Horn Bud Research Articles

Gene expression supports a single origin of horns and antlers in hoofed mammals

Horns, antlers, and other bony cranial appendages of even-toed hoofed mammals (ruminant artiodactyls) challenge traditional morphological homology assessments. Cranial appendages all share a permanent bone portion with family-specific integument coverings, but homology determination depends on whether the integument covering is an essential component or a secondary elaboration of each structure. To enhance morphological homology assessments, we tested whether juvenile cattle horn bud transcriptomes share homologous gene expression patterns with deer antlers relative to pig outgroup tissues, treating the integument covering as a secondary elaboration. We uncovered differentially expressed genes that support horn and antler homology, potentially distinguish them from non-cranial-appendage bone and other tissues, and highlight the importance of phylogenetic outgroups in homology assessments. Furthermore, we found differentially expressed genes that could support a shared cranial neural crest origin for horns and antlers and expression patterns that refine our understanding of the timing of horn and antler differentiation.

Histological characterisation of the horn bud region in 58 day old bovine fetuses.

The presence of horns in domestic ruminants, such as cattle, sheep and goats, has financial and welfare implications. The genetic interactions that lead to horn development are not known. Hornless, or polled, cattle occur naturally. The known causative DNA variants (Celtic, Friesian, Mongolian and Guarani) are in intergenic regions on bovine chromosome 1, but their functions are not known. It is thought that horns may be derived from cranial neural crest stem cells and the POLLED variants disrupt the migration or proliferation of these cells. Relaxin family peptide receptor 2 (RXFP2) is more highly expressed in developing horns in cattle compared to nearby skin and has been shown to play a role in horn development in sheep. However, the role of RXFP2 in horn formation is not understood. Histological analyses of cranial tissues from homozygous horned and polled cattle fetuses at day 58 of development was carried out to determine the differences in the structure of the horn bud region. Condensed cells were only observed in the horn bud mesenchyme of horned fetuses and could be the progenitor horn cells. The distribution of neural crest markers (SOX10 and NGFR) and RXFP2 between horned and polled tissues by immunohistochemistry was also analysed. However, SOX10 and NGFR were not detected in the condensed cells, and therefore, these cells are either not derived from the neural crest, or have differentiated and no longer express neural crest markers. SOX10 and NGFR were detected in the peripheral nerves, while RXFP2 was detected in peripheral nerves and in the horn bud epidermis. Previous research has shown that RXFP2 variants are associated with horn phenotypes in cattle an sheep. Therefore, the RXFP2 variants may affect the development of the epidermis or peripheral nerves in the horn bud.

Early pain experiences in dairy calves on pain sensitivity later in life

In humans, early painful experiences can increase pain sensitivity later in life, but little is known regarding this phenomenon in cattle. This study assessed if a painful event early in life affects later pain sensitivity in 40-d-old calves. Holstein calves (n = 26) were randomly assigned to control or treatment conditions, blocked within sex and birth weight. At 9.5 ± 1.8 d old, treatment calves had 1 horn bud disbudded using caustic paste, whereas control calves had 1 horn sham disbudded; in both cases, multimodal pain control was provided. All calves had the contralateral horn bud disbudded using a hot iron 4 wk later, again with multimodal pain control. Mechanical nociceptive responses were assessed weekly using an algometer applied adjacent to both horn buds and on the rump, beginning 3 d before the first disbudding and ending 30 d after the second disbudding. Following the second disbudding, both groups of calves showed evidence of increased sensitivity (i.e., algometer pressure declined 3.69 ± 0.60 √N to 2.13 ± 0.70 √N) on the contralateral bud, but there was no difference between the control group and the treatment groups. An interaction between treatment and time, likely driven by treatment differences, was found on the rump when tested 5 h after the second disbudding event. These responses are not consistent with the hypothesis that an early pain experience results in increased sensitivity to later painful experiences.

Behavioral changes in the first 3 weeks after disbudding in dairy calves

Hot-iron disbudding, the practice of cauterizing horn bud tissue to prevent horn growth in dairy calves, results in behavioral changes indicative of pain in the first few days after the procedure. However, few studies have quantified behavioral changes in the following weeks, while the burn wounds are still healing. Female Holstein calves were disbudded with a heated iron and pain relief (5.5 mL lidocaine cornual nerve block and 1 mg/kg oral meloxicam) at 4 to 10 d of age (n = 19) or not disbudded (n = 19). Calves wore ear tag accelerometers that reported the dominant behavior being performed at 1-min intervals from 3 to 21 d after disbudding. Compared with age-matched controls, disbudded calves tended to spend more time inactive throughout the observation period, ruminated less in the first 3 to 11 d after disbudding, and sucked more from a milk bottle beginning 5 d after disbudding until the end of the 21-d observation period. In addition to the accelerometer data, live observations of sleeping (using a behavioral proxy), lying, and ruminating were collected using instantaneous sampling at 5-min intervals for 24-h periods 3, 10, and 17 d after disbudding. Disbudded calves slept with their head down more on all live observation days and spent more time lying on the 17th d after disbudding, but ruminating did not differ compared with controls, in contrast to the accelerometer results. More time spent inactive, sleeping, and lying, and less time spent ruminating (as indicated by the accelerometer) can be interpreted as attempts to reduce painful stimulation of the disbudding wounds and allocate energy to healing. It is unclear whether the greater amount of sucking in the disbudded calves is nutritive (milk present) or non-nutritive (milk absent), as the algorithm did not distinguish the type of sucking, and further research is needed to explore the factors underlying this effect. We conclude that disbudding alters daily behavior patterns for at least 3 wk, far beyond the duration of recommended pain medication, raising additional welfare concerns about the procedure.

Investigating the use of local nerve blocks and general anaesthesia in reducing pain during and after disbudding procedure in goat kids

ABSTRACT The aim of this study was to compare the pain responses (as measured by noise and movement) during administration of local anaesthetic and during and after disbudding in goat kids. Eighty, seven- to ten-day-old, Saanen goat kids from one farm were enrolled and randomly assigned to one of four different methods of pain relief. Twenty kids had local anaesthetic (LA) applied at two sites per horn bud (LA group), 20 kids had LA applied to the two locations using a jet injector (JI group) and 20 kids were given a general anaesthetic (GA) using a combination of 0.02 mg/kg medetomidine and 2 mg/kg ketamine followed by a horn bud block applied as per the LA group (GA group). The remaining 20 kids had no treatment other than meloxicam (control group). Although responses between goat kids and at different time periods were variable, in comparison to the control group, GA eliminated the responses associated with injection of lignocaine and the responses during the period of disbudding, and provided a reduction in head scratches and shakes across multiple time periods.

Wound healing and pain sensitivity following caustic paste disbudding in dairy calves

Caustic paste disbudding is increasing in popularity on commercial dairy farms in the US, but little research has explored the pain and welfare implications beyond the acute period of this procedure. Researchers have reported it takes 7 to 9 wk, on average, for hot-iron disbudding wounds to re-epithelialize in dairy calves. Our objective was to describe wound healing and sensitivity following caustic paste disbudding. Jersey and Holstein female calves were disbudded using Dr. Naylor's caustic paste at 3 d of age (n = 18) while control calves received a sham procedure (n = 15). Before disbudding, calves received a local block and an NSAID. Calves ≥34 kg and <34 kg at birth had 0.3 or 0.25 mL of paste applied per unshaved horn bud, respectively. Following disbudding, wounds were scored 2x/wk for the presence/absence of 8 tissue categories including the final stages: new epithelium and fully healed. Control calves were removed from the experiment after 6 wk to be hot-iron disbudded and avoid more invasive procedures. Mechanical nociceptive threshold (MNT) measures were collected weekly to evaluate wound sensitivity until calves were removed from the study or wounds were healed. Wounds were slow to re-epithelialize (16.2 ± 5.7 wk, mean ± SD; range: 6.2-32.5 wk) and contract to be considered fully healed (18.8 ± 6 wk, mean ± SD; range: 8.7-34.1 wk). Compared with non-disbudded controls, paste calves exhibited lower MNT values for all 6 wk (mean ± SE, Control:1.46 ± 0.16; Paste:1.18 ± 0.12 N). These data indicate that wounds from caustic paste disbudding are more sensitive than undamaged tissue for at least 6 wk and take twice as long to heal compared with cautery methods described in the literature. In conclusion, caustic paste disbudding wounds took 18.8 wk to fully heal and were more sensitive than intact horn buds for 6 wk. Future work should examine if aspects of paste application (e.g., amount, time rubbed in, age, pain mitigation), could improve healing time and sensitivity.

Randomised Control Trial Investigating the Efficacy of Meloxicam and Sodium Salicylate Non-Steroidal Anti-Inflammatory Drugs for Calf Cautery Disbudding.

Disbudding calves using hot iron cautery is a routine management procedure to destroy the germinal cells around the horn bud in calves. It is recommended that NSAIDs are used in conjunction with local anaesthesia to reduce pain in calves during and after the procedure. In this study, two treatment groups were examined; calves in the positive control MEL group received subcutaneous meloxicam, and SAL calves received sodium salicylate orally for three days, both in addition to a local anaesthesia. Tri-axis accelerometers were attached to the calves, and DLWG (Daily Live Weight Gain) was measured. There was no significant difference between the treatment groups with regard to DLWG (p = 0.52), MI (motion index (p = 0.66)), lying bouts (p = 0.96) or lying times (p = 0.54). Given these findings, sodium salicylate may offer a lower-cost option for farmers when given at licensed doses compared to meloxicam, as well as providing a reduced-stress method of NSAID administration via an oral route. In addition, this study identified significant differences in activity in the time periods before and after disbudding, with MI (p < 0.01), lying bouts (p = 0.002) and lying times (p < 0.001) indicating changes in behaviour which extended to five days post disbudding.

Identification of Critical Genes for Ovine Horn Development Based on Transcriptome during the Embryonic Period.

Horns, also known as headgear, are a unique structure of ruminants. As ruminants are globally distributed, the study of horn formation is critical not only for increasing our understanding of natural and sexual selection but also for the breeding of polled sheep breeds to facilitate modern sheep farming. Despite this, a significant number of the underlying genetic pathways in sheep horn remain unclear. In this study, to clarify the gene expression profile of horn buds and investigate the key genes in horn bud formation, RNA-sequencing (RNA-seq) technology was utilized to investigate differential gene expression in the horn buds and adjacent forehead skin of Altay sheep fetuses. There were only 68 differentially expressed genes (DEGs) identified, consisting of 58 up-regulated genes and 10 down-regulated genes. RXFP2 was differentially up-regulated in the horn buds and had the highest significance (p-value = 7.42 × 10-14). In addition, 32 DEGs were horn-related genes identified in previous studies, such as RXFP2, FOXL2, SFRP4, SFRP2, KRT1, KRT10, WNT7B, and WNT3. Further, Gene Ontology (GO) analysis showed that the DEGs were mainly enriched with regard to growth, development, and cell differentiation. Pathway analysis revealed that the Wnt signaling pathway may be responsible for horn development. Further, through combining the protein-protein interaction networks of the DEGs, it was found that the top five hub genes, namely, ACAN, SFRP2, SFRP4, WNT3, and WNT7B, were also associated with horn development. Our results suggest that only a few key genes, including RXFP2, are involved in bud formation. This study not only validates the expression of candidate genes identified at the transcriptome level in previous studies but also provides new possible marker genes for horn development, which may promote our understanding of the genetic mechanisms of horn formation.

Topologically associating domains in the POLLED region are the same for Angus- and Brahman-specific Hi-C reads from F1 hybrid fetal tissue.

Horns, a form of headgear carried by Bovidae, have ethical and economic implications for ruminant production species such as cattle and goats. Hornless (polled) individuals are preferred. In cattle, four genetic variants (Celtic, Friesian, Mongolian and Guarani) are associated with the polled phenotype, which are clustered in a 300-kb region on chromosome 1. As the variants are intergenic, the functional effect is unknown. The aim of this study was to determine if the POLLED variants affect chromatin structure or disrupt enhancers using publicly available data. Topologically associating domains (TADs) were analyzed using Angus- and Brahman-specific Hi-C reads from lung tissue of an Angus (Celtic allele) cross Brahman (horned) fetus. Predicted bovine enhancers and chromatin immunoprecipitation sequencing peaks for histone modifications associated with enhancers (H3K27ac and H3K4me1) were mapped to the POLLED region. TADs analyzed from Angus- and Brahman-specific Hi-C reads were the same, therefore, the Celtic variant does not appear to affect this level of chromatin structure. The Celtic variant is located in a different TAD from the Friesian, Mongolian, and Guarani variants. Predicted enhancers and histone modifications overlapped with the Guarani and Friesian variants but not the Celtic or Mongolian variants. This study provides insight into the mechanisms of the POLLED variants for disrupting horn development. These results should be validated using data produced from the horn bud region of horned and polled bovine fetuses.

Pharmacokinetics and efficacy of a novel long-acting bupivacaine formulation for cornual nerve block in calves.

Local anesthetics are commonly used in farm animals to provide analgesia for painful procedures but can cause adverse effects at high systemic concentrations. The pharmacokinetics and efficacy of a long-acting sucrose acetate isobutyrate (SAIB) bupivacaine formulation following cornual nerve block in calves were compared to lidocaine. Fourteen calves were randomly assigned to one of the treatment groups (i) 5% Bupivacaine-SAIB (BUP-SAIB), n = 7; or (ii) 2% lidocaine (LID), n = 7. Cornual nerve block was performed, and duration of effective analgesia was evaluated by nociceptive threshold testing using a hand-held pressure algometer. Blood samples were collected at various time points and plasma concentrations were analyzed by HPLC. Pharmacokinetic parameters were calculated using a non-compartmental model. The mechanical nociceptive thresholds showed that the novel formulation could desensitize the skin around the horn bud for 18.77 ± 8.88 h (range 8-36 h), compared to 0.79 ± 0.34 h (range 0.5-1.5 h) with lidocaine. The mean maximum plasma concentration (Cmax) of bupivacaine was 152.03 (SD 37.34) ng/mL and its Tmax was 0.39 (SD 0.13) h. The half-life of elimination was 32.79 ± 11.00 h and the rate of clearance was 0.12 ± 0.03 L h-1. No toxicity signs were seen after treatment in either group. The novel formulation produced long-lasting analgesia of several times greater duration than that produced by lidocaine. This study showed that the safety and efficacy of the SAIB formulation justifies further studies in a larger population of animals.

Uncovering Novel Features of the Pc Locus in Horn Development from Gene-Edited Holstein Cattle by RNA-Sequencing Analysis.

The Polled Celtic (Pc) mutation locus is a genetically simple single mutation that is the best choice for breeding polled cattle using gene editing. However, the mechanism of the Pc locus for regulating horn development is unclear, so we used gene editing, somatic cell nuclear transfer and embryo transfer to obtain polled Holstein fetal bovine (gestation time 90 days) with a homozygous Pc insertion (gene-edited Holstein fetal bovine, EH) and the wild-type 90 days Holstein fetal bovine (WH) as controls. The hematoxylin-eosin (HE) staining results showed that, compared to the WH, the EH horn buds had no white keratinized projections or vacuolated keratinocytes and no thick nerve bundles under the dermal tissue. Furthermore, DNA sequencing results showed that the Pc locus was homozygously inserted into the fetal bovine genome. A total of 791 differentially expressed genes were identified by transcriptome sequencing analysis. Enrichment analysis and protein interaction analysis results of differentially expressed genes showed that abundant gene changes after Pc insertion were associated with the adhesion molecule regulation, actin expression, cytoskeletal deformation and keratin expression and keratinization. It was also noted that the results contained several genes that had been reported to be associated with the development of horn traits, such as RXFP2 and TWIST1. This study identified these changes for the first time and summarized them. The results suggested that the Pc mutant locus may inhibit neural crest cell EMT generation and keratin expression, leading to failures in neural crest cell migration and keratinization of the horn bud tissue, regulating the production of the polled phenotype.

Best-practice recommendations for cautery disbudding goat kids

Abstract Cautery disbudding is a painful husbandry practice routinely performed on goat kids using a hot cautery iron to destroy the horn buds and prevent horn growth. After a thorough review of the scientific literature on cautery disbudding of goat kids, it is apparent that there is considerable variation in disbudding practice in relation to training, kid-related factors (e.g. age, sex), iron-related factors (e.g. temperature, application time), procedure (e.g. horn bud removal), and pain mitigation, which all can impact the behaviour and welfare of goat kids. This case study provides best-practice recommendations for cautery disbudding goat kids. It is imperative that goat kid-specific training, with allowance for time and opportunities for practice, is provided to improve the technical ability of operators, and to reduce the potential for negative impacts on kid health and welfare. Procedural factors to be taken into consideration in preparation for disbudding include age of goat kid at disbudding, sex, temperature and application time of the iron, and whether the horn bud is removed. Goat kids typically are very vocal with rapid and intense movements during disbudding, and they perform head shaking and scratching following disbudding, which can be alleviated with appropriate pain management. Isoflurane or dexmedetomidine and non-steroidal anti-inflammatory drugs such as meloxicam can reduce pain, whereas lidocaine blocks have varying efficacy and cause pain to administer. Goat kid-focused best-practice recommendations for cautery disbudding can improve the health and welfare outcomes for goat kids.

Comparison of Three Anaesthetic Options to Reduce Acute Pain Response in Kid Goats

ABSTRACT Three options for anesthetizing the skin around the horn bud of dairy goat kids were explored. Forty-five <10-day-old Saanen goat kids from were randomly split into five treatment groups (topical anesthetic cream (TA), vapocoolant spray (VS), local anesthetic applied by jet injector (JI), control – no treatment but painful stimulus applied (C), sham – no treatment and touching sites with a finger. The painful stimulus was multiple needle pricks on the skin around the horn bud. The outcome variables measured were heart rate movement, and vocalization during treatment application and administration of a painful stimulus around the horn bud. Heart rates were greater during application of a VS compared to TA.Neither the TA nor the VS appeared to have any effect on the response to the painful stimulus. Kids in the JI group had a 96% reduced odds of expressing a marked pain response in comparison to TA group and an 83% reduction in the odds of a high movement grade during a painful procedure in comparison to the combined results of the other three treatment groups.

Effect of plane of nutrition and analgesic drug treatment on wound healing and pain following cautery disbudding in preweaning dairy calves

The objective of this study was to determine the effect of a biologically normal plane of nutrition compared with a limited plane on the primary outcome wound healing, and one dose of nonsteroidal anti-inflammatory drug (NSAID) compared with 2 on the secondary outcomes: lying behavior, haptoglobin concentrations, and mechanical nociceptive threshold (MNT) in calves disbudded via cautery iron. Eighty female Holstein calves were enrolled at birth, individually housed, and fed via a Calf Rail system (Förster Technik). A 2 × 2 factorial design was used to assess the effect of plane of nutrition and an additional NSAID. Calves were randomly assigned to a biologically normal plane of nutrition (BN; offered up to 15 L/d) or a limited plane (LP; offered up to 6 L/d) and to receive one or 2 doses of meloxicam. All calves received a lidocaine cornual nerve block and a subcutaneous injection of meloxicam 15 min before cautery disbudding at 18 to 25 d of age, and half the calves received an additional injection of meloxicam (0.5 mg/kg) 3 d after disbudding. Tissue type present, wound diameter, and wound depth were evaluated 2 times per week for 7 to 8 wk as measures of wound healing, lying behavior was recorded beginning 1 to 2 wk before disbudding until 7 to 8 wk after as a behavioral indicator of pain, haptoglobin concentrations were measured once per day for 7 d after disbudding, and MNT was evaluated 2 times/wk for 3 wk. Survival analyses were analyzed using Cox regression models (wound healing) and continuous data were analyzed using mixed-effect linear regression models. Only 12% of horn buds were completely healed by 7 to 8 wk after disbudding and 54% had re-epithelized at this time. At any time, wounds from BN calves were more likely to have had re-epithelization occur compared with wounds from LP calves (hazard ratio: 1.93, 95% CI: 1.18-3.14). Wounds from calves that received only one dose of NSAID were more likely to have re-epithelization occur, compared with wounds from calves given 2 doses (hazard ratio: 1.87, 95% CI: 1.15-3.05). Wounds from BN calves had smaller diameters and depths over time beginning on wk 3 compared with LP calves. Wounds from calves that received an additional NSAID had larger diameters and depths over time beginning on wk 4 and 3 respectively, compared with calves that only received one dose of NSAID. Calves that received an extra NSAID tended to be less sensitive 7, 10, and 17 d after disbudding compared with calves that only received one dose and spent less time lying in the week after disbudding. Calves on the BN milk program were more active compared with LP calves with lower lying times, fewer lying bouts per day, and longer average lying bouts. Our results indicate that a BN milk feeding program for calves can result in faster healing times and more activity, and that providing an extra NSAID 3 d after disbudding appears to slow the healing process but may result in less pain experienced by the calf 1 to 2 wk after the procedure. This study is also among the first to demonstrate that after the complete removal of the horn bud, wounds can take more than 8 weeks to re-epithelize and fully heal.

LincRNA#1 knockout alone does not affect polled phenotype in cattle heterozygous for the celtic POLLED allele

A long intergenic non-coding RNA (lincRNA#1) is overexpressed in the horn bud region of polled (hornless) bovine fetuses, suggesting a potential role in horn bud suppression. Genome editing was used to test whether the absence of this sequence was associated with the horned phenotype. Two gRNAs with high mutation efficiencies targeting the 5′ and the 3′ regions flanking the lincRNA#1 sequence were co-injected with Cas9 as ribonucleoprotein complexes into bovine zygotes (n = 121) 6 h post insemination. Of the resulting blastocysts (n = 31), 84% had the expected 3.7 kb deletion; of these embryos with the 3.7 kb deletions, 88% were biallelic knockouts. Thirty-nine presumptive edited 7-day blastocysts were transferred to 13 synchronized recipient cows resulting in ten pregnancies, five with embryos heterozygous for the dominant PCPOLLED allele at the POLLED locus, and five with the recessive pp genotype. Eight (80%) of the resulting fetuses were biallelic lincRNA#1 knockouts, with the remaining two being mosaic. RT-qPCR analysis was used to confirm the absence of lincRNA#1 expression in knockout fetuses. Phenotypic and histological analysis of the genotypically (PCp) POLLED, lincRNA#1 knockout fetuses revealed similar morphology to non-edited, control polled fetuses, indicating the absence of lincRNA#1 alone does not result in a horned phenotype.

Comparison of alternative methods for thermal disbudding in calves

Calves are frequently disbudded to reduce the number of injuries due to horns to other animals and farm workers. The most commonly used method is thermal disbudding which is increasingly causing animal welfare concerns (Stafford and Mellor, 2011; Sutherland et al., 2013, Cozzi et al., 2015). The objective of our study was to evaluate alternative disbudding methods either by injection of clove oil or its synthetic analogue isoeugenol.Forty Simmental calves (26 male, 14 female) aged between 1 and 5 days were treated using 4 different methods (n = 10): injection of 1.5 mL clove oil, injection of 1.5 mL isoeugenol, injection of 1.5 mL isotonic NaCl solution (control group) and thermal disbudding. For thermal disbudding sedation and local anesthesia were legally required. Horn growth of all calves was repeatedly measured. Computer tomography (CT) of the horn bud region and histological examination of biopsy samples taken from the horn bud region were performed. Additionally, saliva cortisol concentrations were measured before and after intervention.Significant differences in horn growth were found between isoeugenol treatment and control group (P = 0.001), between clove oil treatment and control group (P = 0.001) and between thermal dehorning and control group (P < 0.001). After clove oil injection 10/10 calves showed signs of inflammation and swelling of the horn bud region including the upper eye lid area. CT images of animals after thermal disbudding showed complete destruction on the horn buds but also severe local damage to the frontal bone. CT images of animals after clove oil or isoeugenol injection showed complete or partial destruction of the horn bud. Histological examination was performed at 1 horn bud per animal of 5 per each group. Eight biopsy samples showed normal unchanged epidermis and dermis (3 isoeugenol, 5 control group), in 6 samples normal vital skin side by side with necrotic epidermis and/or dermis was found (2 isoeugenol, 2 clove oil, 3 thermal dehorning). In one sample (thermal dehorning) necrotic bone with osteomyelitis was detected. In 3 samples (thermal dehorning) dystrophic calcification was present. After clove oil treatment necrotic epidermis and dermis as well vital skin with a purulent crust was detected.The highest mean cortisol value was measured in calves treated with clove oil. Fifteen minutes after treatment the mean value of thermal dehorned calves showed the lowest values, however these calves were sedated for the treatment.Thermal disbudding tended to have the highest efficacy of disbudding. Horn growth was also substantially decreased in clove oil and in isoeugenol group. In cases in which the horn growth was not totally suppressed the horns remained very small. Based on our results, disbudding calves with isoeugenol causes less tissue damage than thermal disbudding. Since clove oil caused temporary swelling we consider it less suitable. Future research is required to evaluate the behavioral responses of calves experiencing these methods of disbudding; in addition, more research is needed to optimize the injection volume and technique in a larger group of animal.

Evaluation of Horn Bud Wound Healing Following Cautery Disbudding of Dairy Calves With and Without the Use of Oxytetracycline Aerosol Spray.

Oxytetracycline is commonly applied as a topical agent to burn lesions post cautery disbudding of calves. Judicial use of antibiotics dictates that they should only be used where necessary to reduce the development of resistance in target bacteria. The objective of this study, therefore, was to evaluate the efficacy of topical oxytetracycline spray on wound healing post cautery disbudding of dairy calves over a 6-week period. Dairy calves were disbudded by veterinarians, technicians, or veterinary surgeons, using a standard cautery disbudding protocol. Oxytetracycline spray was randomly applied to the right or left horn bud of each animal (OXY), while the other horn bud received no antibiotic spray (NA). The outcomes measured were wound diameter (WD) and lesion score (LS), either normal healing (NH) or abnormal healing (AH). These assessments were conducted every 14 days following disbudding, until 42 days. A total of 360 animals completed the study. There was a difference in wound diameter and lesion score on day 14 post disbudding between the two groups. Cautery lesions sprayed with oxytetracycline (OXY) were 0.5 ± 0.15 mm smaller than NA lesions (P = 0.001), and there were fewer abnormal healing lesions for OXY compared to the NA (2.5 vs. 11%, respectively; P ≤ 0.001). There were no differences at day 28 and day 42 post disbudding, and on day 42, 34% of wounds had healed in both groups.In summary, the authors were unable to demonstrate a difference in healing between the groups using the described methods.

A deletion at the polled PC locus alone is not sufficient to cause a polled phenotype in cattle

Dehorning is a common practice in the dairy industry, but raises animal welfare concerns. A naturally occurring genetic mutation (PC allele) comprised of a 212 bp duplicated DNA sequence replacing a 10-bp sequence at the polled locus is associated with the hornless phenotype (polled) in cattle. To test the hypothesis that the 10 bp deletion alone is sufficient to result in polled, a CRISPR-Cas9 dual guide RNA approach was optimized to delete a 133 bp region including the 10 bp sequence. Timing of ribonucleoprotein complex injections at various hours post insemination (hpi) (6, 8, and 18 hpi) as well as in vitro transcribed (IVT) vs synthetic gRNAs were compared. Embryos injected 6 hpi had a significantly higher deletion rate (53%) compared to those injected 8 (12%) and 18 hpi (7%), and synthetic gRNAs had a significantly higher deletion rate (84%) compared to IVT gRNAs (53%). Embryo transfers were performed, and bovine fetuses were harvested between 3 and 5 months of gestation. All fetuses had mutations at the target site, with two of the seven having biallelic deletions, and yet they displayed horn bud development indicating that the 10 bp deletion alone is not sufficient to result in the polled phenotype.

The effect of transdermal flunixin meglumine on blood cortisol levels in dairy calves after cautery disbudding with local anesthesia

The objective of this study was to evaluate the effect of the nonsteroidal anti-inflammatory drug transdermal flunixin meglumine (Finadyne Transdermal) on plasma cortisol, average daily weight gain, and standing and lying behavior of calves, when given at the time of disbudding combined with local anesthesia. A sedative was not used to minimize pharmacological interactions. Seventy-one female Holstein Friesian calves aged 13 ± 2 d, with an average weight of 48.9 ± 4.26 kg were enrolled in the study. All calves were randomly assigned to one of 3 treatment groups: (1) control group (CON, n = 27), (2) 1-flunixin group (1-FLU, n = 26) with a single administration of transdermal flunixin meglumine at disbudding, and (3) 2-flunixin group (2-FLU, n = 24) with 2 administrations of transdermal flunixin meglumine, the first treatment at disbudding and the second 6h after disbudding. Although the CON group received a placebo, 1-FLU and 2-FLU received flunixin meglumine transdermally. To account for plasma cortisol changes due to manipulation and handling of the calves, a sham disbudding procedure was performed one week before disbudding took place. Sham disbudding was conducted by using a cold cautery dehorner applied to each horn bud for 10 s. Disbudding was performed in a similar way by using a hot cautery dehorner. Plasma samples were collected to measure the stress biomarker cortisol at 7 different time points. Body weights were measured 4 times in 2 wk. Standing and lying behavior was assessed via 3-dimensional accelerometer. During sham disbudding and disbudding mean plasma cortisol concentrations were 6.09 ± 2.5 ng/mL and 5.16 ± 2.8 ng/mL, respectively. Treatment tended to have an effect on plasma cortisol concentrations during sham disbudding and had an effect on plasma cortisol concentrations during disbudding. Plasma cortisol concentrations were affected by treatment 2 h after disbudding in comparison to CON group. Furthermore, there was a significant effect on plasma cortisol concentrations 6 h after disbudding in contrast to CON. A return to baseline plasma cortisol levels (initial concentrations) was not achieved in CON during disbudding. There was no statistical difference between average daily weight gain and the treatment procedure. Total lying time was not affected by treatment after disbudding. In conclusion, transdermal flunixin meglumine given at the time of disbudding combined with local anesthesia decreased concentrations of the stress biomarker cortisol, but a second dose 6 h after disbudding had no further effect on plasma cortisol levels.

The analgesic effect of preventive administration of meloxicam in calves submitted to hot-iron dehorning

ABSTRACT: Dehorning is a zootechnical practice that causes severe pain in cattle. Although there are several studies evaluating the effects of analgesics in calf dehorning, none of them used validated pain assessment instruments. We evaluated the analgesic effectiveness of meloxicam administered before dehorning, compared to a control group, using the Unesp-Botucatu, numerical, simple descriptive, and visual analogue scales for pain assessment before and 4, 8, and 24 hours after the dehorning in 44 female calves. All calves received 0.04 mg/kg of xylazine IM 20 minutes before dehorning and local anesthetic block with 2% lidocaine with a vasoconstrictor. Calves were divided into two groups: without (GX; n = 22) or with 0.5 mg/kg of meloxicam (GXM; n = 22) administered intravenously before the procedure. Dehorning was performed through the section of the base of the horn bud, followed by thermocautery disbudding. For comparisons over time, mixed linear or generalized mixed linear model were used. The interaction between groups and study phases was used as fixed effects and each calf as a random effect. Bonferroni post hoc test was used. There was an increase in the pain scores at 4h compared to baseline in both groups (GX and GXM) for the four scales. The scores at 4h were higher in GX compared to GXM for all scales. Meloxicam reduced, but did not eliminate, behavioral expressions of pain in calves submitted to hot-iron dehorning. Therefore, it should be included in the analgesic protocol to improve welfare in calves undergoing dehorning.