Comparison of turbidometric immunoassay, refractometry, and gamma-glutamyl transferase to radial immunodiffusion for assessment of transfer of passive immunity in high-risk beef calves.

BACKGROUND: Attainment of adequate transfer of passive immunity (TPI) is critical to health of calves; however, studies comparing available tools for measurement of TPI in individual beef animals are limited. OBJECTIVES: To report agreement between 4 tests evaluating individual TPI status in beef calves. ANIMALS: One hundred ninety-six beef calves born to cows and heifers presenting for calving management or dystocia. METHODS: Retrospective study to assess serum immunoglobulin (IgG) concentrations via turbidimetric immunoassay (TI), gamma-glutamyl transferase (GGT), serum total protein (TP), and single radial immunodiffusion (RID; reference standard). Test agreement was evaluated using Passing-Bablok regression, Bland-Altman analysis, Cohen's kappa, and receiver operating characteristic (ROC) curves with and without covariate adjustment to determine optimal thresholds. RESULTS: Correlation between RID and test results varied: TI, ρ = 0.757; TP, ρ = 0.715; GGT: ρ = 0.413. For the TI compared to RID, regression analysis identified a constant (intercept = -0.51 [CI: -2.63, 3.05]) and proportional (slope = 1.87 [CI: 1.69, 2.08]) bias. Based on ROC, TI concentrations of ≤9.89 and ≤13.76 g/L, and TP concentrations of ≤5.5 and ≤6.0 g/dL, indicated IgG concentrations <18.0 and <25.0 g/L, respectively. CONCLUSIONS AND CLINICAL IMPORTANCE: Within this cohort of calves, TI demonstrated the best correlation with RID; however, significant bias was identified which led to frequent underestimation of IgG concentration. Serum total protein demonstrated less correlation with RID but had less misclassification than TI. Both TI and TP demonstrated less correlation for calves that received colostrum replacement prompting clinical awareness of colostrum type when evaluating individual TPI in beef calves.

Comparison of turbidometric immunoassay and brix refractometry to radial immunodiffusion for assessment of colostral immunoglobulin concentration in beef cattle.

BACKGROUND: Colostral immunoglobulin G (IgG) concentration is critical to the attainment of adequate transfer of passive immunity in cattle, however, studies comparing available tools for measurement of colostral IgG concentration in beef cattle are limited. OBJECTIVES: To report the agreement between 3 commercially available tests for evaluating IgG concentration in beef colostrum. ANIMALS: Two hundred six beef-breed cows hospitalized for calving management or dystocia. METHODS: Retrospective study to assess IgG of whole colostrum measured stall-side via turbidimetric immunoassay (TI) and brix refractometry (BRIX), compared to fat separated (FS) analysis via single radial-immunodiffusion (RID; reference standard), TI-FS and BRIX-FS. Test performance was assessed using Passing Bablock regression, Bland-Altman analysis, and area under the curve to determine optimal thresholds. RESULTS: Correlation between RID and TI-FS, BRIX-FS, or BRIX was similar (Spearman's ρ = 0.717, 0.715, 0.716, respectively) but correlation for TI was poor (ρ = 0.586). Regression analysis identified a substantial constant (-214.75 [CI: -272.03 to -178.07]) and proportional (13.24 [CI: 11.81-15.37]) bias between the RID and TI-FS which was similar for TI. TI-FS concentrations of 28.47, 38.75, and 50.62 g/L, BRIX-FS of ≤21.9%, ≤24.0%, and ≤27.4%, and BRIX of ≤21.3%, ≤23.8%, and ≤26.4% indicated IgG concentrations <50, <100, and <150 g/L, respectively; appropriate cutoffs for TI could not be generated. CONCLUSIONS AND CLINICAL IMPORTANCE: Both TI and TI-FS demonstrated a large constant and proportional bias compared to RID; BRIX and BRIX-FS were well correlated with RID and remain a reliable method for estimation of colostral IgG concentration in beef cattle.

Effects of endogenous progesterone during ovarian follicle superstimulation on embryo quality and quantity in beef cows.

Despite modifications in techniques and protocols used for multiple ovulation embryo transfer in recent decades, transferrable quality embryos (TQE) has remained relatively unchanged. The objective of this study was to evaluate the effects of endogenous progesterone during beef cow superstimulation on embryo quality and quantity. Thirty non-pregnant beef cows were sorted into 1 of 5 replicates and randomly assigned to one of two groups: High Progesterone (HP) or Low Progesterone (LP). All cows, were pre-synchronized utilizing a 5-d CO-Synch + CIDR protocol. Nine days after estrus (d 0) with a corpus luteum present, all cows received ultrasound-guided dominant follicle ablation (DFA) and were administered a CIDR with LP cows also being administered PGF2α. All cows began a timed, 13-d, superovulation CIDR-based protocol and were artificially inseminated (AI) twice. Embryo were recovered and evaluated on each replicate 7 days after first AI. Blood samples were collected to evaluate progesterone (P4) and estradiol concentrations daily when cows were handled. Greater number of total embryos were recovered from the HP than the LP cows (19.26 vs. 10.74, P = 0.01). The HP cows also had greater number Stage 4 embryos along with more Quality Grade 3 and 4 embryos than the LP group (5.76 vs 2.20 P = 0.002; 1.87 vs 0.61, P = 0.01; 8.22 vs 2.89, P = 0.01, respectively). However, LP cows had a greater percentage overall of freezable embryos with a higher percentage of Grade 1 embryos (58.22 vs 37.32, P = 0.03) and a greater percentage of Stage 7 and 6 TQEs (18.47 vs 1.22, P = 0.01; 10.37 vs 3.19, P = 0.03). Serum P4 concentrations were greater on d 2-3 in the HP cows (P = 0.002). In addition, HP cows had greater concentrations of estradiol (P < 0.0001) on d 6. Comparatively, estradiol was greater in concentration in the LP cows (P ≤ 0.04) on d 2-4. In conclusion, removal of endogenous progesterone during superovulation may decrease the total number of embryos but increase the percentage of Grade 1 embryos and percentage of Stage 6 and 7 TQE in a single recovery.

Climatic factors affecting quantity and quality grade of in vivo derived embryos of cattle.

The present study investigated the effects of climatic variables on the quality grade and quantity of in vivo derived cattle embryos in the Midwestern United States. Climatic information included greatest and least daily temperature, average daily wind speed and average temperature-humidity index for each of the 765 records. The response variables included the number of ovarian structures, viable embryos, quality grade 1 embryos, quality grade 2 embryos, quality grade 3 embryos, freezable embryos (sum of quality grade 1 and quality grade 2 embryos), transferable embryos (sum of quality grade 1-3 embryos), degenerate embryos and unfertilized ova. Measures for variables among the breeds of donors and sires grouped by geographical origin were compared. A negative effect of greater temperatures during the early embryonic development stage tended (P < 0.10) to be associated with a decrease in the quality of embryos recovered. Interestingly, the greater the Temperature-Humidity Index (THI) during the early ovarian antral follicular development stage 40-45 days prior to ovulation was associated with a tendency for greater numbers of total number of freezable and transferable embryos recovered per uterine flushing (P < 0.10). Increased wind speed at the early antral follicular phase 40-45 days prior to ovulation was associated with an increase in the percentage of quality grade 1 embryos recovered (P < 0.05). Wind speed during the estrous synchronization period was also associated with a lesser number of embryos recovered (P < 0.05). This retrospective study confirms that climatic variables have significant effects on the in vivo production of cattle embryos and that wind speed should be considered in future analyses of factors affecting embryo quality.

Validation of a gauze sponge sampling methodology to detect Tritrichomonas foetus by real-time PCR.

Bovine trichomoniasis is a concern for the cattle industry. Advances in testing systems have increased the ability to detect the disease in bulls. However, the greatest limitation is proper collection of an adequate sample. The low repeatability observed with most sample collection techniques can cause false-negative results. The aim of our study was to validate a sample collection technique that increases diagnostic sensitivity and is easier and safer to collect than preputial scraping. Commercial bulls (n = 111) of unknown infection status were sampled for detection of Tritrichomonas foetus using 2 different collection methods: 1) preputial scraping with a dry insemination pipette and 2) penile sponging with a 16-ply gauze sponge. Preputial scraping samples were collected by vigorously scraping preputial and penile mucosa using a rigid insemination pipette while applying negative pressure with a syringe. Penile sponge samples were obtained by swabbing the penile and preputial mucosa with a gauze sponge during full extension of the penis. All samples were processed using a commercial medium and submitted under similar conditions for PCR testing. Positive PCR results were detected in 37 of 111 (33%) bulls using the preputial scraping technique; however, 39 of 111 (35%) were positive using the penile sponging technique. The Newton-Raphson algorithm predicted that the sensitivity of the preputial scraping method was 0.919 (95% CI: 0.689-0.983) and the sensitivity of the penile sponging was 0.949 (95% CI: 0.818-0.987). These data indicate that the penile sponging technique is a reliable alternative to the preputial scraping method.

Effects of label-dose permethrin administration in yearling beef cattle: I. Reproductive function and embryo quality of superovulated heifers.

The objective was to study the effects of a commercial pyrethroid-based pour-on product, permethrin, on reproductive performance in superovulated beef heifers by assessing steroid biosynthesis and embryo quality. Nonpregnant, yearling beef heifers (n = 10; 418 ± 33 kg; 5.5 ± 0.2 body conditioning scores) were assigned by body weight and breed to either (1) saline control or (2) permethrin pour-on administered at label dose (PYR). Superovulation was achieved on all heifers using a timed, 17-day, CIDR-based protocol with GnRH and PGF2α and decreasing total dosage of 240-mg FSH administered twice daily for 4 days. Heifers were artificially inseminated twice (at onset of estrus and 12 hours later) by same technician with frozen semen from single bull collection. To determine short- and long-term effects of permethrin on embryo quality and steroid biosynthesis, superovulation was initiated twice with collection of embryos occurring at 17 and 51 days after treatment. Embryos were recovered 6.5 days after first artificial insemination via nonsurgical flush and were evaluated by International Embryo Transfer Society standards. Blood was collected at standing estrus and day of embryo recovery. Estradiol (E2) and progesterone (P4) concentrations were analyzed via RIA. MIXED and GLIMMIX procedures of SAS were used to analyze continuous and categorical data, respectively. Heifer per flush was the experimental unit. Total embryos recovered did not differ because of treatment (P = 0.30), but did decrease in flush 2 compared with flush 1 (P = 0.02). Quality grade, total transferable quality embryos, and overall flush success did not differ because of treatment (P ≥ 0.16). However, transferable quality embryos were decreased in flush 2 compared with flush 1 (P = 0.05). Total unfertilized oocytes were greater in saline control (P = 0.04). The PYR heifers tended to have less total P4 (P = 0.15) and P4 per CL (P = 0.06) at recovery. E2 per ovulated follicle and E2 per total ovarian structure was greater in flush 2 (P ≤ 0.03) but did not differ because of treatment (P ≥ 0.23). In summary, these data indicate that permethrin administration at label dose in superovulated beef heifers has a tendency to reduce P4, but embryo quality is not affected.

Effects of label-dose permethrin administration in yearling beef cattle: I. Bull reproductive function and testicular histopathology.

Pyrethroid administration to a wide variety of laboratory animals has been shown to cause detrimental effects on male fertility, including sperm quality, by means of endocrine disruption. The objective of this experiment was to study the effects of a commercial, permethrin-containing pour-on product on reproductive variables and testicular histopathology of yearling beef bulls. Black Angus bulls (n = 60; aged 369 ± 17 days; 511 ± 33 kg; 6.2 ± 0.5 body condition scores) were assigned to either (1) saline control (CON) or (2) permethrin pour-on administered at label dose (PYR). Blood samples were collected, and industry standard breeding soundness examinations (BSE), via electroejaculation, were performed on all bulls at 5 days before and 14 days after treatment. Progressive sperm motility and eosin-nigrosin-stained sperm were analyzed using high-power phase-contrast microscopy. Plasma testosterone concentrations were analyzed via radioimmunoassay. Bulls were slaughtered at 34 days, and one testicle per bull was randomly collected for histologic examination. Change in sperm motility between BSEs was not different because of treatment; sperm morphology however improved across treatments, but PYR bulls had less improvement in percent of head (P < 0.001) sperm abnormalities compared to CON, resulting in less improvement of primary abnormalities (P = 0.04). Nonetheless, morphological differences did not change the overall outcome for satisfactory breeder status. Change in testosterone concentration did not differ because of treatment. Histopathologic examination identified that testicular degeneration and tubule diameter did not differ as a result of treatment. It should be noted, however, that degeneration score (higher score having more degeneration) was positively correlated with primary abnormalities (P < 0.01; r = 0.35) and negatively correlated with normal sperm cells (P < 0.001; r = -0.43). In summary, these data indicate that a single use of permethrin at label dose in yearling Angus bulls results in minimal detrimental effects on sperm morphology but not to a degree that impacts the ability of bulls to pass a standard BSE.

Identification of a Divergent Lineage Porcine Pestivirus in Nursing Piglets with Congenital Tremors and Reproduction of Disease following Experimental Inoculation.

Congenital tremors is a sporadic disease of neonatal pigs characterized by action-related repetitive myoclonus. A majority of outbreaks of congenital tremors have been attributed to an unidentified virus. The objectives of this project were to 1) detect potential pathogen(s) in samples from piglets with congenital tremors and 2) develop an infection model to reproduce disease. Using next-generation sequencing, a divergent lineage pestivirus was detected in piglets with congenital tremors. The virus was originally most closely related to a bat pestivirus but is now more closely related to a recently published novel porcine pestivirus provisionally named atypical porcine pestivirus. A quantitative real-time PCR detected the virus in samples from neonatal piglets with congenital tremors from two separate farms, but not in samples from unaffected piglets from the same farm. To fulfill the second objective, pregnant sows were inoculated with either serum containing the pestivirus or PBS (control) by intravenous and intranasal routes simultaneously with direct inoculation of fetal amniotic vesicles by ultrasound-guided surgical technique. Inoculations were performed at either 45 or 62 days of gestation. All sows inoculated with the novel pestivirus farrowed piglets affected with congenital tremors while PBS-inoculated control piglets were unaffected. Tremor severity for each piglet was scored from videos taken 0, 1 and 2 days post-farrowing. Tremor severity remained relatively constant from 0 to 2 days post-farrowing for a majority of piglets. The prevalence of congenital tremors in pestivirus-inoculated litters ranged from 57% (4 out of 7 affected piglets) to 100% (10 out of 10 affected piglets). The virus was consistently detected by PCR in tissues from piglets with congenital tremors but was not detected in control piglets. Samples positive by PCR in greater than 90% of piglets sampled included brainstem (37 out of 41), mesenteric lymph node (37 out of 41), tracheobronchial lymph node (37 out of 41), and whole blood (19 out of 20). Although the first description of congenital tremors was in 1922, this is the first reported reproduction of congenital tremors following experimental inoculation with a divergent lineage porcine pestivirus. Studies investigating disease mechanism, epidemiology, and diagnostic assay development are needed to better understand the pathophysiology of congenital tremors due to this pestivirus.