Metaproteomic Analysis of Gut Resistome in the Cecal Microbiota of Fattening Pigs Raised without Antibiotics.

Improper use of antibiotics in swine could reduce commensal bacteria and possibly increase pathogen infections via the gut resistome. This study aimed to compare the metaproteomic profiles of the gut resistome and related metabolism in the cecal microbiota of fattening pigs raised under antibiotic-free (ABF) conditions with those of ordinary industrial pigs (controls [CTRL]). The top three relatively abundant microbes in both groups were Escherichia coli, Ruminococcus, and Lactobacillus, followed by Bacteroides and Bifidobacterium. E. coli, Lactobacillus, and Bacteroides were found to be increased in the CTRL group, whereas Ruminococcus and Clostridium were greater in the ABF group. The highest abundances of antibiotic resistance proteins (log2 expression levels [ELs] of >10) were found to be for tetracycline resistance (Tetr) and aminoglycoside resistance (AMGr) proteins found in Bacteroides, with a significant increase in the CTRL group. High Tetr (ELs of 5.32) was found in Ruminococcus in the CTRL group, although pigs in both groups had never received tetracycline, possibly reflecting the influence of environments in farms. In E. coli, AMGr and ß-lactamase family proteins were observed in both groups (ELs of 3 to 6), whereas multidrug resistance protein MdtL was significantly expressed in the CTRL group (ELs of around 3). In the ABF group, CRISPR-associated endonucleases Cas1 and Cas9, which function to defend against viruses, were markedly observed in Ruminococcus and Lactobacillus, respectively, with ELs of 8.6 and 4.15, respectively. In conclusion, this study demonstrated that CRISPR-associated endonucleases were markedly observed in the ABF group, whereas higher levels of Tetr, AMGr, and multidrug resistance protein MdtL was markedly observed in dominant bacterial species in the CTRL group. IMPORTANCE In order to control and reduce antibiotic use in animals, the Department of Livestock Development, Thailand, has launched a campaign for antibiotic-free livestock production. The present study has shown for the first time that CRISPR-associated endonucleases Cas1 and Cas9, which function to defend against viruses, were markedly observed in Ruminococcus and Lactobacillus, respectively, in ceca of pigs raised without antibiotics (ABF). The highest abundances of antibiotic resistance proteins were for tetracycline (Tetr) and aminoglycoside resistance (AMGr) proteins found in Bacteroides, with a significant increase in the controls. In E. coli, the microbe with the highest relative abundance, AMGr and ß-lactamase family proteins were observed in both groups, whereas multidrug resistance protein MdtL was significantly expressed in the controls. Pigs in both ABF and control groups had never received tetracycline, possibly reflecting the influence of farm environments. We suggest that pigs raised without antibiotics may have more beneficial microorganisms for the gut than pigs raised with antibiotics.

Rapid identification of canine uropathogens by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry and the clinical factors that correlated bacterial species and antimicrobial resistance.

Using the matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS) method for bacterial diagnosis, rapid urine sample preparation can reduce time relapsing of diagnosis and improve discriminatory power in coinfection cases. We aimed to evaluate rapid urine preparation procedures before MALDI-TOF MS application using dog clinical urine samples in comparison with standard microbiological diagnostic methods by agreement analysis. We determined the frequency and distribution of bacteria and bacterial resistance and their correlations to clinical history. Three experimental procedures comprising direct centrifugation, 10% sodium dodecyl sulfate digestion, and ultrasonic preparation were performed for method validation and sensitivity. Sterile urine containing Escherichia coli and/or Staphylococcus aureus were used as simulated samples. By ultrasonic preparation, the microorganisms could be detected 1.46-1.51 × 105 CFU, which was considered the most suitable technique. This preparation was significantly consistent with the routine method based on data from Hospital Information Systems for 50 urine samples from canine cystitis. By standard protocol, Enterobacteriaceae and Staphylococcus pseudintermedius were found in most of the 155 urine samples with cystitis. Extended-spectrum beta-lactamase-producing Enterobacteriaceae was found in 25-30% of the samples. Imipenem resistance was found in 70% of Acinetobacter baumannii cases; almost all were resistant to second-generation fluoroquinolones and tetracyclines. The most efficient antibiotic for treating bacterial urinary tract infection was amoxicillin plus clavulanic acid. A. baumannii and Pseudomonas aeruginosa were susceptible to pradofloxacin. Prolonged urine catheterization was linked to lower urinary tract infections by Enterobacter spp., which also correlated with chronic kidney disease.

Influence of Backfat Thickness and the Interval from Altrenogest Withdrawal to Estrus on Reproductive Performance of Gilts.

Estrus synchronization of gilts can be achieved by feeding the orally active progestogen altrenogest (AT) to cycling gilts at 20 mg/day for 14 to 18 days with gilts usually returning to estrus 4 to 8 days after the last feeding. In practice, gilts failing to exhibit estrus by 6 days after AT withdrawal may compromise weekly breeding targets. The cause of prolonged intervals to estrus are unknown but may involve prolonged suppression due to the release of progesterone (P4), and by extension AT, from adipose tissues. The present study examined relationships between gilt P2 backfat depth (<13.5 mm, 14-16.5 mm, >17 mm groups), the AT withdrawal to estrus interval, and subsequent reproductive performance in gilts. We noted longer intervals to estrus in gilts with greater P2 backfat depths (p < 0.0001), and higher serum P4 concentrations on the last day of AT feeding and at estrus detection (p < 0.05). Total born litter sizes were unaffected by backfat depth, but pigs born alive progressively decreased with increasing backfat depth with the fattest gilts producing the fewest liveborn pigs (p < 0.05). Taken together, these data suggest that adipose tissues may provide a reservoir of steroid, with its release from fatter gilts having potential negative effects on their subsequent reproductive performance.

Metagenomics of antimicrobial and heavy metal resistance in the cecal microbiome of fattening pigs raised without antibiotics.

This study aimed to detect the cecal microbiome, antimicrobial resistance (AMR) and heavy metal resistance genes (MRGs) in fattening pigs raised under antibiotic-free (ABF) conditions compared with ordinary industrial pigs (control, C) using whole-genome shotgun sequencing. ABF pigs showed the enrichment of Prevotella (33%) and Lactobacillus (13%), whereas Escherichia coli (40%), Fusobacterium and Bacteroides (each at 4%) were notably observed in the C group. Distinct clusters of cecal microbiota of ABF and C pigs were revealed; however, microbiota of some C pigs (C1) appeared in the same cluster as ABF and were totally separated from the remaining C pigs (C2). For AMR genes, the highest abundance tet(Q) (35.7%) and mef(A) (12.7%) were markedly observed in the ABF group whereas tet(Q) (26.2%) and tet(W) (10.4%) were shown in the C group. tet(Q) was positively correlated to Prevotella in ABF and C1 samples. In the C2 group, the prominent tet(W) was positively correlated to Fusobacterium and Bacteroides Pigs have never received tetracycline but pregnant sows used chlortetracycline once 7 d before parturition. Chromosomal Cu and Zn resistance genes were also shown in both groups regardless the received Cu and Zn feed additives. A higher abundance of multi-metal resistance genes was observed in the C group (44%) compared with the ABF group (41%). In conclusion, the microbiome clusters in some C pigs were similar to that in ABF pigs. High abundant tetracycline resistance genes interrelated to major bacteria were observed in both ABF and C pigs. MRGs were also observed.IMPORTANCE: Owing to the increased problem of AMR in farm animals, raising farm animals without antibiotics is one method that could solve this problem. Our study showed that only some tetracycline and macrolide resistance genes, tet(Q), tet(W) and mef(A), were markedly abundant in ABF and C groups. The tet(Q) and tet(W) genes interrelated to different predominant bacteria in each group, showing the potential role of major bacteria as reservoirs of AMR genes. In addition, chromosomal Cu and Zn resistance genes were also observed in both pig groups, not depending on the use of Cu and Zn additives in both farms. The association of MRGs and AMR genotypes and phenotypes together with the method to re-sensitize bacteria to antibiotics should be studied further to unveil the cause of high resistance genes and solve the problems.

Serum Anti-Mullerian Hormone and Estradiol Concentrations in Gilts and Their Age at Puberty.

For experiment one, blood samples were obtained from 200 gilts at 90, 120, 150, 180, and 200 days of age. Serum samples from the 30 youngest (166.1 ± 0.7 days) and 30 oldest (198.8 ± 0.6 days) gilts exhibiting estrus by 200 days, and a further 18 gilts that remained anestrus at 200 days, were assayed for serum concentrations of anti-Mullerian hormone (AMH) and estradiol (E2). Gilts younger at puberty had higher (p < 0.05) AMH levels than those older at puberty, and both groups had higher AMH levels than anestrus gilts (p < 0.05). Regardless of age, serum E2 was higher (p < 0.05) in gilts that achieved puberty than in gilts remaining anestrus. At spontaneous pubertal estrus detection, there was no effect of pubertal age on the number of preovulatory ovarian follicles. For experiment two, 152 prepubertal gilts received an intramuscular (IM) injection of 400 IU eCG plus 200 IU hCG and then received fence-line boar contact to detect estrus onset. Serum AMH concentrations were higher (p < 0.05) in the first 25 gilts to exhibit puberty than the last 28 gilts, with the first gilts also having more preovulatory follicles (p < 0.0001). Taken together, these data support an association between serum AMH concentrations and degree of physiological maturity and ovarian follicular development in gilts.

Effects of season and single layer centrifugation on bull sperm quality in Thailand.

OBJECTIVE: The aim of study was to investigate the effects of season and single layer centrifugation (SLC) before cryopreservation on post-thaw bull sperm quality in Thailand. METHODS: Semen was collected from 6 bulls (Bos indicus) in summer, rainy season and winter 2014 through 2016. Semen characteristics, sperm morphology, sperm kinematics, viability, chromatin structure and mitochondrial membrane were evaluated. Meteorological data were available from the local meteorological station. RESULTS: Season had an effect on semen characteristics in the raw ejaculate, with higher proportions of normal spermatozoa and lower abnormalities in winter than in the other two seasons. Sperm kinematics, viability, DNA fragmentation index, and mitochondrial membrane potential were not different between seasons. Sperm samples selected by SLC had greater normal morphology and a lower proportion with bent tails than controls and higher values of progressive motility (PRO), beat cross frequency, linearity, straightness, wobble (WOB), and lower values of slow motility, velocity average path (VAP), velocity curved line, and amplitude of lateral head displacement than controls. In addition, SLCselection had a favorable effect on PRO, VAP, and WOB that differed among seasons. CONCLUSION: Our results suggested that these bulls were well adapted to their location, with season having an effect on sperm morphology. Moreover, SLC could be used prior to cryopreservation, regardless of season, to enhance normal morphology and kinematics of bull sperm samples without adversely affecting other parameters of sperm quality. However, there was considerable variation among bulls in DNA fragmentation index, mitochondrial membrane potential and sperm viability. In addition, SLC had a positive effect on sperm morphology and sperm kinematics, which could be expected to influence fertility.

Molecular Characterization and Antimicrobial Resistance of Livestock-Associated Methicillin-Resistant Staphylococcus aureus Isolates from Pigs and Swine Workers in Central Thailand.

This study presents molecular characteristics of livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) from pigs and swine workers in central Thailand. Sixty-three MRSA isolates were recovered from pigs (n = 60) and humans (n = 3). Two major LA-MRSA lineages, including sequence type (ST) 398 and clonal complex 9 (ST9 and ST4576, a novel single-locus variant of ST9), were identified. ST398 had spa type t034 (n = 55). ST9 and ST4576 had t337 (n = 8) and carried staphylococcal cassette chromosome mec (SCCmec) IX only. MRSA-ST398-t034 contained various SCCmec, including SCCmec V (n = 42), a novel SCCmec composite island (n = 12), and a nontypeable SCCmec (n = 1). All isolates were multidrug resistant and carried common resistance genes found in LA-MRSA. This is the first report of the presence of swine MRSA ST398 and multidrug resistance gene cfr in MRSA ST9 in Thailand. With identical molecular characteristics, pigs could be a source of MRSA ST398 spread to humans. A minor variation of genetic features and resistance gene carriage in both lineages represented a heterogeneous population and evolution of the endemic clones. A monitoring program and farm management, with prudent antimicrobial uses, should be implemented to reduce spreading. Strict hygiene and personal protection are also necessary to prevent transfer of LA-MRSA to humans.

Feeding altrenogest during late lactation improves fertility of primiparous sows nursing smaller litters.

The objective of this study was to determine whether feeding altrenogest (AT) to primiparous sows with smaller litters during the last week of lactation would improve their fertility. At day 21 of a 28-day lactation, 40 primiparous sows nursing ≤ 8 pigs were assigned equally to 2 groups, either to be fed 20 mg per day of AT for the last 7 days of lactation or to serve as untreated controls. To detect estrus, sows had daily fence-line contact with a mature boar. At estrus detection, sows were subject to an ovarian examination with transrectal ultrasound and preovulatory follicles (≥ 0.6 mm) were counted. Sows were artificially inseminated at estrus detection and again 24 hours later. Compared to controls, the AT-fed sows had longer weaning-to-estrus intervals (WEIs; P < 0.001), more pre-ovulatory follicles (P < 0.001), and larger subsequent litter sizes (P = 0.03). Farrowing rates were unaffected by treatment. These data suggest that sows nursing small litters are more likely to initiate a follicular phase during lactation, but that feeding AT prevents this and increases ovulation rate and subsequent litter size.

L'objectif de la présente étude était de déterminer si en fournissant de l'altrenogest (AT) à des truies primipares avec des petites portées durant la dernière semaine de lactation il y aurait amélioration de leur fertilité. Au jour 21 d'une période de lactation de 28 jours, 40 truies primipares allaitant ≤ 8 porcelets ont été assignées également à deux groupes, pour soit être nourries avec 20 mg par jour d'AT pour les sept derniers jours de la lactation ou de servir de témoins négatifs. Afin de détecter l'oestrus, les truies venaient en contact derrière une barrière avec un verrat mature. Au moment de la détection de l'oestrus, les truies étaient soumises à un examen des ovaires par échographie transrectale et les follicules pré-ovulatoires (≥ 0,6 mm) étaient dénombrés. Les truies furent inséminées artificiellement au moment de la détection de l'oestrus et encore une fois 24 h plus tard. Comparativement aux témoins, les truies nourries avec de l'AT avaient un intervalle sevrage-oestrus plus long (WEIs; P < 0,001), avaient plus de follicules pré-ovulatoires (P < 0,001) et des tailles de portées subséquentes plus grandes (P = 0,03). Les taux de mise-bas n'étaient pas affectés par le traitement. Ces résultats suggèrent que les truies allaitant des petites portées sont plus susceptibles d'initier une phase folliculaire durant la lactation, mais qu'en leur donner de l'AT prévient ceci et augmente le taux d'ovulation et la taille subséquente des portées.(Traduit par Docteur Serge Messier).

Alteration of androgen receptor expression, apoptosis and cell proliferation in cryptorchid suckling, nursery and growing-finishing pigs.

Cryptorchidism, a condition of one or two undescended testicles, is a common congenital disease in pigs, causing loss in the pig industry. One of the major factors affecting testicular descent is the androgen receptor (AR), which binds to androgen and then regulates the expression of androgen-responsive genes in the inguinoscrotal phase of testicular descent. AR expression has been reported to regulate apoptosis in testicular stem cells. The present study aimed to immunohistochemically examine AR and Ki-67 protein expression and apoptosis detection in unilateral undescended testicles (UDT) and descended testicles in cryptorchid pigs (DT) of suckling (aged 1-2 weeks), nursery (aged 6 weeks) and growing-finishing pigs (aged 12, 15 and 20 weeks) and in normal testicles (NT) at 1-2 and 12 weeks of age. At 1-2 weeks, decreased expression of AR was observed in UDT and DT compared with NT and was lower than that at 6-20 weeks. The expression of Ki-67, a marker of cell proliferation, in UDT and DT at 12 weeks was lower than that in NT at the same age. In addition, Ki-67 expression in UDT at 6 and 12 weeks was lower than that in UDT at 1-2 and 15-20 weeks. More testicular apoptosis was revealed in UDT at 1-2 weeks than in DT and NT at the same age. At 15-20 weeks, more apoptosis was detected in UDT than in DT. Positive correlation of AR expression in DT at 6 and 12 weeks was also noted, in addition to the association of the expression of AR and Ki-67 in NT at 12 weeks. Taken together, this study unveiled the low expression of AR and high apoptosis detection in UDT, whereas low expression of AR and low apoptosis detection were noted in DT in suckling piglets. Diminished cell proliferation was shown in UDT at 6-12 weeks, whereas high apoptosis was observed in UDT at 15-20 weeks. High expression of AR was shown only in nursery pigs. Distinct expression of AR in DT and NT at 1-2 and 12 weeks indicated that both conditions were not interchangeable.

Autochthonous lactic acid bacteria isolated from pig faeces in Thailand show probiotic properties and antibacterial activity against enteric pathogenic bacteria.

Lactic acid bacteria (LAB) play an important role in pig health and performance that arises from their beneficial impacts on the balance of gastrointestinal microbes, ability to fight enteric pathogens, and capacity to support the immune system. The aim of this study was to evaluate the functional and safety aspects of five previously isolated autochthonous LAB strains, (Lactobacillus plantarum 22F, 25F and 31F, Pediococcus acidilactici 72N and Pediococcus pentosaceus 77F) from pig faeces as potential probiotics for a pig feed supplement. The functional and safety properties of the strains were assessed by in vitro tests. The functional properties tested were their abilities in tolerating low pH values under simulated gastric conditions, their cell surface properties (hydrophobicity, auto- and co-aggregation), antibacterial activity against the common enteric pathogenic bacteria in pigs (such as pathogenic Escherichia coli, Salmonella Choleraesuis and Streptococcus suis), and diacetyl production. The safety of the strains was analyzed based on the absent of haemolysis on blood and bile salt hydrolase activity. Although all strains demonstrated diacetyl production, good survivability and antibacterial activities, L. plantarum 22F and 25F showed the best performance with the strongest antibacterial actions against the indicator pathogens. Of the strains, only P. pentosaceus 77F exhibited haemolysis or bile salt hydrolase activity. Furthermore, a principal component analysis revealed that L. plantarum 22F possessed superior functional and safety aspects compared to the other four autochthonous strains and to reference strains L. plantarum JCM 1149 and P. acidilactici DSM 20284. Further in vivo studies using oral administration of the strains are justified to assess their effectiveness as feed supplements for pigs.

Amenorative effects of exogenous gonadotropins on reproductive profiles of replacement gilts with delayed puberty in a farm in Thailand.

This study was to investigate the effect of gonadotropins on reproductive profiles of replacement gilts with delayed puberty. Totally, 136 Landrace X Yorkshire crossbred gilts, were categorized into control (n = 58) and treatment (n = 78) groups. Gonadotropins (400 U eCG plus 200 IU hCG) were administered in treatment group only. The results revealed that gilts in treatment group had higher number of gilts with estrus (92.3 vs 25.9%, P < 0.001), shorter onset to estrus (4.7 ± 0.3 vs 9.0 ± 0.8 d, P < 0.001), higher number of dominant follicles (18.0 ± 0.2 vs 13.2 ± 0.3 follicles, P < 0.001), and higher farrowing rate (87.5 vs 53.3%, P = 0.002) than those in control group. In conclusion, gonadotropins containing 400 IU eCG plus 200 IU hCG could improve reproductive profiles in replacement gilts with delayed puberty.

Sperm pretreatment with dithiothreitol increases male pronucleus formation rates after intracytoplasmic sperm injection (ICSI) in swamp buffalo oocytes.

Failure of male pronucleus formation has hampered the success of intracytoplasmic sperm injection (ICSI) in swamp buffalo. The aim of the present study was to improve male pronucleus formation by pretreating sperm with various chemicals before ICSI. In Experiments1 and 2, sperm were treated according to one of the following protocols: (1) 0.1% Triton-X 100 (TX) for 1 min, (2) 10 µM calcium ionophore (CaI) for 20 min, (3) freezing and thawing (FT) without any cryoprotectant, or (4) no treatment (control). These sperm treatment groups then either did or did not receive additional sperm treatment with 5 mM dithiothreitol (DTT) for 20 min. Acrosomal integrity (Experiment 1) and DNA fragmentation (Experiment 2) were evaluated in the sperm before ICSI. In Experiment 3, oocytes matured in vitro were subjected to ICSI using pretreated sperm as described above and then were cultured either with or without activation. The TX- and CaI-treated sperm caused an increase in the number of acrosome-loss sperm, whereas the FT treatment and control increased the proportion of acrosome-reacted sperm (P<0.05). The DNA fragmentation did not differ among treatments (P>0.05). At 18 h post-ICSI, pronucleus (PN) formation was found only in activated oocytes. The majority of the activated ICSI oocytes contained intact sperm heads. Normal fertilization was observed in the CaI and FT treatment groups and control group when sperm were treated with DTT before ICSI. In conclusion, DTT treatment of sperm with reacted acrosomes before ICSI together with activation of the ICSI oocytes is important for successful male pronucleus formation.

Comparison of artificial insemination with natural mating on smallholder farms in Thailand, and the effects of boar stimulation and distance of semen delivery on sow reproductive performance.

Following the implementation of artificial insemination (AI) services for smallholder pig farms, we investigated the reproductive performance after AI and its influencing factors. A small-scale boar station with an AI lab was established with two active boars having good genetics and free from reproductive diseases. Individual sow cards were used for reproductive data recording. A total of 171 sows on 92 farms situated within a radius of 50 km from the AI center were included in this study. Sows bred by AI (n = 121) were inseminated twice per estrus by two trained inseminators. A further 50 sows were mated by natural services using local rental boars. The impact of boar stimulation and distance from the AI center to the farm were also determined. Non-return (P = 0.02) and farrowing rates (P = 0.03) were higher for AI than for naturally bred sows (84.0% and 76.0% vs. 74.0% and 70.0% for AI and naturally bred, respectively). For sows bred by AI, boar stimulation increased non-return rate (84.1% vs. 70.0%; P = 0.09), farrowing rate (83.7% vs. 69.2%; P = 0.01) and litter size (11.2 +/- 2.3 vs. 9.7 +/- 1.7; P < 0.01). There was no effect on performance due to distance of semen transport. These results clearly indicate that sow performance on smallholder farms will improve if AI is utilized and boar stimulation is employed.