Cell culture models are used to assess the impacts that dietary changes have on host health; however, there is limited knowledge about canine intestinal cell models compared to those for humans. The objective of this study was to use a canine intestinal epithelial cell (cIEC) line to investigate responses to pro- and anti-inflammatory treatments relevant to health in the dog. It was anticipated that lipopolysaccharides (LPS) would cause a pro-inflammatory response, while butyrate would cause an anti-inflammatory response and mitigate the pro-inflammatory response induced by LPS. cIEC were stimulated with 250 µg/mL of LPS, 1 mM of butyrate, or a combination of LPS and butyrate compared to untreated controls over 8 h (n = 6 per treatment group). LPS treatment significantly reduced transepithelial electrical resistance (TEER) over time and caused increases in the protein abundance and gene expression of the pro-inflammatory interleukin (IL)-8 and chemokine (C-C motif) ligand (CCL)2 compared to untreated controls. Additionally, LPS treatment caused increases in the gene expression of the pro-inflammatory interferon gamma-induced protein (IP)-10, and increased protein abundances of keratinocyte chemotactic (KC)-like in apical and basal cell media compared to untreated cIEC. Butyrate failed to elicit an increase in anti-inflammatory cytokines, although treatment significantly reduced protein expression of the pro-inflammatory CCL2 in apical cell culture media and increased protein expression of IP-10. Butyrate reduced the LPS-induced IL-8 and KC-like increased pro-inflammatory protein abundances in basal cell culture media compared to untreated canine IEC and was found to reduce the LPS-induced intestinal barrier hyperpermeability. However, butyrate and LPS in combination caused an increase in CCL2 gene expression compared to treatment with butyrate and LPS alone. Therefore, this study has shown that butyrate can mitigate some aspects of the LPS-induced inflammatory response in an in vitro model of the canine intestine. Overall, changes in intestinal barrier integrity in response to the treatment were similar to that reported for human cell models; however, the molecular signaling somewhat differed, highlighting the need for further studies and illustrating the value of studying a canine cell model to understand the molecular responses in the dog.

Introduction:Mycoplasma bovis (M. bovis) manifests as diverse clinical pathologies in cattle that significantly impacts cow health and productivity worldwide. Current diagnostic methods rely on active infection to detect antibodies and bacterial DNA, however, these methods could be improved for subclinical infections. This is of particular importance in long-term surveillance programs in countries that have decided to eradicate M. bovis, such as New Zealand. Methods:This study investigated small extracellular vesicles (sEV) from serum samples of M. bovis infected dairy cows to identify protein cargo that may be used as potential diagnostic markers during subclinical infection to complement current testing methods. Small EV were isolated from serum of dairy cows positive for M. bovis infection (n = 45) and their protein cargo compared with those from serum sEV from non-infected dairy cows (n = 49). Proteins were identified using liquid chromatography-ion mobility-tandem mass spectrometry in pooled samples (n = 10/group), resulting in 695 non-redundant top proteins identified across all samples. Results:Differential protein abundance analysis indicated 90 proteins significantly (q-value < 0.05) different in sEV of infected animals compared with controls. Proteins associated with inflammation and the complement system as well as proteins involved in the oxidative stress pathway, were in greater abundance in sEV from M. bovis positive animals compared with M. bovis negative animals. Several histone proteins and antimicrobial peptides exhibited lower abundance in sEV from M. bovis positive compared with negative cows. Conclusion:Although this study has not identified a protein candidate specific enough for use as a single diagnostic marker, our results indicate a shift towards a diseased state in M. bovis infected dairy cows and provide valuable insight into sEV biology during M. bovis infection.

Mycoplasma bovis (M. bovis) is a pathogenic bacterium that causes significant production losses and welfare challenges in cattle. Eradication is challenging as M. bovis can infect host cells intracellularly, reducing detection from immunomodulatory cells. With an aim to improve diagnostics for post-eradication surveillance, small extracellular vesicles (sEVs) were used to identify novel biomarkers of M. bovis infection. Readily accessible in body fluids, these nanoparticles encapsulate protein cargo indicative of the metabolic state of their tissue of origin. Here, we developed a novel three-dimensional in vitro cell culture model using static bioreactors to investigate changes in host cell (bovine endometrial epithelial cells) sEV protein cargo during M. bovis infection. Three-dimensional cell culture modelling better mimics native tissue architecture than two-dimensional modelling, enhancing intercellular function and physiological relevance. Size exclusion chromatography columns were used to isolate sEVs from control and co-culture bioreactor flasks. Liquid chromatography-trapped ion mobility spectrometry-tandem mass spectrometry was used to investigate the proteome of sEVs. Differential proteome profile analysis resulted in 193 proteins that demonstrated significantly different abundance (p < 0.05) in sEVs (co-culture vs. control cells), with changes to host Histone proteins the most prominent. Most M. bovis-derived proteins were those involved in cellular metabolism with enrichment of proteins classified under glycolysis. However, owing to their reduced genome, several glycolytic enzymes in Mycoplasma spp. are multifunctional and contribute to pathogenesis through involvement in adhesion and invasion of host cells. Homologs of immunoreactive M. bovis-lipoproteins were present in the proteome of co-culture sEVs. Host-derived proteins were consistent with those linked with infection and sEV trafficking. Our study indicated that sEV protein cargo was altered by M. bovis infection, with results providing an important insight into sEV biology in the context of bacterial infection.

The transition period, encompassing the 3 wk before and 3 wk after calving, represents the most metabolically and immunologically demanding phase of the dairy cow's lactation cycle. Despite lower milk yield targets, grazing cows experience homeorhetic adaptations and immune perturbations comparable in magnitude to those of high-yielding, TMR-fed cows. However, pasture-based systems impose distinct management constraints that necessitate a pragmatic, rather than precision-focused, approach to nutritional management. This review synthesizes recent evidence on transition physiology in grazing systems and examines strategies that account for inherent pasture variability while maximizing on-farm achievability. The evidence demonstrates that optimal BCS at calving, controlled prepartum energy restriction to 80% to 100% of requirements, and focused mineral supplementation, particularly magnesium and calcium, remain the most cost-effective determinants of transition success. Secondary strategies, including targeted fatty acid supplementation, may provide additional benefits but should complement, not replace, foundational management. Emerging precision technologies offer promise for identifying individual cows at risk before clinical disease manifests, yet their utility in grazing systems depends on integration with variable pasture quality and realistic labor constraints. This review emphasizes that pragmatic, fundamentals-first management, aligned with seasonal pasture dynamics and farm-specific constraints, offers the most reliable pathway to improved transition outcomes, reduced metabolic disease, and sustainable dairy production in grazing systems.

This study examined the long-term effects of increased growth rates between three and seven months of age on gene expression in the mammary gland of nondairy ewes during their second lactation. A total of 19 twin-bearing, two-year-old ewes that either had an increased growth rate between three and seven months of age (heavy; n = 9; 153 ± 2.2 g/d and 47.9 ± 0.38 kg at breeding) or did not have an increased growth rate (control; n = 10; 127 ± 1.9 g/d and 44.9 ± 0.49 kg at breeding) were selected. Mammary gland biopsies were collected at day 128 of pregnancy and day 30 of lactation to examine the expression of 37 genes involved in mammary cell development and milk fat metabolism using NanoString nCounter technology. Ewes in the heavy group tended to exhibit lower expression (P < 0.05) of acyl-CoA synthetase short-chain family member 1 (ACSS1), a gene critical for mitochondrial acetyl-CoA synthesis, energy production, and milk fat synthesis, and a trend toward (P = 0.11) lower expression of signal transducer and activator of transcription 5A (STAT5A), a regulator of mammary epithelial cell differentiation and survival. These lower expressions suggest potential carry-over effects of increased growth rate between three and seven months of age. However, no phenotypic differences were observed in lamb growth or live weight, and no differences were detected in the expression of downstream target genes or modulators of these pathways, suggesting limited functional impact on mammary gland development and lamb performance outcomes. Further investigations, including a functional assessment of lactation and use of comprehensive transcriptomic analyses, would be needed to understand the effects of increased growth rates between three and seven months of age on ewe mammary function and milk composition.

Deer velvet extracts' anti-inflammatory and immune-modulatory effects have been widely demonstrated. However, the effects of deer velvet extracts on neuroinflammation and sickness behaviour have only been suggested. This study aimed to test the effect of two deer velvet supplements, SAT and SANT, on preventing LPS-induced systemic inflammation, neuroinflammatory changes and sickness behaviour in mice. Mice diet was supplemented (4 mg/kg/day) with either SAT or SANT or given normal chow as a control for four weeks before receiving a single dose of either LPS (1 mg/kg) or saline. Velvet supplements reduced sickness scores at 6 and 12 h, but not mice activity as measured by open field test at 24 h after LPS injection. Velvet supplements did not affect plasma cytokines but prevented the overexpression of brain inflammation genes and reduced microglial reactivity after 24 h of LPS injection. Results indicate that SAT and SANT modulate sickness behaviour during peak LPS effects and brain inflammation after 24 h of injections through different pathways.

CONTEXT:Declining fertility is an issue in multiple mammalian species. As the site of fertilisation and early embryo development, the oviduct plays a critical role in embryo survival, yet there is a paucity of information on how the oviduct regulates this process.AIMS:We hypothesised that differences in steroid hormone signalling and/or immune function would be observed in a model of poor embryo survival, the peripubertal ewe.METHODS:We examined expression of steroid hormones in systemic circulation, oviductal expression of oestrogen receptorαand genes important in steroid hormone signalling, and immune function in pregnant and cyclic peripubertal and adult ewes on day 3 after oestrus.KEY RESULTS:Concentrations of progesterone, but not oestradiol, were decreased in the peripubertal ewe compared to the adult ewe. Oestrogen receptorαprotein expression was increased in the peripubertal ewe, but pathway analysis of gene expression revealed downregulation of the oestrogen signalling pathway compared to the adult ewe. Differential expression of several genes involved in immune function between the peripubertal and adult ewe was consistent with an unfavourable oviductal environment in the peripubertal ewe lamb. Oestradiol concentration was positively correlated with the expression of multiple genes involved in the regulation of immune function.CONCLUSIONS:Differences in the immune environment of the oviduct, potentially linked to differential modulation by steroid hormones, may partially underly the poor fertilisation and early embryo survival observed in the peripubertal ewe.IMPLICATIONS:A unfavourable oviductal environment may play an important role in limiting reproductive success.

A delayed recovery of the reproductive tract from natural inflammatory processes associated with postpartum involution will compromise further reproductive function. Following a literature review, we selected serum amyloid A (SAA) and α1-acid glycoprotein (α1-AGP) to assess as potential circulating markers of acute uterine inflammation, as concentrations of these 2 acute phase proteins were reported to be elevated early postpartum in dairy cows with active uterine infection. Convenience serum samples from an induced model of uterine infection were used to measure concentrations of these markers. Infection was induced by infusing either 107 or 109 cfu of Trueperella pyogenes (n = 9 cows each; bacteria group n = 18) or saline as a control (n = 18) into the uterus at 48 d postpartum. Although infection stimulated an increase in uterine polymorphonuclear neutrophils, SAA and α1-AGP concentrations in serum were not different between infusion groups. Cows were subsequently classified into uterine health groups based on the presence of endometritis, with or without the presence of T. pyogenes in uterine culture in response to uterine infusion. Mean SAA concentrations were greater in cows that were either endometritis negative – infection positive (n = 9), endometritis positive – infection negative (n = 5), or endometritis positive – infection positive, compared with the endometritis negative – infection negative (n = 11) cows. There was no difference between uterine health groups for α1-AGP concentrations. Mean SAA and α1-AGP concentrations changed over time, reaching maximum concentrations on d 3 and 7 post-infusion, respectively, before decreasing thereafter to d 17 post-infusion.

Placental nutrient transport capacity influences fetal growth and development; however, it is affected by environmental factors, which are poorly understood. The objective of this study was to understand the impact of the ovine placentome morphological subtype, tissue type, and maternal parenteral supplementation of arginine mono-hydrochloride (Arg) on nutrient transport capacity using a gene expression approach. Placentomal tissues of types A, B, and C morphologic placentome subtypes were derived from 20 twin-bearing ewes, which were infused thrice daily with Arg (n = 9) or saline (Ctrl, n = 11) from 100 to 140 days of gestation. Samples were collected at day 140 of gestation. Expression of 31 genes involved in placental nutrient transport and function was investigated. Differential expression of specific amino acid transporter genes was found in the subtypes, suggesting a potential adaptive response to increase the transport capacity. Placentomal tissues differed in gene expression, highlighting differential transport capacity. Supplementation with Arg was associated with differential expressions of genes involved in amino acid transport and angiogenesis, suggesting a greater nutrient transport capacity. Collectively, these results indicate that the morphological subtype, tissue type, and maternal Arg supplementation can influence placental gene expression, which may be an adaptive response to alter the transport capacity to support fetal growth in sheep.

Context: Dairy calf-rearing practises have the potential to influence profitability through milk requirements at rearing, and affect lifetime productivity and market access from an animal-welfare perspective. Aims: The aim of this research was to compare calf growth and milk requirements by using conventional artificial rearing (AR) and restricted milk allocation with AR and high milk allocation or suckled calves. Methods Forty-five Friesian x Jersey calves were allocated to be either AR on 6 L/calf.day, (ARC) or 12 L/calf.day, (ARH), or suckled with dams in a cow-calf contact system using partial contact (15 h/day, CCC). AR calves were fed, and intake measured, using an automatic feeder, whereas CCC calves had access to their dams between 1500 hours and 0600 hours. Calves were transitioned off milk, once they reached at least 75 kg liveweight (LW), by gradually reducing their milk allocation (AR) or reducing access to their dam (CCC). Key results Milk consumption for ARH was greater than for ARC calves (382 vs 450 L/calf, P < 0.05), whereas for CCC calves milk-yield difference between dams for the control and suckling groups during the suckling period and over the full season was 706 and 1048 L/cow respectively. There was no difference in weaning weight of calves (87 +/- 1.7 kg LW), but, owing to differences in pre-weaning growth rate, age to weaning was youngest (P < 0.05) for CCC (55 days), followed by ARH (62 days) and ARC (73 days). Respective growth rates between birth and weaning (P < 0.001) for CCC, ARH and ARC calves were 0.955, 0.873 and 0.755 +/- 0.028 kg/day. Although there was a growth check among CCC calves during the weaning period, there were no post-weaning differences in growth rate among the groups. Conclusions Increasing milk allowance had the benefit of improving calf pre-weaning growth rate, giving the option of weaning calves either earlier or when heavier. However, milk yield losses under suckling systems may be too high to warrant the elevated pre-weaning growth of calves. Implications The costs-benefits of high milk allocation or suckling systems need to be ascertained over the lifetime of the animals to assess long-term survival and productivity outcomes.

The Manuka tree is unique to Aotearoa New Zealand and was recognised by early Maori traditions as a taonga (treasure) due to its wide variety of uses and is the first globally recognised taonga. Manuka honey (MH) is reported to have exceptional antioxidant and antimicrobial properties. The objective of this review is to summarise the current published peer-reviewed literature for benefits of the use of MH as a Rongoa (traditional medicine) in ruminant livestock and companion animals to support the potential use of MH as a therapeutic for companion animals and livestock. The key finding was that there is very little peer-reviewed literature providing scientific evidence for the use of MH as a therapeutic in livestock and companion animals. There are a wide range of products now commercially available that have MH as an active ingredient. However, further scientific studies are required to evaluate the direct effects of MH relative to its effects when used in conjunction with other compounds, dose-response effects, and the effect of the Unique Manuka Factor and methylglyoxal levels on efficacy in order to provide scientific evidence to support the efficacy of MH as a functional food or therapeutic in livestock and companion animal species.

Cryptosporidiosis is a worldwide diarrheal disease caused by the protozoan Cryptosporidium. The primary symptom is diarrhea, but patients may exhibit different symptoms based on the species of the Cryptosporidium parasite they are infected with. Furthermore, some genotypes within species are more transmissible and apparently virulent than others. The mechanisms underpinning these differences are not understood, and an effective in vitro system for Cryptosporidium culture would help advance our understanding of these differences. Using COLO-680N cells, we employed flow cytometry and microscopy along with the C. parvum-specific antibody Sporo-Glo™ to characterize infected cells 48 h following an infection with C. parvum or C. hominis. The Cryptosporidium parvum-infected cells showed higher levels of signal using Sporo-Glo™ than C. hominis-infected cells, which was likely because Sporo-Glo™ was generated against C. parvum. We found a subset of cells from infected cultures that expressed a novel, dose-dependent auto-fluorescent signal that was detectable across a range of wavelengths. The population of cells that expressed this signal increased proportionately to the multiplicity of infection. The spectral cytometry results confirmed that the signature of this subset of host cells closely matched that of oocysts present in the infectious ecosystem, pointing to a parasitic origin. Present in both C. parvum and C. hominis cultures, we named this Sig M, and due to its distinct profile in cells from both infections, it could be a better marker for assessing Cryptosporidium infection in COLO-680N cells than Sporo-Glo™. We also noted Sig M’s impact on Sporo-Glo™ detection as Sporo-Glo™ uses fluoroscein–isothiocynate, which is detected where Sig M also fluoresces. Lastly, we used NanoString nCounter® analysis to investigate the transcriptomic landscape for the two Cryptosporidium species, assessing the gene expression of 144 host and parasite genes. Despite the host gene expression being at high levels, the levels of putative intracellular Cryptosporidium gene expression were low, with no significant difference from controls, which could be, in part, explained by the abundance of uninfected cells present as determined by both Sporo-Glo™ and Sig M analyses. This study shows for the first time that a natural auto-fluorescent signal, Sig M, linked to Cryptosporidium infection can be detected in infected host cells without any fluorescent labeling strategies and that the COLO-680N cell line and spectral cytometry could be useful tools to advance the understanding of Cryptosporidium infectivity.

Placental function is a key determinant of fetal growth and development that can be influenced by maternal and fetal environmental factors. The molecular mechanisms by which the placenta senses and responds to environmental cues are poorly understood. This exploratory study aimed to characterize the effect of birth rank (single vs. twin) and placentome morphologic subtype on expression of genes involved in nutrient transport, angiogenesis, immunity and stress response. Cotyledonary tissue was collected from type A, B and C placentomes from five single and six twin fetuses at 140 days of gestation. GLUT1 and GLUT3 were the most highly expressed genes consistent with the high demand for glucose to support fetal growth. Expression of BCKDHβ and IGF-2 was 1.3- and 1.5-fold higher, respectively, and PCYT1A was 3-fold lower in singles compared to twins (P < 0.05) while no other differences in gene expression were observed between birth ranks. Expression of EAAT2 and LAT2 was higher while PCYT1A was lower in A compared to B type cotyledons. Expression of GUCY1B1/3 and IGF-1 was higher while CD98 and LAT2 were lower in type B compared to C cotyledons (P < 0.05). Compared to type C cotyledons, expression of EAAT2, IGF-1, IGF-2, LAT1 was higher, while TEK was lower in type A cotyledons. The effects of birth rank on placental gene expression in this study indicated that placental nutrient transport and/or function differs between single and twin pregnancies in sheep. Differences in gene expression between the placentome subtypes suggests that changes in placentome morphology are associated with shifts in amino acid transport and metabolism, oxidative stress and angiogenesis and/or blood flow. This study highlights that placental gene expression differs in response to birth rank and placentome morphologic subtype which suggests that both maternal and fetal factors may influence placental function in sheep. These associations provide insights into gene pathways for more targeted future investigations as well as potential adaptations to improve placental efficiency to support fetal growth in twin pregnancies.

Mycobacterium avium subspecies paratuberculosis (MAP) causes chronic progressive granulomatous enteritis leading to diarrhea, weight-loss, and eventual death in ruminants. Commercially available vaccine provides only partial protection against MAP infection and can interfere with the use of current diagnostic tests for bovine tuberculosis in cattle. Here, we characterized immune responses in calves to vaccines containing four truncated MAP antigens as a fusion (Ag85A 202-347 -SOD 1-72 -Ag85B 173-330 -74F 1-148+669-786 ), either displayed on protein particles, or expressed as a soluble recombinant MAP (rMAP) fusion protein as well as to commercially available Silirum ® vaccine. The rMAP fusion protein elicited the strongest antigen-specific antibody responses to both PPDA and recombinant antigen and strong and long-lasting T-cell immune responses to these antigens, as indicated by increased production of IFN-γ and IL-17A in antigen-stimulated whole blood cultures. The MAP fusion protein particle vaccine induced minimal antibody responses and weak IFN-γ responses but stimulated IL-17A responses to recombinant antigen. The immune response profile of Silirum ® vaccine was characterized by weak antibodies and strong IFN-γ and IL-17A responses to PPDA. Transcription analysis on antigen-stimulated leukocytes from cattle vaccinated with rMAP fusion protein showed differential expression of several immune response genes and genes involved in costimulatory signaling, TLR4 , TLR2 , PTX3 , PTGS2 , PD-L1 , IL1B , IL2 , IL6 , IL12B , IL17A , IL22 , IFNG , CD40 , and CD86 . Moreover, the expression of several genes of immune pathways correlated with cellular immune responses in the rMAP fusion protein vaccinated group. These genes have key roles in pathways of mycobacterial immunity, including autophagy, manipulation of macrophage-mediated killing, Th17- and regulatory T cells- (Treg) mediated responses. Calves vaccinated with either the rMAP fusion protein or MAP fusion protein particle vaccine did not induce reactivity to PPDA and PPDB in a comparative cervical skin test, whereas Silirum ® induced reactivity to these tuberculins in most of the vaccinated animals. Overall, our results suggest that a combination of recombinant MAP antigens in the form of a soluble fusion protein vaccine are capable of inducing strong antigen-specific humoral and a balanced Th1/Th17-cell immune response. These findings, together with the absence of reactivity to tuberculin, suggest this subunit vaccine could provide protective immunity against intracellular MAP infection in cattle without compromising the use of current bovine tuberculosis surveillance test.

Chronic postpartum uterine infection detrimentally affects subsequent fertility. Nonsteroidal anti-inflammatory drugs (NSAID) are used to alleviate pain and treat inflammatory conditions in transition dairy cows with varying success. To screen the efficacy of NSAID in the absence of animal experiments, we have established an in vitro model to study uterine inflammation. Inflammation was induced in cultured bovine endometrial epithelial cells by challenging cells with an inflammation cocktail: lipopolysaccharide and proinflammatory cytokines, interleukin-1β (IL1β) and tumor necrosis factor α (TNFα). Release of the inflammation markers, serum amyloid A (SAA) and α-1-acid glycoprotein (αAGP), was measured by ELISA. Concentration of these markers was used to indicate the effectiveness in dampening inflammation of 5 NSAID: meloxicam, flunixin meglumine, aspirin, ketoprofen, and tolfenamic acid. Three NSAID, meloxicam, flunixin meglumine, and tolfenamic acid, were successful at dampening the release of SAA and αAGP into cell-culture supernatant, and the corresponding treated cells were selected for down-stream mRNA expression analysis. Expression of 192 genes involved in regulation of inflammatory pathways were investigated using Nanostring. Of the genes investigated, 81 were above the mRNA expression-analysis threshold criteria and were included in expression analysis. All SAA genes investigated (SAA2, SAA3, M-SAA3.2) were upregulated in response to the inflammation cocktail, relative to mRNA expression in control cells; however, AGP mRNA expression was below the expression analysis threshold and was, therefore, excluded from analysis. Treatment with NSAID downregulated genes involved in regulating chemokine signaling (e.g., CXCL2, CXCR4, CXCL5, and CXCL16) and genes that regulate the eicosanoid pathway (e.g., LTA4H, PTGS2, PLA2G4A, and PTGDS). Of the 5 NSAID investigated, meloxicam, flunixin meglumine, and tolfenamic acid are recommended for further investigation into treatment of postpartum uterine inflammation. The results from this study confirm the immunomodulatory properties of the endometrial epithelium in response to inflammatory stimuli and suggest that NSAID may be beneficial in alleviating uterine inflammation.

In silico prediction of epitopes is a potentially time-saving alternative to experimental epitope identification but is often subject to misidentification of epitopes and may not be useful for proteins from archaeal microorganisms. In this study, we mapped B- and T-cell epitopes of a model antigen from the methanogen Methanobrevibacter ruminantium M1, the Big_1 domain (AdLP-D1, amino acids 19-198) of an adhesin-like protein. A series of 17 overlapping 20-mer peptides was selected to cover the Big_1 domain. Peptide-specific antibodies were produced in mice and measured by ELISA, while an in vitro splenocyte re-stimulation assay determined specific T-cell responses. Overall, five peptides of the 17 peptides were shown to be major immunogenic epitopes of AdLP-D1. These immunogenic regions were examined for their localization in a homology-based model of AdLP-D1. Validated epitopes were found in the outside region of the protein, with loop like secondary structures reflecting their flexibility. The empirical data were compared with epitope predictions made by programmes based on a range of algorithms. In general, the epitopes identified by in silico predictions were not comparable to those determined empirically.

Methane is produced in the rumen of ruminant livestock by methanogens, accounting for approximately 14.5% of anthropogenic greenhouse gas emissions in terms of global warming potential. The rumen contains a diversity of methanogens species, and only a few of these have been cultured. Immunomagnetic capture technology (ICT) is a simple and effective method to capture and concentrate target organisms in samples containing complex microflora. We hypothesized that antibody-coated magnetic beads could be used to demonstrate antibody specificity and cross-reactivity to methanogens in rumen samples. Sheep polyclonal antibodies raised against four isolates of rumen dwelling methanogens, Methanobrevibacter ruminantium strain M1, Methanobrevibacter sp. AbM4, Methanobrevibacter sp. D5, and Methanobrevibacter sp. SM9 or an equal mix of all four isolates, were used to coat paramagnetic beads. ICT was used together with flow cytometry and qPCR to optimize key parameters: the ratio of antibody to beads, coupling time between antibody and paramagnetic beads to produce immunomagnetic beads (IMBs), and optimal incubation time for the capture of methanogen cells by IMBs. Under optimized conditions, IMBs bound strongly to their respective isolates and showed a degree of cross-reactivity with isolates of other Methanobrevibacter spp. in buffer and in rumen fluid, and with resident methanogens in rumen content samples. The evidence provided here indicates that this method can be used to study the interaction of antibodies with antigens of rumen methanogens, to understand antigen cross-reactivity and antibody binding efficiency for the evaluation of antigens used for the development of a broad-spectrum anti-methanogen vaccine for the abatement of methane production.

The physiology of the dairy cow while transitioning from pregnancy to lactation is complex, with multifactorial processes studied extensively for the role they play in manifestation of disease along with associated economic losses and compromised animal welfare. Manuscripts outlining associations among nutrition, production, physiology, and genetics variables and transition cow disorders are common in literature, with blood analytes that are central to energy metabolism (e.g., nonesterified fatty acids; NEFA, β-hydroxybutyrate; BHB) often reported. Immunity and inflammation have increasingly been explored in the pathogenesis and persistence of disorders, with cytokines and acute phase proteins well documented. However, most of these studies have involved cows fed total mixed rations, which may not always reflect profiles of blood analytes and other physiological indicators of transition cow health in grazing cows consuming fresh pasture. Considering the comparatively lesser characterization of these analytes and markers in pasture-based, seasonal-calving dairy cows, we compiled a database consisting of 2,610 cow lactations that span 20 yr of transition cow research in New Zealand. Using this database, analyte profiles from approximately 28 d precalving to 35 d postcalving were identified in dairy cows with a range of genetics, milk production potentials, and pasture-based farm management systems. These profiles characterize changes in energy reserves and metabolism (NEFA, BHB, glucose, insulin, growth hormone, insulin-like growth factor-1, leptin, body condition score, body weight), liver function (globulin, aspartate aminotransferase, glutamate dehydrogenase, gamma-glutamyl transpeptidase, bilirubin, cholesterol, liver triacylglycerides), protein metabolism (albumin, total protein, albumin:globulin ratio, creatinine, urea, creatine kinase), mineral balance (calcium, magnesium, phosphate, potassium, sodium, chloride, bicarbonate), inflammation (IL-1β, IL-6, haptoglobin, reactive oxygen species, total antioxidant capacity), and uterine health (polymorphonuclear cells, macrophage cells, vaginal discharge score). Temporal changes are generally consistent with previously characterized homeorhetic changes experienced by the dairy cow during the transition from pregnancy to lactation in both pastoral and housed systems. Some of the profiles had not previously been presented for pastoral systems, or in some cases, presented for either system. Our results indicate that moderate-yielding dairy cows undergo similar homeorhetic changes to high-yielding housed cows; however, differences in diet composition result in greater BHB concentrations than expected, based on their milk production and NEFA concentrations. In addition, most cows were able to transition to a state of higher energy requirement following calving, albeit with an increased metabolic challenge in the liver, and only a small percentage of cows were classified with severe hepatic lipidosis or severe hyperketonemia. Increases in metabolic function of the liver were accompanied by changes in indicators of the immune system and changes in mineral balance that, combined, probably reflect the innate response to the transition from gestation to lactation.

Abstract This study evaluated the influence of feeding low and high preweaning allowances of unpasteurized whole milk (MA) on intake, selected blood metabolites, antibody response, mammary gland growth, and growth of New Zealand (NZ) dairy heifers to 7 mo of age. At 10 ± 2 d of age (study day 0), group-housed (six·pen−1) heifer calves (Holstein-Friesian × Jersey) were allocated to low (4 L whole milk·calf−1·d−1; n = 7 pens) or high (8 L whole milk·calf−1·d−1; n = 7 pens) MA for the next 63 d. Calves were gradually weaned between days 63 ± 2 and 73 ± 2. Calves in each pen had ad-libitum access to clean water, pelleted calf starter, and chopped grass hay from day 1 to 91 ± 2 d. At 92 ± 2 d, all calves were transferred to pasture, grazed in a mob, and their growth and selected blood metabolites were measured until day 209. All animals were weighed weekly during the indoor period (to day 91) and then at days 105, 112, 128, 162, 184, and 209. Skeletal growth measurements and blood samples to analyze selected metabolites were collected at the start of the experiment, weaning, and then postweaning on day 91, and day 201. Specific antibodies against Leptospira and Clostridia were quantified in weeks 7, 13, and 27. Mammary glands were scanned using ultrasonography at the start of the experiment, weaning, and day 201. Feeding high vs. low amounts of MA increased the preweaning growth in heifer calves (P = 0.02) without negatively affecting postweaning average daily gain (ADG) (P = 0.74). Compared with heifers fed with low MA, high MA fed heifers had a greater increase in antibodies against Leptospira and Clostridia by 13 wk of age (P = 0.0007 and P = 0.06, respectively). By 27 wk of age, the antibody response was the same in heifers offered low or high MA. There was no effect of MA on the total size of the mammary gland, measured by ultrasonography, at weaning and 7 mo of age. However, the greater MA was associated with more mammary parenchyma (P = 0.01) and less mammary fat pad (P = 0.03) in back glands at 7 mo of age compared with heifers fed lower MA. In conclusion, feeding a high vs. a low amount of unpasteurized whole milk increased the preweaning growth of New Zealand replacement heifers without negatively affecting their ADG during postweaning under grazing conditions. Feeding more (8 vs. 4 L·d−1) unpasteurized whole milk positively affected antibody responses early in life and mammary gland composition by 7 mo of age in dairy heifers reared for pasture-based dairy systems.