Function In Cattle Research Articles

Progressive ataxia of Charolais cattle highlights a role of KIF1C in sustainable myelination

Hereditary spastic paraplegias (HSPs) are clinically and genetically heterogeneous human neurodegenerative diseases. Amongst the identified genetic causes, mutations in genes encoding motor proteins such as kinesins have been involved in various HSP clinical isoforms. Mutations in KIF1C are responsible for autosomal recessive spastic paraplegia type 58 (SPG58) and spastic ataxia 2 (SPAX2). Bovines also develop neurodegenerative diseases, some of them having a genetic aetiology. Bovine progressive ataxia was first described in the Charolais breed in the early 1970s in England and further cases in this breed were subsequently reported worldwide. We can now report that progressive ataxia of Charolais cattle results from a homozygous single nucleotide polymorphism in the coding region of the KIF1C gene. In this study, we show that the mutation at the heterozygous state is associated with a better score for muscular development, explaining its balancing selection for several decades, and the resulting high frequency (13%) of the allele in the French Charolais breed. We demonstrate that the KIF1C bovine mutation leads to a functional knock-out, therefore mimicking mutations in humans affected by SPG58/SPAX2. The functional consequences of KIF1C loss of function in cattle were also histologically reevaluated. We showed by an immunochemistry approach that demyelinating plaques were due to altered oligodendrocyte membrane protrusion, and we highlight an abnormal accumulation of actin in the core of demyelinating plaques, which is normally concentrated at the leading edge of oligodendrocytes during axon wrapping. We also observed that the lesions were associated with abnormal extension of paranodal sections. Moreover, this model highlights the role of KIF1C protein in preserving the structural integrity and function of myelin, since the clinical signs and lesions arise in young-adult Charolais cattle. Finally, this model provides useful information for SPG58/SPAX2 disease and other demyelinating lesions.

Genome variants associated with RNA splicing variations in bovine are extensively shared between tissues

BackgroundMammalian phenotypes are shaped by numerous genome variants, many of which may regulate gene transcription or RNA splicing. To identify variants with regulatory functions in cattle, an important economic and model species, we used sequence variants to map a type of expression quantitative trait loci (expression QTLs) that are associated with variations in the RNA splicing, i.e., sQTLs. To further the understanding of regulatory variants, sQTLs were compare with other two types of expression QTLs, 1) variants associated with variations in gene expression, i.e., geQTLs and 2) variants associated with variations in exon expression, i.e., eeQTLs, in different tissues.ResultsUsing whole genome and RNA sequence data from four tissues of over 200 cattle, sQTLs identified using exon inclusion ratios were verified by matching their effects on adjacent intron excision ratios. sQTLs contained the highest percentage of variants that are within the intronic region of genes and contained the lowest percentage of variants that are within intergenic regions, compared to eeQTLs and geQTLs. Many geQTLs and sQTLs are also detected as eeQTLs. Many expression QTLs, including sQTLs, were significant in all four tissues and had a similar effect in each tissue. To verify such expression QTL sharing between tissues, variants surrounding (±1 Mb) the exon or gene were used to build local genomic relationship matrices (LGRM) and estimated genetic correlations between tissues. For many exons, the splicing and expression level was determined by the same cis additive genetic variance in different tissues. Thus, an effective but simple-to-implement meta-analysis combining information from three tissues is introduced to increase power to detect and validate sQTLs. sQTLs and eeQTLs together were more enriched for variants associated with cattle complex traits, compared to geQTLs. Several putative causal mutations were identified, including an sQTL at Chr6:87392580 within the 5th exon of kappa casein (CSN3) associated with milk production traits.ConclusionsUsing novel analytical approaches, we report the first identification of numerous bovine sQTLs which are extensively shared between multiple tissue types. The significant overlaps between bovine sQTLs and complex traits QTL highlight the contribution of regulatory mutations to phenotypic variations.

Effect of early calf-hood nutrition on the transcriptomic profile of subcutaneous adipose tissue in Holstein-Friesian bulls

BackgroundAdipose tissue is a major endocrine organ and is thought to play a central role in the metabolic control of reproductive function in cattle. Plane of nutrition during early life has been shown to influence the timing of puberty in both male and female cattle, though the exact biological mechanisms involved are currently unknown. The aim of this study was to investigate the effect of early calf-hood nutrition on the transcriptomic profile of subcutaneous adipose tissue in Holstein-Friesian bulls to identify possible downstream effects on reproductive physiology.ResultsHolstein-Friesian bull calves with a mean (±S.D.) age and bodyweight of 19 (±8.2) days and 47.5 (±5.3) kg, respectively, were assigned to either a high (n = 10) or low (n = 10) plane of nutrition. Calves were fed in order to achieve an overall growth rate of 1.08 and 0.57 kg/day for the high and low plane of nutrition treatments, respectively. At 126 days of age, the bulls were euthanized, subcutaneous adipose tissue samples were harvested and RNAseq analysis was performed. There were 674 genes differentially expressed in adipose tissue of calves on the low compared with the high plane of nutrition (P < 0.05; FDR < 0.05; fold change > 2.0). High plane of nutrition positively altered the expression of genes across an array of putative biological processes but the most dominant cellular processes affected were cellular energy production and branched chain amino acid degradation. A high plane of nutrition caused upregulation of genes such as leptin (LEP) and adiponectin (ADIPOQ), which are known to directly affect reproductive function.ConclusionsThese results provide an insight into the effect of augmenting the plane of nutrition of Holstein-Friesian bull calves in the prepubertal period on the transcriptome of adipose tissue.

Sex Steroid-Mediated Control of Oviductal Function in Cattle.

In cattle, the oviduct is a tubular organ that connects the ovary and the uterus. The oviduct lumen stages a dynamic set of cellular and molecular interactions to fulfill the noble role of generating a new individual. Specific anatomical niches along the oviduct lumen provide the appropriate microenvironment for final sperm capacitation, oocyte capture and fertilization, and early embryo development and transport. To accomplish such complex tasks, the oviduct undergoes spatially and temporally-regulated morphological, biochemical, and physiological changes that are associated with endocrine events of the estrous cycle. Specifically, elevated periovulatory concentrations of estradiol (E2) and progesterone (P4) influence gene expression and morphological changes that have been associated positively to fertility in beef cattle. In this review, we explore how E2 and P4 influence oviductal function in the beginning of the estrous cycle, and prepare the oviductal lumen for interactions with gametes and embryos.

Evolution of knowledge on ovarian physiology and its contribution to the widespread application of reproductive biotechnologies in South American cattle.

As our understanding of ovarian function in cattle has improved, our ability to control it has also increased. Luteal function in cattle has been studied in detail, and prostaglandin F2α has been used for several years for the elective induction of luteal regression. More recently, follicle wave dynamics has been studied and protocols designed to induce follicular wave emergence and ovulation have reduced, and even eliminated, the need for estrus detection. The addition of progestin-releasing devices, estradiol, GnRH and equine chorionic gonadotropin (eCG) have provided opportunities for fixed-time AI (FTAI) and possibilities for increased pregnancy rates. In embryo transfer programs, these same treatments have eliminated the need for estrus detection, permitting fixed-time embryo transfer and the initiation of superstimulatory treatments without regard to day of the estrous cycle. Collectively, new treatment protocols have facilitated the application of assisted reproductive technologies, and this is especially true in South America. Over the last 20 years, the use of AI in South America has increased, due largely to the use of FTAI. There has been more than a 10-fold increase in the use of FTAI in Brazil with more than 11 million treatments in 2016, representing 85% of all AI. Similar trends are occurring in Argentina and Uruguay. Production of in vivo-derived (IVD) embryos has remained relatively stable over the years, but in vitro embryo production (IVP) has increased dramatically over the past 10 to 15 years, especially in Brazil where more than 300,000 IVP embryos were produced in 2010. World-wide, more than 666,000 bovine IVP embryos were produced in 2016, of which more than 57% were produced in South America. The use of assisted reproductive technologies has facilitated the dissemination of improved genetics and increased reproductive performance; other South American countries are now following suit.

The mycotoxin metabolite deepoxy- deoxynivalenol increases apoptosis and decreases steroidogenesis in bovine ovarian theca cells†

The mycotoxin deoxynivalenol (DON) has been shown to inhibit ovarian granulosa cell function in cattle in vitro, but it is not known whether DON or its metabolite deepoxy-DON (DOM-1) affects theca cell function. The objectives of this study were to determine the effects of DON and of DOM-1 on theca cell steroidogenesis and apoptosis, and to determine the main pathways through which they act. Bovine theca cells were cultured in a nonluteinizing serum-free culture system, and challenged with DON or DOM-1 for 4 days to measure steroidogenesis and apoptosis, for 1-8 h to measure immediate-early genes, and for 5-60 min to measure phosphorylation of intracellular signaling proteins. Addition of DON decreased progesterone secretion at doses as low as 0.5 ng/ml but had no effect on testosterone secretion. Addition of DOM-1 inhibited progesterone and testosterone secretion at 0.5 ng/ml. Treatment of cells with 1 ng/ml DOM-1 increased the proportion of apoptotic cells, whereas DON had no effect. Addition of DON or DOM-1 stimulated phosphorylation of EIF2AK2, MAPK3/1, and AKT. However, DON inhibited and DOM-1 stimulated MAPK14 phosphorylation. DON increased the levels of mRNA encoding early-immediate genes EGR1, EGR3, and FOS, whereas DOM-1 was without effect. DOM-1 but not DON increased abundance of mRNA of the endoplasmic reticulum (ER) stress-related proteins, PRKRA and ATF4. We conclude that DOM-1 has a major impact on theca function in cattle, and possibly induces theca cell apoptosis through ER stress.

Effect of melatonin on bovine theca cells in vitro

Melatonin affects granulosa cell function in several species but its function in theca cells is less clear, particularly in monotocous animals. Thus, the objectives of this study were to determine the effects of melatonin on theca cell steroidogenesis, gene expression and cell proliferation in a monotocous species, namely cattle. Ovaries were collected from a local bovine abattoir, from which theca cells were isolated from large (8-22mm) follicles and treated with various hormones in serum-free medium for 24h or 48h. Melatonin caused a dose-dependent inhibition (P<0.05) of LH+insulin-like growth factor 1 (IGF1)-induced androstenedione and progesterone production. Also, melatonin inhibited (P<0.05) LH+IGF1-induced expression of steroidogenic acute regulatory protein (StAR) mRNA (via real-time polymerase chain reaction) in theca cells, but it had no effect (P>0.10) on cytochrome P450 11A1 (CYP11A1) and cytochrome P450 17A1 (CYP17A1) mRNA abundance. In LH+IGF1-treated theca cells, melatonin decreased caspase 3 (CASP3) mRNA to levels similar to those observed in LH-treated theca cells. In contrast, melatonin increased (P<0.05) the number of bovine theca cells in both LH- and LH+IGF1-treated cultures. In conclusion, melatonin may act as an endocrine regulator of ovarian function in cattle by stimulating theca cell proliferation and inhibiting differentiation via inhibition of hormone-induced steroidogenesis.

Aromatase inhibitors: A new approach for controlling ovarian function in cattle

Numerous treatments and protocols have been used to control the reproductive cycle in cattle, with varying effectiveness and many involving the administration of steroid hormones. Steroid hormones, such as estradiol, are perceived as having a negative impact on consumer health. This internationally shared opinion has led to a ban on the use of steroid hormones in food producing animals in many countries (i.e., European Union, New Zealand, and Australia). Letrozole, a non-steroidal aromatase inhibitor, inactivates the aromatase enzyme responsible for the synthesis of estrogens by reversibly binding to the “heme” group of the P450 subunit. Letrozole is approved as an adjuvant or first-line treatment for hormone-dependent breast cancer in post-menopausal women, but has been used increasingly for ovulation induction in the treatment of infertility in women. Using the bovine model to determine the effects on ovarian function, letrozole treatment was found to extend the lifespan of the dominant follicle and thereby delay emergence of the next follicle wave and/or ovulation. Letrozole treatment also had a luteotrophic effect; that is, larger CL and/or higher circulating concentrations of progesterone were detected in letrozole-treated heifers. Results of the initial studies in cattle provided the impetus for the development of aromatase inhibitor-based synchronization and fertility treatment in cattle. Biologically active concentrations of letrozole were achieved via intravenous, intramuscular or intravaginal administration, but the intravaginal route of administration is of particular interest because it permits extended and defined treatment periods, is minimally invasive, and reduces animal handling. Recent results revealed that irrespective of the stage of the cycle, a 4-day letrozole-based protocol induced ovulation in a significantly greater proportion of animals and with significantly greater synchrony than the control treatment. Evidence and reasons for the increasing use of programmed breeding and fixed-time artificial insemination are discussed in this review as a background to current development of an innovative aromatase inhibitor-based protocol as a safe and effective method of controlling the estrous cycle and ovulation in cattle.

Influence of a Roundup formulation on glyphosate effects on steroidogenesis and proliferation of bovine granulosa cells in vitro

Glyphosate (N-phosphonomethyl-glycine) is a non-selective systemic herbicide widely used worldwide. The purpose of this study is to determine if glyphosate alone (GLPH) or in formulation with Roundup (G-RU) can affect granulosa cell proliferation and steroid production. Four experiments were conducted. In Exp. 1, 10 and 300 μg/mL of GLPH had no effect (P > 0.05) on cell numbers, estradiol or progesterone production, whereas 10 and 300 μg/mL of G-RU dramatically decreased (P < 0.05) cell numbers and estradiol and progesterone production. In Exp. 2, G-RU at 0.1 μg/mL had no significant effect whereas G-RU at 10 μg/mL decreased (P < 0.05) GC numbers, progesterone and estradiol production. In the absence of IGF1 but presence of FSH, 1 μg/mL of G-RU decreased (P < 0.05) estradiol production, whereas in the presence of IGF1 and FSH, 1 μg/mL of G-RU increased (P < 0.05) cell numbers, progesterone and estradiol production. In Exp. 3, IGF1 significantly increased cell numbers (by 2.8-fold) and estradiol (by 17.8-fold) and progesterone (by 6.1-fold) production. GLPH at 10 μg/mL alone had no significant effect on FSH-induced (i.e., basal) or FSH plus IGF1-induced cell numbers, estradiol or progesterone production. However, G-RU at 10 μg/mL significantly inhibited FSH plus IGF1-induced cell numbers, estradiol and progesterone production by 65%–91%. In Exp. 4, 48 h treatment of G-RU had no significant effect on viability of attached cells. In conclusion, the present studies demonstrate that GLPH and particularly G-RU may have the potential to impair reproductive function in cattle.

Role of Components of the Insulin-like Growth Factor System in the Early Stages of Ovarian Follicular Persistence in Cattle

Cystic ovarian disease (COD) is one of the main causes of infertility in dairy cattle. It has been postulated that the insulin-like growth factor (IGF) system may contribute to follicular persistence and development of COD. The initiation of the IGF response is a result of interactions between IGF-binding proteins (IGFBPs) and IGFBP proteases, mainly pregnancy-associated plasma protein A (PAPP-A). IGFBPs bind IGFs with high affinity and consequently regulate their access to IGF receptors (IGFRs). The aim of this research was to determine variations in components of the IGF system in the ovaries of cows with persistent follicles induced by long-term administration of progesterone. Proteins of the IGF system were evaluated at 0 (expected day of ovulation), 5, 10 and 15 days of follicular persistence to determine whether the changes occur early in the development of COD. The concentrations of IGF1 and IGFBP4 in follicular fluid were similar in all groups with follicular persistence and in control antral follicles. IGFR1 and IGFBP4 expression in situ were higher in granulose cells in persistent follicles than in control follicles. No differences were found in PAPP-A concentration within follicular fluid in persistent follicles relative to control antral follicles. These data support the hypothesis that the IGF system is altered in the initial stages of development of follicular persistence and has a determinant role in ovarian function in cattle.

Genome-wide methylation analysis reveals differentially methylated loci that are associated with an age-dependent increase in bovine fibroblast response to LPS

BackgroundDifferences in DNA methylation are known to contribute to the development of immune-related disorders in humans but relatively little is known about how methylation regulates immune function in cattle. Utilizing whole-transcriptome analyses of bovine dermal fibroblasts, we have previously identified an age and breed-dependent up-regulation of genes within the toll-like receptor 4 (TLR4) pathway that correlates with enhanced fibroblast production of IL-8 in response to lipopolysaccharide (LPS). Age-dependent differences in IL-8 production are abolished by treatment with 5-aza-2-deoxycytidine and Trichostatin A (AZA-TSA), suggesting epigenetic regulation of the innate response to LPS. In the current study, we performed reduced representation bisulfite sequencing (RRBS) on fibroblast cultures isolated from the same animals at 5- and 16-months of age to identify genes that exhibit variable methylation with age. To validate the role of methylation in gene expression, six innate response genes that were hyper-methylated in young animals were assessed by RT-qPCR in fibroblasts from animals at different ages and from different breeds.ResultsWe identified 14,094 differentially methylated CpGs (DMCs) that differed between fibroblast cultures at 5- versus 16-months of age. Of the 5065 DMCs that fell within gene regions, 1117 were located within promoters, 1057 were within gene exons and 2891 were within gene introns and 67% were more methylated in young cultures. Transcription factor enrichment of the promoter regions hyper-methylated in young cultures revealed significant regulation by the key pro-inflammatory regulator, NF-κB. Additionally, five out of six chosen genes (PIK3R1, FES, NFATC1, TNFSF13 and RORA) that were more methylated in young cultures showed a significant reduction in expression post-LPS treatment in comparison with older cultures. Two of these genes, FES and NFATC1, were similarly down-regulated in Angus cultures that also exhibit a low LPS response phenotype.ConclusionsOur study has identified immune-related loci regulated by DNA methylation in cattle that may contribute to differential cellular response to LPS, two of which exhibit an identical expression profile in both low-responding age and breed phenotypes. Methylation biomarkers of differential immunity may prove useful in developing selection strategies for replacement cows that are less susceptible to severe infections, such as coliform mastitis.

Pre-hatching embryo-dependent and -independent programming of endometrial function in cattle.

The bovine pre-implantation embryo secretes bioactive molecules from early development stages, but effects on endometrial function are reported to start only after elongation. Here, we interrogated spatially defined regions of the endometrium transcriptome for responses to a day 7 embryo in vivo. We hypothesize that exposure to an embryo changes the abundance of specific transcripts in the cranial region of the pregnant uterine horn. Endometrium was collected from the uterotubal junction (UTJ), anterior (IA), medial (IM) and posterior (IP) regions of the uterine horn ipsilateral to the CL 7 days after estrus from sham-inseminated (Con) or artificially inseminated, confirmed pregnant (Preg) cows. Abundance of 86 transcripts was evaluated by qPCR using a microfluidic platform. Abundance of 12 transcripts was modulated in the Preg endometrium, including classical interferon-stimulated genes (ISG15, MX1, MX2 and OAS1Y), prostaglandin biosynthesis genes (PTGES, HPGD and AKR1C4), water channel (AQP4) and a solute transporter (SLC1A4) and this was in the UTJ and IA mainly. Additionally, for 71 transcripts, abundance varied according to region of the reproductive tract. Regulation included downregulation of genes associated with proliferation (IGF1, IGF2, IGF1R and IGF2R) and extracellular matrix remodeling (MMP14, MMP19 and MMP2) and upregulation of anti-adhesive genes (MUC1) in the cranial regions of uterine horn. Physical proximity to the embryo provides paracrine regulation of endometrial function. Embryo-independent regulation of the endometrial transcriptome may support subsequent stages of embryo development, such as elongation and implantation. We speculate that successful early embryo-dependent and -independent programming fine-tune endometrial functions that are important for maintenance of pregnancy in cattle.

In vitro effects of the Fusarium mycotoxins fumonisin B1 and beauvericin on bovine granulosa cell proliferation and steroid production

Fusarium mycotoxins are natural contaminants of various commodities representing significant problem worldwide. Since the co-occurrence of beauvericin (BEA) and fumonisin B1 (FB1) in grains is frequent, the present study was carried out to evaluate the individual and combined effects of FB1 and BEA on cell proliferation, steroid production and gene expression using bovine granulosa cells (GC). When tested alone FB1 did not show (P ≥ 0.05) effects on cell proliferation at any dose. Whereas BEA at 3, 6, and 10 μM significantly decreased (P < 0.05) cell numbers. FB1 alone had no significant effect (P ≥ 0.05) on progesterone production at any tested doses, whereas FB1 at 1, 1.5 and 3 μM slightly inhibited (P < 0.05) estradiol production. At concentrations ≥3 μM, BEA was found to strongly decrease (P < 0.05) steroid production, and FB1 did not influence these effects of BEA. At 10 μM, both mycotoxins decreased (P < 0.001) serum-induced GC proliferation. At 30 μM, BEA showed inhibitory effects on FSH plus IGF1-induced CYP11A1 and CYP19A1 mRNA abundance (P < 0.05), whereas FB1 at 30 μM had no effect on CYP11A1 and CYP19A1 gene expression. Taken together these results demonstrate that the Fusarium mycotoxins BEA and FB1 may impair reproductive function in cattle.

Effects of angiogenin on granulosa and theca cell function in cattle

Angiogenin is a member of the ribonuclease A superfamily of proteins that has been implicated in stimulating angiogenesis but whether angiogenin can directly affect ovarian granulosa or theca cell function is unknown. Therefore, the objective of these studies was to determine the effect of angiogenin on proliferation and steroidogenesis of bovine granulosa and theca cells. In experiments 1 and 2, granulosa cells from small (1 to 5 mm diameter) follicles and theca cells from large (8 to 22 mm diameter) follicles were cultured to evaluate the dose-response effect of recombinant human angiogenin on steroidogenesis. At 30 and 100 ng/ml, angiogenin inhibited (P<0.05) granulosa cell progesterone production and theca cell androstenedione production but did not affect (P>0.10) granulosa cell estradiol production or theca cell progesterone production, and did not affect numbers of granulosa or theca cells. In experiments 3 and 4, granulosa and theca cells from both small and large follicles were cultured with 300 ng/ml of angiogenin to determine if size of follicle influenced responses to angiogenin. At 300 ng/ml, angiogenin increased large follicle granulosa cell proliferation but decreased small follicle granulosa cell progesterone and estradiol production and large follicle theca cell progesterone production. In experiments 5 and 6, angiogenin stimulated (P<0.05) proliferation and DNA synthesis in large follicle granulosa cells. In experiment 7, 300 ng/ml of angiogenin increased (P<0.05) CYP19A1 messenger RNA (mRNA) abundance in granulosa cells but did not affect CYP11A1 mRNA abundance in granulosa or theca cells and did not affect CYP17A1 mRNA abundance in theca cells. We conclude that angiogenin appears to target both granulosa and theca cells in cattle, but additional research is needed to further understand the mechanism of action of angiogenin in granulosa and theca cells, as well as its precise role in folliculogenesis.

Impact of Supplementing Cattle with Omnigen-AF at the Receiving or Fin-ishing Phase on Beef Shelf-Life

ObjectivesA proprietary feed additive designed to augment the innate immune function in cattle, OmniGen-AF (Phibro Animal Health Cooperation, Teaneck, NJ), might extend beef shelf-life by incorporating antioxidants via phenolic-rich compounds. Thus, the objective of this study was to evaluate the effect of supplementing cattle at the receiving or finishing phase on beef steak shelf-life after 8, 22, and 29 d of aging.Materials and MethodsThree treatment groups included a control group with no supplementation, OmniGen-AF supplementation (4 g/100lb BW/hd per d) for 28 d after receiving, and supplementation for 210 d during finishing. The study had a total of 288 steers that were randomly assigned to one of the three treatment groups (96 hd/treatment) which were randomly sorted into groups of 8 steers for a total of 12 pens/treatment. At harvest, 24 USDA Choice carcasses were selected within each dietary treatment (n = 72) and left and right side Longissimus lumborum samples were collected for analysis.ResultsThe inclusion of OmniGen-AF had no effect on tenderness (P = 0.31). Meat from cattle supplemented throughout the finishing phase had more C18:1, C19:0, C18:2, C18:3ω3, total, UFA, MUFA, and trans fatty acids in relation to meat from cattle supplemented through the receiving phase while meat from non-supplemented cattle had intermediate amounts (P < 0.05). Steaks from non-supplemented cattle had the most C18:2TT in comparison to the other two treatment groups (P = 0.04). There was more C20:5ω3 fatty acid in beef from the non-supplemented group than the receiving group, with the finishing group being intermediate (P = 0.02). The SFA:UFA ratio was greater in beef from the receiving group, intermediate in beef from the non-supplemented group, and lowest in beef from the finishing group (P = 0.04). The PUFA concentration was greater in beef from the finishing and non-supplemented groups (P < 0.01). Dietary treatment had no effect on discoloration under retail display conditions at any aging period (P = 0.34). Although color stability seemed to be unaffected by the supplementation, feeding OmniGen-AF throughout the entire finishing period tended (P = 0.10) to decrease TBARS values (a measure of oxidation) versus cattle supplemented only through the receiving phase or those never supplemented (2.80 vs. 3.07 and 3.06 mg malonaldehyde/kg tissue, respectively). Despite this trend, meat from cattle fed OmniGen-AF throughout finishing did not show meaningful differences (P = 0.92) in superoxide dismutase activity compared to meat from cattle that were not supplemented.ConclusionSupplementing cattle with a greater concentration of OmniGen-AF or increasing the antioxidant components in the feed supplement could be explored to further maximize beef shelf-life following long periods of aging.

A combination of lactic acid bacteria regulates Escherichia coli infection and inflammation of the bovine endometrium

Uterine function in cattle is compromised by bacterial contamination and inflammation after calving. The objective of this study was to select a combination of lactic acid bacteria (LAB) to decrease endometrium inflammation and Escherichia coli infection. Primary endometrial epithelial cells were cultured in vitro to select the most favorable LAB combination modulating basal tissue inflammation and E. coli infection. Supernatants were obtained to determine expression of pro-inflammatory cytokines, and E. coli infection was evaluated after harvesting the tissue and plate counting. The selected LAB combination was tested in uterus explants to assess its capacity to modulate basal and acute inflammation (associated with E. coli infection). The combination of Lactobacillus rhamnosus, Pediococcus acidilactici, and Lactobacillus reuteri at a ratio of 25:25:2, respectively, reduced E. coli infection in vitro with (89.77%) or without basal tissue inflammation (95.10%) compared with single LAB strains. Lactic acid bacteria treatment reduced CXCL8 and IL1B expression 4.7- and 2.2-fold, respectively, under acute inflammation. Ex vivo, the tested LAB combination reduced acute inflammation under E. coli infection, decreasing IL-8, IL-1β, and IL-6 up to 2.2-, 2.5-, and 2.2-fold, respectively. In the total inflammation model, the LAB combination decreased IL-8 1.6-fold and IL-6 1.2-fold. Ultrastructural evaluation of the tissue suggested no direct interaction between the LAB and E. coli, although pathological effects of E. coli in endometrial cells were greatly diminished or even reversed by the LAB combination. This study shows the promising potential of LAB probiotics for therapeutic use against endometrial inflammation and infection.

Does prostacyclin (PGI2) support porcine corpus luteum function? – data from in vitro study

Background. Prostacyclin (PGI2) of luteal origin is involved in the control of corpus luteum (CL) development and function in cattle. PGI2 may regulate the process of angiogenesis and may stimulate progesterone (P4) secretion by luteal cells via its specific receptors, PTGIR. In contrast to cattle, the role of PGI2 in the pig CL has not yet been described.Aim. The present study aimed to investigate the effect of PGI2 on 1) P4 secretion by luteal cells, and 2) the expression of angiogenesis-related genes in endothelial cells of the porcine CL.Methods. CL collected from gilts on day 5-7 of the estrous cycle were used for enzymatic isolation of luteal (Experiment 1) and endothelial (Experiment 2) cells. In Exp. 1, cultured luteal cells were incubated with increasing (0, 0.01, 0.1, 1, 5 µM) doses of PGI2 analogues: iloprost (ILO) and carbaprostacyclin (cPGI2) for 8 h. To determine the effective doses of PGI2 analogues, P4 concentration in culture medium was examined by RIA. Thereafter, luteal cells were treated with ILO and cPGI2 at the concentration of 1 and 5 µM in the presence or absence of PTGIR antagonist (CAY10441). After 8 h of incubation the medium was collected for P4 determination. In Exp. 2, isolated endothelial cells were treated for 24 h with ILO and cPGI2 at doses of 1 and 5 µM. Then, cells were collected for analysis of Ang-1 and -2 mRNA expression using qPCR.Results. Both, ILO and cPGI2 affected P4 secretion by luteal cells. Elevated levels of P4 were observed in medium after treatment of luteal cells with 1 µM of ILO and 0.1, 1 and 5 µM of cPGI2 compared with control values (p&lt;0.05). The addition of CAY10441 inhibited the stimulatory effect of ILO on P4 secretion, while did not change P4 production by luteal cells incubated with cPGI2. Moreover, PGI2 analogues differentially affected (p&lt;0.05) the expression of proangiogenic factors. ILO stimulated Ang-2, whereas cPGI2 positively affected Ang-1 mRNA expression in endothelial cells at concentrations of 1 µM and 5 µM, respectively.Conclusion. PGI2 affects P4 secretion during luteal phase of the estrous cycle and may regulate the process of angiogenesis in the porcine CL.

Activities for leptin in bovine trophoblast cells

Leptin is involved in various reproductive processes in humans and rodents, including placental development and function. The specific ways that leptin influences placental development and function in cattle are poorly understood. This work was completed to explore how leptin regulates hormone, cytokine and metalloprotease transcript abundance, and cell proliferation in cultured bovine trophoblast cells. In the first set of studies, cells were cultured in the presence of graded recombinant bovine leptin concentrations (0, 10, 50, 250 ng/mL) for 6 or 24 h. Transcript profiles were examined from extracted RNA. Leptin supplementation did not affect abundance of the maternal recognition of pregnancy factor, interferon-tau (IFNT), but leptin increased (P < 0.05) abundance of chorionic somatomammotropin hormone 2 (CSH2; ie, placental lactogen) at both 6 and 24 h at each concentration tested. At 24 h, the greatest CSH2 abundance (P < 0.05) was detected in cells supplemented with 50 ng/mL leptin. Transcript abundance of the remodeling factor, metalloprotease 2 (MMP2), was greater (P < 0.05) in leptin-treated cells at 24 h but not at 6 h. The 24 h MMP2 response was greatest (P < 0.05) at 250 ng/mL. Transcript abundance for MMP9 was not altered by leptin treatment. In a separate set of studies, cell proliferation assays were completed. Leptin supplementation did not affect bovine trophoblast cell line proliferation at any dose tested. In conclusion, leptin supplementation did not affect bovine trophoblast cell proliferation or IFNT expression, but leptin increases CSH2 and MMP2 transcript abundance. Both of these factors are involved with peri-implantation and postimplantation placental development and function, and this implicates leptin as a potential mediator of early placental development and function in cattle.

Identification of a novel β-adrenergic octopamine receptor-like gene (βAOR-like) and increased ATP-binding cassette B10 (ABCB10) expression in a Rhipicephalus microplus cell line derived from acaricide-resistant ticks.

BackgroundThe cattle tick Rhipicephalus (Boophilus) microplus is an economically important parasite of livestock. Effective control of ticks using acaricides is threatened by the emergence of resistance to many existing compounds. Several continuous R. microplus cell lines have been established and provide an under-utilised resource for studies into acaricide targets and potential genetic mutations associated with resistance. As a first step to genetic studies using these resources, this study aimed to determine the presence or absence of two genes and their transcripts that have been linked with acaricide function in cattle ticks: β-adrenergic octopamine receptor (βAOR, associated with amitraz resistance) and ATP-binding cassette B10 (ABCB10, associated with macrocyclic lactone resistance) in six R. microplus cell lines, five other Rhipicephalus spp. cell lines and three cell lines representing other tick genera (Amblyomma variegatum, Ixodes ricinus and Hyalomma anatolicum).MethodsEnd-point polymerase chain reaction (PCR) was used for detection of the βAOR gene and transcripts in DNA and RNA extracted from the tick cell lines, followed by capillary sequencing of amplicons. Quantitative real-time PCR (qPCR) was performed to determine the levels of expression of ABCB10.ResultsβAOR gene expression was detected in all Rhipicephalus spp. cell lines. We observed a second amplicon of approximately 220 bp for the βAOR gene in the R. microplus cell line BME/CTVM6, derived from acaricide-resistant ticks. Sequencing of this transcript variant identified a 36 bp insertion in the βAOR gene, leading to a 12-amino acid insertion (LLKTLALVTIIS) in the first transmembrane domain of the protein. In addition, nine synonymous SNPs were also discovered in R. appendiculatus, R. evertsi and R. sanguineus cell lines. Some of these SNPs appear to be unique to each species, providing potential tools for differentiating the tick species. The BME/CTVM6 cell line had significantly higher ABCB10 (P = 0.002) expression than the other R. microplus cell lines.ConclusionsThe present study has identified a new βAOR gene and demonstrated a higher ABCB10 expression level in the BME/CTVM6 cell line, indicating that tick cell lines provide a useful experimental tool for acaricide resistance studies and further elucidation of tick genetics.Electronic supplementary materialThe online version of this article (doi:10.1186/s13071-016-1708-x) contains supplementary material, which is available to authorized users.

Toxicological effects of fumonisin B1 alone and in combination with other fusariotoxins on bovine granulosa cells

There is now overwhelming evidence of global contamination of commodities with Fusarium mycotoxins. Fumonisin B1 (FB1) is a Fusarium mycotoxin frequently occurring in corn in combination with deoxynivalenol (DON), α-zearalenol (α-ZEA) and β-zearalenol (β-ZEA). The aim of this study was to determine if FB1, alone and combined with DON or α-ZEA or β-ZEA, can affect cell proliferation and steroid production of bovine granulosa cells (GC). A species–specific model with bovine granulosa cells (GC) was used to study the potential endocrine disruptor effects of FB1 alone and in co-exposure. In the presence of β-ZEA (30 ng/mL), FB1 at 30 ng/mL showed a stimulatory effect on GC numbers. Insulin-like growth factor-1 (IGF1)-stimulated cell proliferation was decreased after exposure to β-ZEA alone at 5.0 μg/mL and FB1 with α-ZEA and β-ZEA at the same concentration. Regarding steroid production, FB1 at 30 ng/mL and 100 ng/mL amplified the inhibitory effect of β-ZEA (30 ng/mL) on estradiol (E2) production, while FB1 alone increased (P < 0.05) IGF1-induced E2 production. α-ZEA alone decreased (P < 0.05) E2 production, whereas β-ZEA alone and in combination with FB1 decreased (P < 0.05) E2 production. These studies indicate for the first time that the Fusarium mycotoxin FB1 along with other mycotoxins can affect GC proliferation and steroid production, which ultimately could influence reproductive function in cattle.