Differential Expression of Circadian Clock Genes in the Bovine Neuroendocrine Adrenal System.

Knowledge of circadian rhythm clock gene expression outside the suprachiasmatic nucleus is increasing. The purpose of this study was to determine whether expression of circadian clock genes differed within or among the bovine stress axis tissues (e.g., amygdala, hypothalamus, pituitary, adrenal cortex, and adrenal medulla). Tissues were obtained at an abattoir from eight mature nonpregnant Brahman cows that had been maintained in the same pasture and nutritional conditions. Sample tissues were stored in RNase-free sterile cryovials at -80 °C until the total RNA was extracted, quantified, assessed, and sequenced (NovaSeq 6000 system; paired-end 150 bp cycles). The trimmed reads were then mapped to a Bos taurus (B. taurus) reference genome (Umd3.1). Further analysis used the edgeR package. Raw gene count tables were read into RStudio, and low-expression genes were filtered out using the criteria of three minimum reads per gene in at least five samples. Normalization factors were then calculated using the trimmed mean of M values method to produce normalized gene counts within each sample tissue. The normalized gene counts important for a circadian rhythm were analyzed within and between each tissue of the stress axis using the GLM and CORR procedures of the Statistical Analysis System (SAS). The relative expression profiles of circadian clock genes differed (p < 0.01) within each tissue, with neuronal PAS domain protein 2 (NPAS2) having greater expression in the amygdala (p < 0.01) and period circadian regulator (PER1) having greater expression in all other tissues (p < 0.01). The expression among tissues also differed (p < 0.01) for individual circadian clock genes, with circadian locomotor output cycles protein kaput (CLOCK) expression being greater within the adrenal tissues and nuclear receptor subfamily 1 group D member 1 (NR1D1) expression being greater within the other tissues (p < 0.01). Overall, the results indicate that within each tissue, the various circadian clock genes were differentially expressed, in addition to being differentially expressed among the stress tissues of mature Brahman cows. Future use of these findings may assist in improving livestock husbandry and welfare by understanding interactions of the environment, stress responsiveness, and peripheral circadian rhythms.

The Gluteus Medius Experiences Significant Excursion With Hip Flexion.

Purpose: To evaluate the effect of hip flexion and rotation on excursion of the gluteus medius (Gmed) and minimus (Gmin) myotendinous unit. Methods: Seven hips from 4 cadaveric specimens (males, 68.5 ± 18.3 years old) were dissected to isolate the Gmed posterior and lateral insertions and the Gmin proximal and distal insertions. Sutures were placed from tendon insertions through origins created in the iliac fossa to simulate the myotendinous unit. A load of 10N was applied. Myotendinous excursion was measured at 10° hip extension and 0°, 45°, and 90° of hip flexion in neutral rotation, and from maximum internal and external rotation in 90° flexion. The amount of abduction and rotation was also measured at each flexion position with 20N applied to each tendon. Repeated-measures analysis of variance with Tukey post hoc was used for statistics. Results: Gmed-lateral excursion ranged from 2.4 ± 0.4 mm in 10° of hip extension to 23.0 ± 1.5 mm in 90° of flexion (P < .001), and Gmed-posterior excursion ranged from 0.92 ± 0.5 mm in 10° of extension to 38.1 ± 1.1 mm in 90° of flexion (P < .001). Gmin excursion shortened with hip flexion from 4.2 ± 0.3 mm in 10° of extension to -0.2 ± 1.5 mm in 90° of flexion (Gmin-prox: P = .525, Gmin-distal: P < .001). At 90° flexion from maximum internal to maximum external rotation, Gmin-distal and proximal demonstrated a 92.6% and 51.3% increase in excursion, respectively (P < .001). Gmed-lateral and Gmed-posterior demonstrated 49.4% and 23.1% increase in excursion with external rotation, respectively (P < .001). Conclusions: The Gmed myotendinous unit undergoes significant excursion with hip flexion, whereas both Gmed and Gmin had significant excursion with femoral external rotation in 90° flexion. Clinical Relevance: It is important to understand whether active or passive hip flexion or rotation in the early postoperative period causes excessive strain to an abductor tendon repair. We found that consideration should be given to limit flexion after Gmed repair and external rotation after Gmed or Gmin repairs.

Biomechanical characteristics of a new looping stitch versus the classic Krackow stitch for distal biceps fixation.

Background: The purpose of this study was to quantify the biomechanical characteristics of a new looping stitch, developed with the concepts of a looping, locking stitch that decreases needle penetrations of the tendon, and compare it to a classic Krackow stitch for distal biceps suture-tendon fixation. Methods: The Krackow stitch with No. 2 braided suture and the looping stitch with a No. 2 braided suture loop attached to a 25-mm-length by 1.3-mm-width polyblend suture tape were compared. The Looping stitch was performed with single strand locking loops and wrapping suture around the tendon, resulting in half the needle penetrations through the graft compared to the Krackow stitch. Ten matched pairs of human distal biceps tendons were used. One side of each pair was randomly assigned to either the Krackow or the looping stitch, and the contralateral side was used for the other stitch. For biomechanical testing, each construct was preloaded to 5 N for 60 seconds, followed by cyclic loading to 20 N, 40 N, and 60 N for 10 cycles each, and then loaded to failure. The deformation of the suture-tendon construct, stiffness, yield load, and ultimate load were quantified. Comparisons between the Krackow and looping stitches were performed with a paired t-test using P < .05 as statistically significant. Results: The Krackow stitch and looping stitch had no significant difference in stiffness, peak deformation, or nonrecoverable deformation after 10 cycles of loading to 20 N, 40 N, and 60 N. There was no difference between the Krackow stitch and looping stitch in load applied to displacement of 1 mm, 2 mm, and 3 mm. The ultimate load showed that the looping stitch was significantly stronger compared to the Krackow stitch (Krackow stitch: 223.7 ± 50.3 N; looping stitch: 312.7 ± 53.8 N) (P = .002). The failure modes were either suture breakage or tendon cut through. For the Krakow stitch, there was 1 suture breakage and 9 tendons cut through. For the looping stitch, there were five suture breakages, and five tendons cut through. Conclusions: With fewer needle penetrations, incorporation of 100% of the tendon diameter, and a higher ultimate load to failure compared to the Krackow stitch, the Looping stitch may be a viable option to reduce deformation, failure, and cut-out of the suture-tendon construct.

Comparison of Telomere Length in Age-Matched Primiparous and Multiparous Brahman Cows.

Physiological and psychological stressors have been associated with the attrition of telomeres, which are the protective caps of chromosomes. This study compares the telomere length (TL) in 4-year-old Brahman cows grouped by the first parity (n = 8) and the second parity (n = 11). The cows were bled via jugular venipuncture, weighed, and had their body condition scores recorded at Day -28 prior to calving and at Day + 7 and Day + 28 post-calving. The duration of labor (Dlabor) and parturition ease were recorded. The peripheral leukocytes were isolated, the leukocyte blood count with differential was recorded, and the genomic DNA was extracted. The relative quantity of telomere products, which is proportional to the average TL, was determined via multiplex quantitative PCR using the ratio (T/S ratio) of bovine telomere and ß-globulin DNA. Standards of the bovine telomere (1012-107 dilution series) and ß-globulin (109-104 dilution series) genes were utilized to produce relative copy numbers. The samples were assayed in triplicate and were included if the triplicate Cq difference was less than 0.25 cycles. The parity was the fixed effect, and the random effects included the sire and day repeated with the cow as the subject. Statistical significance was not observed in the leukocyte number or type (p > 0.1). A reduction in the TL of approximately 9225 telomeric copies was found between Parity 1 and Parity 2 (p = 0.02). A trend was found between the TL and Dlabor (p = 0.06). The stress of parturition and raising the first calf of a cow's life may be responsible for TL attenuation. Parity may be considered a stressor of cow longevity.

Perfusion and Ultrasonication Produce a Decellularized Porcine Whole-Ovary Scaffold with a Preserved Microarchitecture.

The application of decellularized scaffolds for artificial tissue reconstruction has been an approach with great therapeutic potential in regenerative medicine. Recently, biomimetic ovarian tissue reconstruction was proposed to reestablish ovarian endocrine functions. Despite many decellularization methods proposed, there is no established protocol for whole ovaries by detergent perfusion that is able to preserve tissue macro and microstructure with higher efficiency. This generated biomaterial may have the potential to be applied for other purposes beyond reproduction and be translated to other areas in the tissue engineering field. Therefore, this study aimed to establish and standardize a protocol for porcine ovaries' decellularization based on detergent perfusion and ultrasonication to obtain functional whole-ovary scaffolds. For that, porcine ovaries (n = 5) were perfused with detergents (0.5% SDS and 1% Triton X-100) and submitted to an ultrasonication bath to produce acellular scaffolds. The decellularization efficiency was evaluated by DAPI staining and total genomic DNA quantification. ECM morphological evaluation was performed by histological, immunohistochemistry, and ultrastructural analyses. ECM physico-chemical composition was evaluated using FTIR and Raman spectroscopy. A cytocompatibility and cell adhesion assay using murine fibroblasts was performed. Results showed that the proposed method was able to remove cellular components efficiently. There was no significant ECM component loss in relation to native tissue, and the scaffolds were cytocompatible and allowed cell attachment. In conclusion, the proposed decellularization protocol produced whole-ovaries scaffolds with preserved ECM composition and great potential for application in tissue engineering.

Hexavalent Chromium Disrupts Oocyte Development in Rats by Elevating Oxidative Stress, DNA Double-Strand Breaks, Microtubule Disruption, and Aberrant Segregation of Chromosomes.

Environmental and occupational exposure to hexavalent chromium, Cr(VI), causes female reproductive failures and infertility. Cr(VI) is used in more than 50 industries and is a group A carcinogen, mutagenic and teratogenic, and a male and female reproductive toxicant. Our previous findings indicate that Cr(VI) causes follicular atresia, trophoblast cell apoptosis, and mitochondrial dysfunction in metaphase II (MII) oocytes. However, the integrated molecular mechanism of Cr(VI)-induced oocyte defects is not understood. The current study investigates the mechanism of Cr(VI) in causing meiotic disruption of MII oocytes, leading to oocyte incompetence in superovulated rats. Postnatal day (PND) 22 rats were treated with potassium dichromate (1 and 5 ppm) in drinking water from PND 22-29 and superovulated. MII oocytes were analyzed by immunofluorescence, and images were captured by confocal microscopy and quantified by Image-Pro Plus software, Version 10.0.5. Our data showed that Cr(VI) increased microtubule misalignment (~9 fold), led to missegregation of chromosomes and bulged and folded actin caps, increased oxidative DNA (~3 fold) and protein (~9-12 fold) damage, and increased DNA double-strand breaks (~5-10 fold) and DNA repair protein RAD51 (~3-6 fold). Cr(VI) also induced incomplete cytokinesis and delayed polar body extrusion. Our study indicates that exposure to environmentally relevant doses of Cr(VI) caused severe DNA damage, distorted oocyte cytoskeletal proteins, and caused oxidative DNA and protein damage, resulting in developmental arrest in MII oocytes.

Inter-Individual Variation in DNA Methylation Patterns across Two Tissues and Leukocytes in Mature Brahman Cattle.

Quantifying the natural inter-individual variation in DNA methylation patterns is important for identifying its contribution to phenotypic variation, but also for understanding how the environment affects variability, and for incorporation into statistical analyses. The inter-individual variation in DNA methylation patterns in female cattle and the effect that a prenatal stressor has on such variability have yet to be quantified. Thus, the objective of this study was to utilize methylation data from mature Brahman females to quantify the inter-individual variation in DNA methylation. Pregnant Brahman cows were transported for 2 h durations at days 60 ± 5; 80 ± 5; 100 ± 5; 120 ± 5; and 140 ± 5 of gestation. A non-transport group was maintained as a control. Leukocytes, amygdala, and anterior pituitary glands were harvested from eight cows born from the non-transport group (Control) and six from the transport group (PNS) at 5 years of age. The DNA harvested from the anterior pituitary contained the greatest variability in DNA methylation of cytosine-phosphate-guanine (mCpG) sites from both the PNS and Control groups, and the amygdala had the least. Numerous variable mCpG sites were associated with retrotransposable elements and highly repetitive regions of the genome. Some of the genomic features that had high variation in DNA methylation are involved in immune responses, signaling, responses to stimuli, and metabolic processes. The small overlap of highly variable CpG sites and features between tissues and leukocytes supports the role of variable DNA methylation in regulating tissue-specific gene expression. Many of the CpG sites that exhibited high variability in DNA methylation were common between the PNS and Control groups within a tissue, but there was little overlap in genomic features with high variability. The interaction between the prenatal environment and the genome could be responsible for the differences in location of the variable DNA methylation.

Improved Resource Utilization Using WALANT Hand Surgery in Adolescents.

BACKGROUND: Cost, efficiency, patient preference, and safety have driven utilization of wide awake, local anesthesia, no tourniquet (WALANT) in hand surgery. This is not well documented in adolescents. We hypothesize that the use of WALANT with adolescents reduced time spent in the operating room (OR) and in the hospital when compared with patients who underwent surgery with traditional anesthesia (TA). METHODS: After institutional review board approval, we performed a retrospective review of patients aged 10 to 17 who underwent surgery at a regional hospital system including the level 1 pediatric trauma hospital. Operative notes were assessed for use of WALANT. We excluded those operations not traditionally amenable to WALANT. Using a propensity matched cohort, hospital time, OR time, and perioperative complications were recorded and compared to evaluate efficiency and perioperative safety. RESULTS: There were 28 cases in the WALANT group and 28 cases in the TA group after excluding cases not amenable to WALANT, and cases were propensity matched. Although the operative time (incision to closure) was similar, for WALANT patients, the in-room to procedure time (15 vs 22 minutes), procedure end to out-room time (5 vs 10 minutes), total room time (52.81 vs 63.68), and length of hospital stay (222 vs 342 minutes) were shorter than patients in the TA group. CONCLUSION: Our case series demonstrates time-savings both in the OR and in the hospital overall. Avoiding TA when WALANT is feasible may result in significant savings to hospital systems, patients, and payers while also freeing up anesthesia staff and perioperative nurses.

DNA methylation patterns and gene expression from amygdala tissue of mature Brahman cows exposed to prenatal stress.

Prenatal stress can alter postnatal performance and temperament of cattle. These phenotypic effects may result from changes in gene expression caused by stress-induced epigenetic alterations. Specifically, shifts in gene expression caused by DNA methylation within the brain's amygdala can result in altered behavior because it regulates fear, stress response and aggression in mammals Thus, the objective of this experiment was to identify DNA methylation and gene expression differences in the amygdala tissue of 5-year-old prenatally stressed (PNS) Brahman cows compared to control cows. Pregnant Brahman cows (n = 48) were transported for 2-h periods at 60 ± 5, 80 ± 5, 100 ± 5, 120 ± 5, and 140 ± 5 days of gestation. A non-transported group (n = 48) were controls (Control). Amygdala tissue was harvested from 6 PNS and 8 Control cows at 5 years of age. Overall methylation of gene body regions, promoter regions, and cytosine-phosphate-guanine (CpG) islands were compared between the two groups. In total, 202 genes, 134 promoter regions, and 133 CpG islands exhibited differential methylation (FDR ≤ 0.15). Following comparison of gene expression in the amygdala between the PNS and Control cows, 2 differentially expressed genes were identified (FDR ≤ 0.15). The minimal differences observed could be the result of natural changes of DNA methylation and gene expression as an animal ages, or because this degree of transportation stress was not severe enough to cause lasting effects on the offspring. A younger age may be a more appropriate time to assess methylation and gene expression differences produced by prenatal stress.

Prenatal transportation stress did not impact ovarian follicle count for three generations of female Brahman offspring.

As prenatal transportation stress altered behavior and adrenal glucocorticoid secretion of calves, we hypothesized that prenatal transportation stress would decrease ovarian reserve size and negatively impact female offspring fertility. The impact of prenatal transportation stress on ovarian follicle numbers in female offspring for three generations was studied. Brahman cows were transported for 2 h on day 60 ± 5, 80 ± 5, 100 ± 5, 120 ± 5, and 140 ± 5 of gestation. Ovaries were collected from offspring of transported or non-transported dams at multiple ages. Primordial, primary, secondary, and antral follicles were histologically analyzed. Antral follicle numbers were determined by ultrasound in a subset of offspring. Numbers of primordial, primary, secondary, and antral follicles were analyzed using the MIXED procedure, while the CORR procedure of SAS was used to determine the correlation between follicles observed by ultrasonography and histology. There were no differences (P > 0.05) in the number of primordial, primary, secondary, antral, or total follicles observed histologically due to treatment. Younger females had significantly greater numbers of follicles than older females (P < 0.0001). Antral follicles tended to be correlated with total histological ovarian follicles (P = 0.10). There was no difference in the number of antral follicles observed at ultrasound due to treatment (P = 0.3147), or generation (P = 0.6005) when controlling for age at observation. These results show that short-term transportation stress during early- to mid-gestation did not impact fertility as measured by ovarian follicle numbers in female Brahman offspring for three generations.

Modifying the National Research Council weight gain model to estimate daily gain for stockers grazing bermudagrass in the southern United States.

The energy requirements, feed intake, and performance of grazing animals vary daily due to changes in weather conditions, forage nutritive values, and plant and animal maturity throughout the grazing season. Hence, realistic simulations of daily animal performance can be made only by the models that can address these changes. Given the dearth of simple, user-friendly models of this kind, especially for pastures, we developed a daily gain model for large-frame stockers grazing bermudagrass sCynodon dactylon (L.) Pers.], a widely used warm-season perennial grass in the southern United States. For model development, we first assembled some of the classic works in forage-beef modeling in the last 50 yr into the National Research Council (NRC) weight gain model. Then, we tested it using the average daily gain (ADG) data obtained from several locations in the southern United States. The evaluation results showed that the performance of the NRC model was poor as it consistently underpredicted ADG throughout the grazing season. To improve the predictive accuracy of the NRC model to make it perform under bermudagrass grazing conditions, we made an adjustment to the model by adding the daily departures of the modeled values from the data trendline. Subsequently, we tested the revised model against an independent set of ADG data obtained from eight research locations in the region involving about 4,800 animals, using 30 yr (1991-2020) of daily weather data. The values of the various measures of fit used, namely the Willmott index of 0.92, the modeling efficiency of 0.75, the R2 of 0.76, the root mean square error of 0.13 kg d-1, and the prediction error relative to the mean observed data of 24%, demonstrated that the revised model mimicked the pattern of observed ADG data satisfactorily. Unlike the original model, the revised model predicted more closely the ADG value throughout the grazing season. The revised model may be useful to accurately reflect the impacts of daily weather conditions, forage nutritive values, seasonality, and plant and animal maturity on animal performance.

Maternal nutrient restriction in late pregnancy programs postnatal metabolism and pituitary development in beef heifers.

Maternal undernutrition during pregnancy followed by ad libitum access to nutrients during postnatal life induces postnatal metabolic disruptions in multiple species. Therefore, an experiment was conducted to evaluate postnatal growth, metabolism, and development of beef heifers exposed to late gestation maternal nutrient restriction. Pregnancies were generated via transfer of in vitro embryos produced using X-bearing sperm from a single Angus sire. Pregnant dams were randomly assigned to receive either 100% (control; n = 9) or 70% (restricted; n = 9) of their total energy requirements from gestational day 158 to parturition. From post-natal day (PND) 301 until slaughter (PND485), heifers were individually fed ad libitum in a Calan gate facility. Calves from restricted dams were lighter than controls at birth (P<0.05) through PND70 (P<0.05) with no difference in body weight from PND105 through PND485 (P>0.10). To assess pancreatic function, glucose tolerance tests were performed on PND315 and PND482 and a diet effect was seen with glucose area under the curve being greater (P<0.05) in calves born to restricted dams compared to controls. At slaughter, total internal fat was greater (P<0.05) in heifers born to restricted dams, while whole pituitary weight was lighter (P<0.05). Heifers from restricted dams had fewer growth hormone-positive cells (somatotrophs) compared to controls (P<0.05). Results demonstrate an impaired ability to clear peripheral glucose in heifers born to restricted dams leading to increased deposition of internal fat. A reduction in the number of somatotrophs may contribute to the adipogenic phenotype of heifers born to restricted dams due to growth hormone's known anabolic roles in growth, lipolysis, and pancreatic islet function.

Genome-wide DNA methylation alteration in prenatally stressed Brahman heifer calves with the advancement of age.

Possible phenotypic impairments associated with maternal stress during gestation in beef cattle may be explained by epigenetic effects. This study examined the impact of prenatal transportation stress on DNA methylation of lymphocytes of Brahman cows over the first 5 years of life. Methylation analysis through reduced representation bisulphite sequencing was conducted on DNA from lymphocytes from 28 paired samples from 6 prenatally stressed (PNS) and 8 control (Control) females obtained initially when they were 28 days of age and 5 years of age. There were 14,386 CpG (C = cytosine; p = phosphate; G = guanine) sites differentially methylated (P < 0.01) in 5-yr-old Control cows compared to their lymphocyte DNA at 28 days of age, this number was slightly decreased in 5-yr-old PNS with 13,378 CpG sites. Only 2,749 age-related differentially methylated CpG sites were seen within PNS females. There were 2,637 CpG sites differentially methylated (P < 0.01) in PNS cows relative to Controls at 5 years of age. There were differentially methylated genes in 5-yr-old cows that contributed similarly to altered gene pathways in both treatment groups. Canonical pathways altered in PNS compared to Control cows at 5 years of age were mostly related to development and growth, nervous system development and function, and immune response. Prenatal stress appeared to alter the epigenome in Brahman cows compared to Control at 5 years of age, which implies a persistent intervention in DNA methylation in lymphocytes, and may confer long-lasting effects on gene expression, and consequently relevant phenotypic changes.

Modification of the summative equation to estimate daily total digestible nutrients for bermudagrass pasture.

In forage-animal nutrition modeling, diet energy is estimated mainly from the forage total digestible nutrients (TDN). As digestibility trials are expensive, TDN is usually estimated using summative equations. Early summative equations assumed a fixed coefficient to compute digestible fiber using the lignin-to-neutral detergent fiber (NDF) ratio. Subsequently, a structural coefficient (φ) was added to the summative equations to reflect an association between lignin and cell wall components. Additional modifications to the summative equations assumed a constant φ value, and they have been used as a standard method by many commercial laboratories and scientists. For feeds with nutritive values that do not change much over time, a constant φ value may suffice. However, for forages with nutritive values that keep changing during the grazing season owing to changes in weather and plant maturity, a constant φ value may add a systematic bias to prediction because it is associated with the variable lignin-to-NDF ratio. In this study, we developed a model to estimate φ as a function of the day of the year by using the daily TDN values of bermudagrass [Cynodon dactylon (L.) Pers.], a popular warm-season perennial grass in the southern United States. The variable φ model was evaluated by using it in the TDN equation and comparing the estimated values with the observed ones obtained from several locations. Values of the various measures of fit used-the Willmott index (WI), the modeling efficiency (ME), R2, root mean square error (RMSE), and percent error (PE)-showed that using the variable φ vis-à-vis the constant φ improved the TDN equation significantly. The WI, ME, R2, RMSE, and PE values of 0.94, 0.80, 0.80, 2.5, and 4.7, respectively, indicated that the TDN equation with the variable φ model was able to mimic the observed values of TDN satisfactorily. Unlike the constant φ, the variable φ predicted more closely the forage nutritive value throughout the grazing season. The variable φ model may be useful to forage-beef modeling in accurately reflecting the impacts of plant maturity and weather on daily forage nutritive value and animal performance.

Temperament influences mitochondrial capacity in skeletal muscle from 8 through 18 mo of age in Brahman heifers.

Temperamental cattle tend to yield carcasses of poorer quality, and Brahman cattle are reportedly more temperamental than non-indicus cattle breeds. A potential link between temperament and product quality may be mitochondrial activity. We hypothesized that mitochondrial measures would be greater in temperamental compared with calm heifers and that the relationships between temperament and mitochondria would persist as heifers age. Serum cortisol and skeletal muscle (longissimus thoracis [LT] and trapezius [TRAP]) mitochondrial profiles and antioxidant activities were quantified from the same calm (n = 6) and temperamental (n = 6) Brahman heifers at 8, 12, and 18 mo of age. Data were analyzed using a mixed model ANOVA in SAS (9.4) with repeated measures. Serum cortisol was greater in temperamental compared with calm heifers throughout the study (P = 0.02). Mitochondrial volume density (citrate synthase [CS] activity) increased over time (P < 0.0001) but was similar between temperament and muscle groups. Mitochondrial function (cytochrome c oxidase activity) was greatest in the temperamental LT at 8 mo of age (P ≤ 0.0006), greatest in the temperamental TRAP at 18 mo of age (P ≤ 0.003), and did not differ by temperament at 12 mo of age. Integrative (relative to tissue wet weight) mitochondrial oxidative phosphorylation capacity with complex I substrates (PCI), PCI plus complex II substrate (PCI+II), noncoupled electron transfer system capacity (ECI+II), and E with functional complex II only (ECII) were greater in the TRAP than LT for calm heifers at all ages (P ≤ 0.002), but were similar between muscle groups in temperamental heifers. Overall, calm heifers tended to have greater intrinsic (relative to CS activity) PCI and flux control of PCI+II (P ≤ 0.1) than temperamental heifers, indicating greater utilization of complex I paired with greater coupling efficiency in calm heifers. Within the LT, integrative PCI+II was greater (P = 0.05) and ECI+II tended to be greater (P = 0.06) in temperamental compared with calm heifers. From 8- to 18-mo old, glutathione peroxidase (GPx) activity decreased (P < 0.0001) and superoxide dismutase activity increased (P = 0.02), and both were similar between muscle groups. The activity of GPx was greater in temperamental compared with calm heifers at 8 (P = 0.004) but not at 12 or 18 mo of age. These results detail divergent skeletal muscle mitochondrial characteristics of live Brahman heifers according to temperament, which should be further investigated as a potential link between temperament and product quality.

l-Carnitine Supplementation during In Vitro Maturation and In Vitro Culture Does not Affect the Survival Rates after Vitrification and Warming but Alters Inf-T and ptgs2 Gene Expression.

l-carnitine is a potent antioxidant used for in vitro culture systems. Controversial results have been reported using l-carnitine in culture medium at different stages of in vitro bovine embryo production. Cumulus-oocyte complexes (n = 843) were in vitro-fertilized and cultured and added (treatment group) or not added (control group) with l-carnitine. At day three of culture, each group was subdivided into two subgroups receiving no l-carnitine (group 1), 3.8 mM l-carnitine added during in vitro maturation (group 2), 1.5 mM added during the in vitro culture (group 3), and 3.8 mM and 1.5 mM added during the maturation and culture, respectively (group 4). At day 8, blastocyst embryos were examined for mitochondrial activity, the presence of lipid droplets, total cell number, gene expression, and cryotolerance by vitrification. The data were analyzed with a one-way analysis of variance. l-carnitine added in the late in vitro culture significantly reduced mitochondrial activity and lipid content, and upregulated ifn-τ and ptgs2 gene expression compared to controls (p < 0.05). l-carnitine supplementation did not significantly affect the embryo rate production or survival rate after vitrification and warming (p > 0.05). l-carnitine supplementation significantly improved embryo potential to develop viable pregnancies in agreement with a study reporting improved pregnancy rates.

Primary Extramedullary, Extradural Cervical Spine Seminoma.

While extragonadal seminomas resulting in spinal cord compression are rarely reported in the literature, most have been treated with surgical decompression followed by radiation therapy. In this report, we present the unique and interesting case of a 38-year-old man who initially presented as an outpatient with a chief complaint of axial neck pain and lateral thoracic wall pain. After an extensive malignancy workup, he was diagnosed with a primary cervical spine seminoma and was treated with a C6-T1 laminectomy with posterior spinal instrumentation from C5 to T2. He has since undergone chemotherapy with cisplatin, vinblastine, and bleomycin, and at 24-month follow-up, he remains asymptomatic with no signs of recurrent disease.

Superior Capsular Reconstruction with Achilles Tendon Allograft.

Superior capsular reconstruction (SCR) has become an increasingly popular choice in the treatment of massive, irreparable rotator cuff tears, pseudoparalysis, and in cases in which revision of previous rotator cuff repair is indicated. The SCR procedure is intended to restore the superior stabilizing forces of a deficient rotator cuff. This technique is accomplished by substituting an autograft or allograft between the superior glenoid and the greater tuberosity of the humerus, thus keeping the humeral head centered in the glenoid during shoulder forward flexion and abduction. Since its advent, numerous techniques have been described for this procedure. A fascia lata autograft was initially described; however, many surgeons in the United States have advocated for the use of a humeral dermal allograft. Yet, biomechanical studies have demonstrated elongation and thinning of this material. Thus, the Achilles tendon allograft may be an attractive choice for SCR, given its previous success with ligamentous and tendinous reconstructions. In this article, we present our technique of SCR using an Achilles tendon allograft.

Efficient correction of a deleterious point mutation in primary horse fibroblasts with CRISPR-Cas9.

Phenotypic selection during animal domestication has resulted in unwanted incorporation of deleterious mutations. In horses, the autosomal recessive condition known as Glycogen Branching Enzyme Deficiency (GBED) is the result of one of these deleterious mutations (102C > A), in the first exon of the GBE1 gene (GBE1102C>A). With recent advances in genome editing, this type of genetic mutation can be precisely repaired. In this study, we used the RNA-guided nuclease CRISPR-Cas9 (clustered regularly-interspaced short palindromic repeats/CRISPR-associated protein 9) to correct the GBE1102C>A mutation in a primary fibroblast cell line derived from a high genetic merit heterozygous stallion. To correct this mutation by homologous recombination (HR), we designed a series of single guide RNAs (sgRNAs) flanking the mutation and provided different single-stranded donor DNA templates. The distance between the Cas9-mediated double-stranded break (DSB) to the mutation site, rather than DSB efficiency, was the primary determinant for successful HR. This framework can be used for targeting other harmful diseases in animal populations.

"Purse-String" Capsular Closure for Decreasing Dislocation Rates in Proximal Femur Replacements.

Hip joint dislocation is the most common complication after a proximal femur replacement. As the utilization of proximal femur replacements continues to increase, it becomes imperative for surgeons to find the optimal method to decrease postoperative dislocation and its sequelae. These cases often involve extensive soft-tissue deficits that require reconstruction to provide postoperative strength and stability. Patients report good functional outcomes; however, dislocation remains a concern. Although "described" previously in the literature, the authors illustrate the "purse-string" hip joint capsular closure technique to help other surgeons understand it and apply to their practice as deemed necessary. We also present the senior author's results with using a modified version of the "purse-string" hip joint capsular closure technique.