Calves severely affected by bovine respiratory disease have reduced protection against histone toxicity and exhibit lower complement activity.

Bovine respiratory disease (BRD) remains the greatest challenge facing the beef industry. Calves affected by BRD can manifest illness ranging from subclinical infection to acute death. In pathologies similar to BRD, extracellular histones have been implicated as major contributors to lung tissue damage. Histones are basic proteins responsible for DNA organization in cell nuclei, however when released extracellularly during cell injury or via neutrophil activation they become cytotoxic. Cattle suffering severe cases of BRD demonstrate reduced capacity to protect against the cytotoxic effects of histones, however, the protective mechanism(s) of serum remain(s) unknown. Therefore, the objective was to identify components within serum that contribute to protection against histone toxicity. Serum proteins from animals considered protective (P; N = 4) and nonprotective (NP; N = 4) against the toxic effects of histones were precipitated by the addition and incubation of exogenous histones. Proteins that interact with histones from both groups were isolated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and identified via label free "shotgun" proteomics. Sixteen candidate proteins increased by ≥2-fold change in P vs. NP animals were identified, with several associated with the complement system. A subsequent study was conducted to evaluate complement system activity and serum protective capacity against exogenous histones in feedlot heifers. Serum samples were collected from 118 heifer calves (BW at arrival = 229 ±â€…2.4 kg) at feedlot arrival. Animals were retrospectively assigned to groups consisting of: calves not requiring treatment with antibiotics for BRD (CONT; N = 80), calves treated once (1TRT; N = 21), calves treated twice (2TRT; N = 5), calves treated thrice (3TRT; N = 3), or calves that died from BRD within 1 wk of entering the feedlot (DA; N = 9). Serum from DA animals was less protective than CONT (P = 0.0005) animals against histone toxicity. Complement activity of DA animals was reduced compared to CONT (P = 0.0044) animals. Additionally, the use of both assays as a ratio resulted in increased ability to detect DA animals. Results suggest that cattle predisposed to severe cases of respiratory disease may have impaired complement activity presumably contributing to reduced protective capacity against histone toxicity.

Bovine respiratory disease (BRD) remains the leading cause of feedlot calf sickness and death. In respiratory disease affecting humans and mice, major tissue damage is caused by release of histones. Histones are proteins found in the nucleus of cells that condense DNA, however, cells that become damaged release histones extracellularly. Research has shown that calves with severe cases of BRD are less able to protect against the toxic effects of histones residing outside of the cell. It is speculated that components within the blood may interact with histones and confer protection from histone toxicity. This study evaluated serum from protective and nonprotective cattle against histone toxicity and identified 16 proteins that were elevated in protective animals. Several proteins were associated with the complement system of the innate immune system. To evaluate immune complement activity and protective capacity against histone toxicity, serum was collected from heifers at feedlot arrival. Calves suffering from a severe case of BRD demonstrated reduced capacity to protect against histone toxicity. Complement activity of calves severely affected with BRD was reduced as well. Results suggest that cattle susceptible to severe cases of BRD may have impaired complement activity likely contributing to reduced protective capacity against histone toxicity.

Factors important for bull purchasing decisions and management in extensive rangeland production systems of New Mexico: a producer survey.

There were 463,000 head of beef cows in New Mexico as of January 1, 2021 (NASS, 2020), down roughly 4% from 2020 (NASS, 2019). Frequent drought often results in herd reduction and loss of valuable genetic progress. Bull selection is critical due to their influence on herd development. A survey was conducted to identify traits important to bull selection in New Mexico. Surveys were collected digitally (n = 83) and via the mail (n = 74). Responses were largely by cow/calf producers averaging 57 ± 1 years old with 24 ± 1 years' experience. Survey respondents represented 4,384,296 acres of private owned, private leased, and leased public rangeland and irrigated pasture meadow in New Mexico and surrounding states. Average cow/calf operation size was 294 ± 39 head and average bull herd size was 21 ± 3 head. Average price paid for a bull in the past 2 years was $3,981 ± 213. Physical characteristics, individual bull performance information, and genetic information are all important traits to New Mexico bull buyers; however, most producers (56%) indicated that structural soundness was the most important factor influencing their selection decisions. Amongst expected progeny differences (EPDs), New Mexico producers consider the calving ease direct (CED) and birth weight (BW) EPDS to be most important (40% and 35%, respectively). Producers also indicated that multitrait selection indexes used by the American Angus Association were important to their selection decisions, with the beef value ($B) and weaned calf value ($W) indexes being cited most often (35% and 31%, respectively). Elements important to bull purchase include the bull's sale preview (87%), body condition score (86%), feed efficiency/average daily gain information (85%), and actual scrotal circumference (82%). Following purchase of a new bull, most (60%) keep the bull separate from the cow herd until the following breeding season, while the remaining 40% of producers turn newly purchased bulls out within 30 days of purchase. Sixty eight percent of producers evaluate semen quality annually or prior to the start of the breeding season. Interestingly, 39% of producers indicated they used reproductive technologies like artificial insemination and synchronization of estrus while most (80%) test for trichomoniasis. The primary factor influencing culling decisions is age, followed by soundness and fertility.

Bull selection and management in extensive rangeland production systems of California: a producer survey.

California's approximately 660,000 head of beef cattle are highly dependent on range bulls used to produce offspring that can perform across the state's diverse ecological regions. Bulls need to be functional on rugged coastal landscapes, rolling foothills, deserts, and in high-elevation terrain. Few data exist that indicate factors related to selection, maintenance, and longevity of bulls used in rangeland landscapes. Objectives herein, were to assess factors influencing bull purchasing, management, and culling decisions of California beef producers. Surveys were mailed to the California Cattlemen's Association membership (N = 1,410) with ~ 16% response rate (N = 227). Mean age and years of bull selection experience of respondents was 61 ± 1 yr and 27 ± 1 yr, respectively. Respondents managed cattle on a total of 694,949 hectare of owned, private leased, and leased public rangelands in California and surrounding states. Cow-calf herd size was 333 ± 92 head and bull battery averaged 18 ± 2 head with average bull longevity of 5 ± 1.3 yr. The average price paid for bulls in the last 2 yr was $5007 ± 163.33, while the highest price paid in the last 5 yr was $7291 ± 335.40. Survey responses were used to define current factors driving management after purchase and for subsequent breeding seasons. After bull purchase, 48% of producers turned bulls out directly with females, while 52% held bulls until the following breeding season. Additionally, most producers (70%) did not manage bulls to reduce condition after purchase. Semen quality analysis, a major component of a breeding soundness exam, was evaluated annually by 45% of respondents, while 20% of respondents never evaluated semen quality. Respondents indicated bull age (35%) and structural soundness (29%) as the most common factors for culling bulls. This research shows that despite the variability in operation demographics, there were similarities in beef bull selection and management across the state. Additionally, these data suggest the need for additional research focused on bull selection and management to maximize producer investment in reproduction.

A Case Study Using Accelerometers to Identify Illness in Ewes following Unintentional Exposure to Mold-Contaminated Feed.

Sensor technologies can identify modified animal activity indicating changes in health status. This study investigated sheep behavior before and after illness caused by mold-contaminated feed using tri-axial accelerometers. Ten ewes were fitted with HerdDogg biometric accelerometers. Five ewes were concurrently fitted with Axivity AX3 accelerometers. The flock was exposed to mold-contaminated feed following an unexpected ration change, and observed symptomatic ewes were treated with a veterinarian-directed protocol. Accelerometer data were evaluated 4 days before exposure (d -4 to -1); the day of ration change (d 0); and 4 days post exposure (d 1 to 4). Herddogg activity index correlated to the variability of minimum and standard deviation of motion intensity monitored by the Axivity accelerometer. Herddogg activity index was lower (p < 0.05) during the mornings (0800 to 1100 h) of days 2 to 4 and the evening of day 1 than days -4 to 0. Symptomatic ewes had lower activity levels in the morning and higher levels at night. After accounting for symptoms, activity levels during days 1 to 4 were lower (p < 0.05) than days -4 to 0 the morning after exposure. Results suggest real-time or near-real time accelerometers have potential to detect illness in ewes.

BEEF SPECIES-RUMINANT NUTRITION CACTUS BEEF SYMPOSIUM: Maternal immune modulation prior to embryo arrival in the uterus is important for establishment of pregnancy in cattle1.

In 1953, Sir Peter Medawar first recognized the allogeneic properties of a developing conceptus and rationalized that an "immune-tolerant" physiological state must exist during pregnancy. Early theories speculated that the conceptus evaded the maternal immune system completely, but 40 yr after Medawar's observations, Wegmann proposed that the maternal immune system shifts the cytokine profile away from inflammatory cytokine production when an embryo is present. The economic consequences and production losses of subfertile animals have been well documented in studies evaluating calving distribution. Despite advances in understanding infertility or subfertility, few technologies exist to identify subfertile animals or improve fertility beyond hormonal intervention associated with synchronization protocols. Work in rodents and some livestock species indicates that the uterine immune cell population shifts dramatically after copulation and these early immune-modulated events establish a receptive uterine environment. Clearly, as evident in embryo transfer, the presence of a conceptus is sufficient to establish communication for pregnancy establishment but does not rule out the importance of other physiological events to prime the maternal immune system prior to blastocyst arrival in the uterus. In support of this concept, work in our laboratory and by others has demonstrated that autologous intrauterine transfer of peripheral immune cells prior to embryo transfer can increase pregnancy rates and accelerate conceptus development in women and cattle. Understanding aberrant immune regulation in subfertile animals may provide markers for subfertility or targets for clinical intervention to enhance fertility, particularly when using reproductive technologies.

ASAS-SSR Triennial Reproduction Symposium: Looking Back and Moving Forward-How Reproductive Physiology has Evolved: WNTs role in bovine folliculogenesis and estrogen production.

Appreciation of mechanisms that affect steroidogenesis is critical to identifying compromising signals that may decrease reproductive efficiency. Follicle maturation and steroidogenesis requires coordinated actions from the pituitary gonadotropins and local ovarian signaling molecules. ß-Catenin (CTNNB1), the lynchpin molecule of canonical wingless-type mouse mammary tumor virus integration site (WNT) signaling, is required for maximal gonadotropin stimulation of steroid production from granulosa (GC) and luteal cells. WNTs are locally secreted glycoproteins involved in ovarian development and folliculogenesis. In cultured bovine GC, WNT2 and AKT mRNAs and CTNNB1 protein increase after FSH stimulation. Likewise, CTNNB1 protein is greater in large antral follicles with high intrafollicular estradiol concentrations, suggesting the hormonal milieu responsible for increased estradiol content modulates CTNNB1 accumulation. In addition, concurrent treatment of FSH and WNT3A in GC results in reduced steroidogenic enzymes and ovarian differentiation factors. It is likely that FSH regulation of WNT signaling establishes a negative feedback loop to ensure CTNNB1 remains controlled. To explore the mechanism resulting in this inhibitory effect, AKT pathway modulators were utilized and unveiled a requirement for AKT activity in FSH-mediated CTNNB1 accumulation. Cells treated with FSH, IGF-1, and IGF-1 + FSH had increased CTNNB1 protein accumulation compared with controls. Similarly, estradiol medium concentrations increased in treated cells compared with non-treated controls, while co-treatment of FSH and IGF-1 with the AKT inhibitor LY294002 reduced CTNNB1 and estradiol production. Subsequent studies evaluated whether FSH regulation of CTNNB1 occurs through a specific phosphorylation event. In bovine GC, phosphorylation of CTNNB1 at Ser-552 was demonstrated in FSH-treated cells, whereas IGF-1 treatment did not phosphorylate CTNNB1 Ser-552. Data indicate that in cattle phosphorylation on CTNNB1 Ser-552 is a protein kinase A (PKA) dependent, protein kinase B (AKT) independent event. Data suggest that CTNNB1 regulated by AKT is a fundamental component of FSH-induced estrogen production. However, AKT's role in estradiol synthesis does not appear to be through phosphorylation of CTNNB1 Ser-552. The complex interplay between FSH and ovarian WNT/CTNNB1 signaling is key to regulation of follicle maturation and steroidogenesis.

Evaluation of steroidogenic capacity after follicle stimulating hormone stimulation in bovine granulosa cells of Revalor 200® implanted heifers.

BACKGROUND: Heifers not used as breeding stock are often implanted with steroids to increase growth efficiency thereby altering hormone profiles and potentially changing the environment in which ovarian follicles develop. Because bovine granulosa cell culture is a commonly used technique and often bovine ovaries are collected from abattoirs with no record of implant status, the objective of this study was to determine if the presence of an implant during bovine granulosa cell development impacts follicle stimulating hormone-regulated steroidogenic enzyme expression. Paired ovaries were collected from 16 feedlot heifers subjected to 1 of 3 treatments: non-implanted (n = 5), Revalor 200 for 28 d (n = 5), or Revalor 200 for 84 d (n = 6). Small follicle (1 to 5 mm) granulosa cells were isolated from each pair and incubated with phosphate buffered saline (n = 16) or 100 ng/mL follicle stimulating hormone (n = 16) for 24 h. RESULTS: Granulosa cells of implanted heifers treated with follicle stimulating hormone produced medium concentrations of progesterone similar (P = 0.22) to non-implanted heifers, while medium estradiol concentrations were increased (P < 0.10) at 28 and 84 d compared to non-implanted heifers indicating efficacy of treatment. Additionally, real-time PCR analysis in response to follicle stimulating hormone treatment demonstrated a decrease in steroidogenic acute regulatory protein (P = 0.05) mRNA expression in heifers implanted for 84 d and an increase in P450 side chain cleavage mRNA in granulosa cells of heifers implanted for 28 (P < 0.10) or 84 d (P < 0.05) compared to non-implanted females. However, no difference in expression of 3-beta-hydroxysteroid dehydrogenase (P = 0.57) and aromatase (P = 0.23) were demonstrated in implanted or non-implanted heifers. CONCLUSIONS: These results indicate follicles which develop in the presence of high concentrations of androgenic and estrogenic steroids via an implant tend to demonstrate an altered capacity to respond to follicle stimulating hormone stimulation. Thus, efforts should be made to avoid the use of implanted heifers to study steroidogenesis in small follicle granulosa cell culture systems.

Canonical WNT signaling inhibits follicle stimulating hormone mediated steroidogenesis in primary cultures of rat granulosa cells.

Beta-catenin (CTNNB1), a key component of wingless-type mouse mammary tumor virus integration site family (WNT) signaling, participates in follicle stimulated hormone-mediated regulation of estrogen (E2) production. The purpose of these studies was to determine if CTNNB1's contribution to FSH-mediated steroidogenesis in primary rat granulosa cells was due in part to extracellular stimulation of the canonical WNT signaling pathway. To achieve this purpose, primary cultures of rat granulosa cells were exposed to vehicle or a canonical member of the WNT signaling pathway, WNT3A, before co-culture and in the presence or absence of FSH for 24 h. Activation of the canonical WNT signaling pathway was determined by dose-dependent induction of Axin2 mRNA expression and stimulation of the CTNNB1/T cell factor promoter-reporter TOPflash. WNT pathway induction was demonstrated at doses of 50 and 500 ng/mL of WNT3A. Granulosa cells treated with WNT3A in combination with FSH had enhanced CTNNB1/T cell factor transcriptional activity above cells treated with WNT3A alone. Steroidogenic enzymes and ovarian differentiation factor mRNAs were quantified via quantitative PCR. Expression of steroidogenic enzyme mRNAs aromatase (Cyp19a1), P450 side chain cleavage (Cyp11a1), and steroidogenic acute regulatory protein (Star) were increased following FSH treatment. Co-incubation of WNT3A and FSH reduced the ability of FSH to stimulate steroidogenic enzymes and subsequent E2 and progesterone (P4) production. Concomitant activation of FSH and WNT pathways results in marked reduction of ovarian differentiation factors, LH receptor (Lhcgr) and inhibin-alpha (Inha). Therefore, WNT inhibits FSH target genes and steroid production associated with maturation and differentiation of the ovarian follicle.

Effects of early conceptus signals on circulating immune cells: lessons from domestic ruminants.

While there are few similarities between mechanisms for extending corpus luteum (CL) function during early pregnancy in ruminants and primates, there is increasing evidence that conceptus-immune crosstalk in ruminants and primates affects the function of circulating immune cells at the very earliest stages of pregnancy. Most notable are changes in immune cell phenotypes with increased numbers of cells exhibiting the T regulatory phenotype and suppression of Th1 cytokines that promote tolerance to paternal alloantigens. Until recently, interferon τ produced by the ruminant trophectoderm was thought to act exclusively on the uterine endometrium; however, it is now clear that this unique embryonic interferon escapes the uterus and alters gene expression in the CL and in peripheral blood leukocytes (PBL). In fact, a large number of interferon-stimulated genes are now known to be increased during early pregnancy in PBL. What is not known is how this conceptus-immune system cross-talk affects maternal immune status outside the reproductive tract. It is attractive to hypothesize that some of these effects are designed to counter-balance progesterone-induced immunosuppression so as not to place the dam at a greater risk of infection on top of the tremendous stresses already induced by pregnancy. Furthermore, recent evidence suggests that pregnancy induced changes in peripheral immune cells may aid in orchestrating establishment of pregnancy. Existing evidence points toward a greater convergence of systemic immune responses to early pregnancy signaling between ruminants and primates.