network 50 (ResNet50), ResNet101, and ResNet152 as the backbones. The performance of the proposed SSL model was then compared to that of baseline models.  The proposed BYOL SSL model outperformed supervised learning models across all classes. Among the SSL models, ResNet50 consistently achieved high accuracy, reaching 0.992 in most classes, which aligns well with the patterns observed in the pre-trained uniform manifold approximation and projection representations. Fine-tuned SSL models exhibit high performance, achieving 95% accuracy and a 0.960 area under the curve of the receiver operating characteristics (ROC) curve even when fine-tuned with 1% of the data in the downstream process. Furthermore, 20% of the data for training with SSL models yielded ≥95% in all other statistical metrics, including accuracy, recall, precision, specification, F1 score, and ROC curve. As a result, multi-class classification prediction demonstrated that model performance exceeded 91% for the F1 score, except for the early stage of Trypanosoma evansi, which showed an F1 score of 87%. This may be due to the model being exposed to high levels of variation during the developmental stage.  This approach can significantly enhance active surveillance efforts to improve disease control and prevent outbreaks, particularly in resource-limited settings. In addition, SSL addresses significant challenges, such as data variability and the requirement for extensive class labeling, which are common in biology and medical fields. 

Computed tomographic (CT) images can elucidate the variations of cardiac orientation that this information among dog breeds has never been reported. This study aimed to explore the heart orientations of dogs with different thoracic types and study their effects on vertebral heart score (VHS) measurements using CT images.  Thoracic CT images of 115 mature dogs without thoracic abnormalities were retrospectively examined. The dogs were classified into four groups: Normal Broad, Abnormal Broad, Normal, and Deep. All dogs were also classified based on their heart deviations. The VHSs were evaluated using lateral VHS, dorsal VHS, and adjusted VHS, and all were compared.  In the normal broad and abnormal broad groups, the lateral VHS and lateral long-axis dimensions were significantly lower than those obtained from the dorsal and adjusted VHSs. In addition, heart deviations were mostly observed in the normal broad and abnormal broad groups. Nevertheless, little evidence was found in the normal and deep groups. The lateral VHS and lateral long-axis dimensions were significantly reduced by heart deviation more than the dorsal and adjusted VHSs.  Cardiac orientations among dog breeds can affect VHSs of lateral projection, especially in the broad thoracic group. Clinical evaluation of the VHS in the broad thoracic dogs should be performed on the dorsal view for more accurate measurement of heart size. 

Papillomaviruses (PVs) infections have been documented in numerous animal species across different regions worldwide. They often exert significant impacts on animal health and livestock production. Scientists have studied natural products for over half a century due to their diverse chemical composition, acknowledging their value in fighting cancer. Acorns (Quercus ilex) are believed to have several unexplored pharmacological properties. This study aimed to evaluate the in vivo safety and cancer chemopreventive activity of an infusion extract of Q. ilex in a transgenic mouse model of human PV (HPV)-16, which developed squamous cell carcinomas through a multistep process driven by HPV16 oncogenes.  Q. ilex extract was prepared by heating in water at 90°C and then characterized by mass spectrometry. Phenolic compounds from this extract were administered in drinking water to female mice in three different concentrations (0.03, 0.06, and 0.09 g/mL) over a period of 28 consecutive days. Six groups (n = 6) were formed for this study: group 1 (G1, wildtype [WT], water), group 2 (G2, HPV, water), group 3 (G3, WT, 0.09 g/mL), group 4 (G4, HPV, 0.03 g/mL), group 5 (G5, HPV, 0.06 g/ mL), and group 6 (G6, HPV, 0.09 g/mL). Throughout the experiment, humane endpoints, body weight, food intake, and water consumption were recorded weekly. Following the experimental period, all mice were sacrificed, and blood, internal organs, and skin samples were collected. Blood was used to measure glucose and microhematocrit and later biochemical parameters, such as creatinine, urea, albumin, alanine aminotransferase, and total proteins. Histological analysis was performed on skin and organ samples.  The administration of Q. ilex extract resulted in a statistically significant increase in relative organ weight among HPV transgenic animals, indicating adaptive biological response to the tested concentrations. Moreover, a reduction in characteristic skin lesions was observed in animals treated with the 0.06 and 0.09 g/mL extract.  These results provide a favorable chemopreventive profile for Q. ilex extract at concentrations of 0.06 and 0.09 g/mL. This study highlights the potential of Q. ilex extract as a safe and effective therapeutic strategy against HPV16- associated lesions in transgenic mouse models. The limitation of our study was the durability of transgenic animals. As a more sensitive species, we must always be careful with the durability of the test. We intend to study concentrations of 0.06 and 0.09 g/mL for longer to further investigate their possible effects. 

Several approaches have been employed to mitigate methane emissions from livestock, with varied results. This study evaluated the effects of shade-dried ground garlic leaf (GL) powder and papaya leaf (PL) powder as crop waste on feed intake, growth performance, ruminal microbial counts, gut epithelial barrier functions, and meat quality in goats.  Forty male adult Beetal goats were randomly divided into five treatment groups: (1) Control (basal diet only); (2) basal diet supplemented with 6% bromodichloromethane (BCM); (3) basal diet supplemented with 30% GL powder; (4) basal diet supplemented with 26% PL powder; and (5) basal diet supplemented with 30% GL powder and 26% PL powder (GP).  Average weight gain, feed conversion ratio, fecal score, and albumin improved in the GP. Aspartate transferase increased significantly in BCM, GL, and PL and was insignificant in the GP group compared with the C group. There was a 13% decrease in methanogen count in PL compared with C, but this difference was not significant between BCM and GP. Ruminal bacteria and protozoa were lowest in GL. Ruminal papilla height and surface area increased in the supplemented groups compared with C (p < 0.05). In vitro experiments using isolated ruminal epithelia revealed a 39% increase in short-circuit current in GP compared with C (p < 0.05). For meat parameters, the pH 24 h decreased significantly in GL compared to BCM.  Dietary supplementations with GL and PL alone or in combination improved growth parameters and gut performance and reduced rumen methanogen levels without altering meat quality parameters. Proper diet formulation and further research on other ruminants may help reduce greenhouse gas emissions from livestock. 

Dengue fever is a recurring arboviral disease. The presence of livestock and domestic animals potentially increases the risk of dengue fever in an area due to the shared habitats of vectors and humans. Therefore, this study aimed to determine the vulnerability map of dengue disease and identify the influence of livestock and domestic animals on the number of cases in Bantul Regency.  An observational study was conducted in 3 Kapanewon (subdistricts) in the Bantul regency, known as the dengue-endemic area. The locations of 302 cases were recorded using the Global Positioning System. Dengue case density was analyzed using Kernel Density Estimation, and vulnerability was assessed using an overlay in ArcGIS Desktop 10.8. Furthermore, buffer analysis was conducted to determine the relationship between case density and the presence of livestock and pet pens.  Banguntapan, Kasihan, and Sewon subdistricts had high vulnerability areas of 424.12 Ha (14.97%), 334.76 Ha (10.46%), and 196.12 Ha (7.05%), respectively. The villages with dengue hotspots were Banguntapan and Potorono (Banguntapan Subdistrict) and Tirtonirmolo (Kasihan Subdistrict). The highest number of patients (180 cases) occurred at a buffer distance of <100 m from houses to livestock pens, closely related to the flight distance of Aedes spp. mosquitoes, the dengue vector.  The three subdistricts were predominantly characterized by low dengue vulnerability. However, livestock and domestic animal pens are significant risk factors. This information is crucial for effectively controlling and managing dengue disease in Bantul Regency. 

Gastrointestinal parasites (GIPs) pose a major health challenge for cattle in Indonesia. GIP infections affect the production and reproductive performance of cattle, resulting in economic losses. However, the prevalence and distribution of infections have not been comprehensively profiled at the national level. This study aimed to estimate the prevalence of GIP infections in cattle in Indonesia.  Overall, 667 articles were identified from six databases in English and Bahasa Indonesia. After removing duplicates and screening titles and abstracts based on the inclusion criteria (i.e., GIP prevalence in cattle in Indonesia), 67 articles were included in the data review. Data were pooled using a random-effects model in STATA software. Heterogeneity was tested using Cochran’s Q-value and I2 statistics, whereas publication bias was assessed using Egger’s regression test.  The overall pooled prevalence of GIP in Indonesia was 46% (95% confidence interval 37%–55%), with a total population of 17,278 cattle screened. The I2 value was 99.59%, Cochran’s Q-value was 15,957.25, and p = 0.001. The results of the regional meta-analysis based on the provinces in the three zones of Indonesia showed estimated prevalence rates of 54.0%, 52.7%, and 53.7% in Western, Central, and Eastern Indonesia, respectively. The parasite with the highest prevalence was Eimeria spp. (37.7%), followed by nematodes (34.4%) and trematodes (Fasciola spp., 21.4%).  The findings reveal a high prevalence of GIPs in cattle across Indonesia, with significant variability across regions and parasite types. Eimeria spp., nematodes, and trematodes represent the most prevalent infections and underscore the urgent need for region-specific control strategies, including improved livestock management practices, routine screening, and integrated parasitic control programs. 

Malignant diseases are among the most common and deadly illnesses that are often spread due to lifestyle choices. These diseases are caused by unchecked cell growth, which can be curable if detected early. Cancer treatment is dependent on various internal and external factors. Newcastle disease virus (NDV) has emerged as a promising virotherapeutic agent due to its oncolytic activity and safety profile. This study investigated the ability of virulent NDV to infect, replicate, and kill digestive tumor cells in esophageal and colorectal cancers.  NDV was used at several concentrations (multiplicities of infection [MOI]: 1, 3, 5, 10, and 20) on two models of tumor cells: colorectal carcinoma (HRT) and esophageal carcinoma (SK-GT). The investigation focused on the cytotoxic effects of NDV in these cell lines.  The results indicated that SK-GT carcinoma cells (esophageal and colorectal carcinoma) exhibited a high cytotoxic response to NDV, which was directly proportional to the MOI concentration. The half-maximal inhibitory concentration of NDV was 5.736 for the SK-GT cell line and 9.878 for the HRT cell line.  NDV can replicate and kill cancer cells in esophageal and colorectal cancers. We recommend conducting in vivo studies on transplanted digestive system tumors in mouse models to evaluate their anti-tumor activity in vivo, as the present study was limited to in vitro models. 

Postbiotics are functional bioactive compounds or bioactive molecules with beneficial effects on health and functional activities in humans or livestock, produced by probiotic bacteria or yeast. Several postbiotics, including enzymes, short-chain fatty acids, amino acids, extracellular polysaccharides, microbial cell fragments, and teichoic acids, are currently being widely studied. This study aimed to explore the potential of secondary metabolites of Schleiferilactobacillus harbinensis LH 991 and Pichia kudriavzevii B-5P as lactic acid bacteria (LAB) and yeast isolated from Budu (fermented fish) which can act as postbiotics through in vitro rumen fermentation.  The method used a completely randomized design 5 × 4, with five treatments and four replications. The substrate diet consisted of 60% forage and 40% concentrate. The culture mixture was 1.3 × 10¹¹ CFU/mL with a 50%:50% ratio of S. harbinensis LH 991 and P. kudriavzevii B-5P. The inoculum concentrations used in this study were 0% (control), 1%, 2%, 3%, and 4%. Treatments are arranged based on differences in inoculum concentration as follows: T0: control (0%); T1: 1%; T2: 2%; T3: 3%; and T4: 4%.  The T4 group showed a significant increase (p < 0.01) in short-chain fatty acids (SCFA), including acetate, propionate, butyrate, valerate, isobutyrate, and isovalerate acids, compared with the other treatments. Meanwhile, T4 shows that there is no significant (p > 0.01) effect on in vitro digestibility (in vitro dry matter digestibility, in vitro organic matter digestibility, and in vitro crude fiber digestibility). However, a highly significant (p < 0.01) effect was on volatile fatty acid total, NH3, and microbial crude protein synthesis.  It is concluded that the treatment with a 4% inoculum concentration (T4) containing a mixture of S. harbinensis LH 991 and P. kudriavzevii B-5P as LAB and yeast isolated from Budu (fermented fish) in 50%:50% ratio increased SCFA and rumen fermentation significantly, whereas it did not affect in vitro digestibility. 

Mosaicism, which is characterized by the presence of wild-type and more than one mutant allele, poses a serious problem in zygotic gene modification through the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 system. Therefore, we used pig embryos to compare the gene editing efficiencies achieved by combining electroporation and lipofection using different aminopeptidase N (APN)-targeting guide RNA (gRNA) sequences.  Six gRNAs (gRNA1–6) with different target sequences were designed to target APN. Zona pellucida (ZP)-intact zygotes collected 10 h after the start of in vitro fertilization (IVF) were electroporated with each gRNA to compare their gene editing efficiency. The gRNA sequences that achieved the lowest and highest mutation rates (gRNA4 and gRNA6, respectively) were selected for additional lipofection to assess gene editing efficiency following combined treatment. As ZP removal is essential for lipofection, ZP-free zygotes were electroporated with gRNA4 or gRNA6 10 h after IVF initiation, followed by lipofection with the same gRNAs 24 or 29 h after IVF initiation. The electroporated ZP-intact and ZP-free zygotes were used as controls.  gRNA4 and gRNA6 exhibited the lowest and highest mutation rates, respectively. gRNA4-targeted ZP-free embryos subjected to additional lipofection 29 h after IVF initiation exhibited significantly higher total and biallelic mutation rates than ZP-intact embryos that received only electroporation. Additional lipofection of gRNA6-targeted embryos had no obvious effect on mutation rates.  Electroporation combined with lipofection using gRNAs with low mutation rates may improve gene editing efficiency in pig embryos. However, the effects may vary based on the timing of gene editing.