Identification of Cytokeratins in Bovine Sperm Outer Dense Fibre Fractions

Outer dense fibres (ODF) are important substructures of mammalian sperm tails that are involved in the regulation of sperm motility. In this study, we investigated the identity of several sodium dodecyl sulphate (SDS)‐insoluble ODF proteins. Bovine ODF were purified by separating sperm heads and tails using ultrasound and Percoll^® density gradient centrifugation. Sperm flagella were treated with the detergent cetyltrimethylammonium bromide (CTAB). CTAB‐insoluble material, which reportedly represents the ODF fraction, was collected, and electron microscopy confirmed a highly purified ODF fraction. We found after solubilization of this fraction with SDS that high amounts of insoluble material were retained after centrifugation. SDS‐insoluble material was collected and quantitatively dissolved in 8 M urea. SDS‐gel electrophoresis in the presence of urea revealed polypeptides with apparent molecular masses of approximately 25, 43, and 50 kDa. Subsequent immunoblotting with anti‐cytokeratin antibodies detected two urea‐soluble, SDS‐insoluble proteins with apparent molecular masses of 45 and 66 kDa. The 45‐kDa protein was identified as cytokeratin 19. An antibody reacting with a palette of cytokeratins (CK 1–18 and CK 20), KL1, was the only antibody that reacted with the 66‐kDa polypeptide. We conclude that sperm ODF fractions contain at least one each of type I and type II intermediate filaments. As keratins and intermediate filaments are described as rope‐like structures, we suggest that these intermediate filaments play an important structural or tension‐bearing role in sperm flagella.     

Excision of the distal sesamoid bone for treatment of infection of the digit in a heifer.

An 8-month-old Holstein heifer was evaluated for right hind limb lameness of 3 weeks' duration. Diagnoses were osteomyelitis and fracture of the distal sesamoid bone, septic arthritis of the distal interphalangeal joint, and osteomyelitis of the third phalanx. After excision of a section of tissue from the plantar aspect of the digit and partial section of the deep digital flexor tendon, the distal sesamoid bone was excised. The third phalanx was curetted, and the surgical wound was lavaged. Twenty months after surgery, the heifer was fully weightbearing on the affected digit. Surgical exploration of the digit should be considered an alternative to claw amputation in cattle that have severe digital infections.     

Lumbar spondylectomy in the subhuman primate.

Removal of the 2nd or 3rd lumbar vertebrae (or both) has been accomplished in the subhuman primate (Macaca mulatta). Variations between this animal and the dog in posture, vertebral column anatomy, and spinal cord blood supply made no apparent difference in the results when compared with those in previous experiments. All macaques were able to clinb and to use their hind legs in a normal manner within 3 days after surgical operation. Once hair had regrown over the surgical incision, persons not familiar with the animals were not able to identify the animals that had undergone surgical operation. If a single vertebra was removed, use of spinous process and a single vertebral body plate was sufficient to stabilize the vertebral column. Except for 1 rhesus macaque whose spinous process plates were removed 20 days after the operation, vertebral columns of macaques (n = 3) that underwent single spondylectomy healed in nearly normal alignment. With the removal of 2 lumbar vertebrae, the previously described stabilization procedure was not sufficient to prevent kyphotic deformity of the vertebral column. Spinous process plates from 1 macaque were removed after 64 days. However, all these macaques (n = 3) could climb and use their legs as well as others in the colony.     

Effects of Confluent, Roscovitine Treatment and Serum Starvation on the Cell-cycle Synchronization of Bovine Foetal Fibroblasts

The present study was designed to examine the effects of cell-cycle synchronization protocols, such as confluent, roscovitine treatment and serum starvation, in bovine foetal fibroblasts on synchronization accuracy at G0/G1, viability, apoptosis, necrosis and ploidy for use as a nuclei donor. The cells in 5-10 passages were randomly allocated into three treated groups. Cells were cultured either in Dulbecco's modified Eagle's medium (DMEM) + 10% foetal bovine serum (FBS) until 90% confluent (group 1, confluent), in DMEM + 10% FBS + 30 microM roscovitine for 12 h (group 2, roscovitine), or in DMEM + 0.5% FBS for 5 days (group 3, serum starvation). Most of the cells (>80%) in all groups were arrested at the G0/G1 stage. Although the rates did not differ, cells in group 1 showed an increased cell population arrested at the G0/G1 phase. Significantly (p < 0.05) higher rates of apoptosis occurred in group 3 than in group 1 and 2 (10% vs 6% and 6%, respectively). No differences in chromosomal abnormality were observed among groups. However, by increasing the number of cell culture passages up to 15, significantly (p < 0.05) higher chromosomal abnormality was observed than in 5 and 10 passages (39% vs 28% and 23%, respectively) in group 1. The results clearly indicated that bovine foetal fibroblasts could be effectively synchronized at G0/G1 stages by all the three different treatments, confluent, roscovitine and serum starvation. However, cells in confluent showed reduced apoptosis and necrosis when they underwent 5-10 passages, exhibiting increased percentage of cells with stable chromosome diversity. Hence, cells in confluent merit further studies before they could be used as nuclear donors.     

Validating the effectiveness of alternative euthanasia techniques using penetrating captive bolt guns in mature swine (Sus scrofa domesticus)

Euthanasia of mature swine is challenging. Temporal and behind-the-ear locations are two sites that have been identified as alternatives to the more commonly used frontal placement. In stage one, the effectiveness of two penetrating captive bolt gun styles (cylinder or pistol) was evaluated using frontal, temporal, and behind-the-ear placement in anesthetized mature swine (n = 36; weight: 267 ± 41 kg). For stage one, when evaluating treatment efficacy by sex, the cylinder-style equipment was 100% effective in achieving death when applied to all cranial locations (frontal, temporal, and behind-the-ear) for sows; however, the pistol-style equipment was only 100% effective when applied at the behind-the-ear location for sows. For boars, the cylinder-style equipment was 100% effective when applied to the frontal and behind-the-ear location, but the pistol-style equipment was not effective for any cranial location in boars. Therefore, the pistol–frontal, pistol–temporal, pistol–behind-the-ear, and cylinder–temporal were not included for boars, and pistol–frontal and pistol–temporal were not included for sows in stage two. In stage two, commercial, mixed-breed, mature swine (n = 42; weight: 292 +/− 56 kg) were randomly assigned to one of four treatments based on the inclusion criteria described in stage one. A three-point traumatic brain injury (TBI) score (0 = normal; 1 = some abnormalities; 2 = grossly abnormal, unrecognizable) was used to evaluate six neuroanatomical structures (cerebral cortex, cerebellum, hypothalamus, thalamus, pons, and brain stem), and the presence of hemorrhage was also noted. All treatments were 100% effective in stage two. A significant interaction between gun style and placement was determined on predicting total TBI as the cylinder style produced a higher total TBI score compared with the pistol type of the magnitude of +2.8 (P < 0.01). The cylinder style tended to produce a greater TBI score than the pistol in the temporal location (+1.2; P = 0.08). No difference was noted for TBI score behind-the-ear between the cylinder- and pistol-style gun (P > 0.05). TBI tended to be less in boars compared with sows (−0.6; P = 0.08). Hemorrhage was observed in frontal, parietal, occipital, and temporal lobes. This study demonstrated that the cylinder-style captive bolt gun more effectively resulted in brain trauma and death compared with a pistol-style gun and the behind-the-ear and temporal placement showed promise as an alternative placement site for euthanizing mature pigs on-farm.     

Conjunctival habronemiasis in a square‐lipped rhinoceros (Ceratotherium simum)

A captive female square‐lipped rhinoceros born in 1993 had been showing intermittent signs of bilateral conjunctivitis and conjunctival proliferation since 1998. Periodic improvement was noted, especially in winter, but overall the condition had deteriorated over the years. Treatment with various topical, intralesional, and systemic antibiotics and glucocorticosteroids was largely ineffective, as were repeated dewormings. No primary cause for these lesions was found in biopsies taken in 2000 and 2006, although a severe infiltrate of numerous eosinophils was observed in the latter. As the condition worsened, secondary corneal changes were noted, and eventually vision was lost due to proliferative conjunctival tissue. Aggressive resection of the proliferating tissue in 2013 restored vision and submitted biopsies yielded a diagnosis of severe allergic conjunctivitis, eosinophilic granuloma, and habronematid (Habronema or Draschia) larval infection. As no other rhinoceros in the herd was affected, including two calves born to the patient who were in close contact with their mother, it was concluded the presentation was most likely due to a hypersensitivity reaction to the dead or dying larvae. Fly repellent is now regularly applied around the eye of this rhinoceros, and a protective face mask has been fitted. Ongoing periodic relapses are treated with oral ivermectin, topical antibiotics, and steroids.