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.     

Prevalence and spatial distribution of bovine tuberculosis in brushtail possums on a forest-scrub margin

Tuberculosis caused by Mycobacterium bovis was diagnosed in 36 of 68 (53%) brushtail possums (Trichosurus vulpecula) trapped in August 1992 from a population of exceptionally low density (trap catch <3%) on a forest-scrub margin in Westland, New Zealand. The prevalence of tuberculosis in possums, based solely on gross lesions, was at least twice that previously recorded in New Zealand, and was about seven times that recorded from the same population in 1980. More male (66%) than female (33%) possums had grossly visible tuberculous lesions. The distribution of infection appeared continuous along the forest-scrub margin. Both stoats (Mustela erminea) and one of six hares (Lepus europaeus occidentalis) trapped were also infected with M. bovis.     

Observations on three outbreaks of Leptospira interrogans serovar pomona infection in lambs

Sickness and deaths associated with leptospirosis were reported in three lamb flocks in the Manawatu region. The clinical, pathological and serological features of Leptospiru interrogans serovar pomona infection are described. The mucous membranes of affected animals were pale and jaundiced. Haemoglobinuria, haemoglobinaemia and centrilobular hepatocellular necrosis were also consistent findings. All clinically sick lambs had very high titres in the microscopic agglutination test for L. pomona. Circumstantial evidence of pigs being the source of infection was found in one of the three cases.     

The action of sulphanilamide on shell deposition

1. The action of sulphanilamide on egg shell deposition has been studied in the domestic fowl. The length of time that eggs remain in the shell gland was measured; the relative thickness and protoporphyrin pigment concentration for egg shells removed from the shell gland were determined; egg shell structure was examined using a replica technique.

2. Sulphanilamide appeared to act mainly by reducing the rate of shell deposition, time spent in the shell gland was unaffected.

3. The thickness of shell components was reduced in a roughly proportional manner in thin shells produced during sulphanilamide treatment.

4. It is proposed that other thin shells in which the component layers are all reduced to a similar extent are also the result of a decreased deposition rate.     

Comparison of a baiting method and PCR for the detection of Phytophthora fragariae var. rubi in certified raspberry stocks*

In winter 2002/2003, a total of 136 root samples from 57 different raspberry stocks in Scotland were examined for the presence of raspberry root rot caused by the fungus‐like pathogen Phytophthora fragariae var. rubi. All stocks had been planted as propagation material entered at different grades in the Scottish certification scheme or had applied for plant passports. For detection, a modified ‘Duncan bait test’ was compared to a nested PCR method. The two tests identified the same infected stocks: PCR detected 10 positive samples from four different stocks, while the bait test picked up two additional positive samples coming from the same four stocks. The two tests had a similar level of reliability in this examination and a recommendation for one or the other depends mainly on the technical equipment and skills available in the laboratory.     

Efficacy of C/B‐OPP rescue for the treatment of relapsed canine lymphoma (1998–2004)

Introduction:  MOPP chemotherapy is useful for relapsed canine lymphoma. This study evaluates the efficacy of this protocol after substitution of CCNU (lomustine) or BiCNU (carmustine) for mechlorethamine (C/B‐OPP).
Methods:  Patient signalment, response to chemotherapy, toxicity and survival data were abstracted from medical records of dogs from receiving C/B‐OPP between 1998 and 2004.
Results:  Fifty‐eight dogs received C/B‐OPP rescue chemotherapy during the study period. The median remission duration after initial chemotherapy, consisting of CHOP‐based therapy in 91% of dogs, was 133 days (range, 10 to 932 days). Thirty‐eight of fifty‐eight dogs (66%) responded to C/B‐OPP rescue after relapse (22 CR, 16 PR), for a median of 48 days (range, 2 to 359 days). Overall, C/B‐OPP extended survival by a median of 90 days (range, 2 to 426 days). Twenty‐four dogs (41%) experienced one or more episodes of Grade II or higher gastrointestinal toxicity. Forty‐one dogs (71%) experienced one or more episodes of Grade II or higher hematologic toxicity. Twelve dogs (20%) developed regenerative anemia with diarrhea consistent with gastrointestinal hemorrhage. Treatment delays due to hematologic toxicity occurred in 37 dogs (63%). There were 16 nonfatal treatment‐related episodes of sepsis requiring hospitalization. 5 dogs died due to sepsis and/or chemotherapy‐related complications.
Conclusions:  C/B‐OPP chemotherapy has activity against relapsed canine lymphoma which is similar to that of traditional MOPP rescue therapy. Moderate to severe hematologic toxicity was observed. Further work is warranted to optimize drug doses and scheduling.     

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.     

Influence of amount and frequency of protein supplementation to steers consuming low-quality,cool-season forage: intake,nutrient digestibility,and ruminal fermentation

This experiment evaluated the influence of protein supplementation frequency (SF) and amount offered on intake, nutrient digestibility, and ruminal fermentation by rumen-fistulated beef steers consuming low-quality [2.9% crude protein (CP); dry matter (DM) basis], cool-season forage. Seven Angus × Hereford steers (300 ± 27 kg) fitted with ruminal cannulas were randomly assigned to 1 of 7 treatments in an incomplete 7 × 4 Latin square. Treatments, in a 2 × 3 factorial design plus a non-supplemented control (CON), consisted of 2 levels of supplemental soybean meal, 100% (F) or 50% (H) of the estimated rumen-degradable protein requirement, provided daily (D), once every 5 d (5D), or once every 10 d (10D). Experimental periods were 30 d and dry matter intake (DMI) was measured from days 19 to 28. On days 21 (all supplements provided) and 30 (only daily supplements provided; day immediately prior to supplementation for 5D and 10D treatments) ruminal fluid was collected for ruminal pH, ammonia-N (NH₃), volatile fatty acids (VFA), and determination of ruminal fermentation variables. Forage and total DM, organic matter (OM), and nitrogen (N) intake increased with supplementation (P ≤ 0.04). However, a linear effect of SF × amount of supplement interaction was observed for forage and total DM, OM, and N intake (P ≤ 0.04), with each variable decreasing as SF decreased, but the decrease being greater with F vs. H. Apparent total tract DM, OM, and neutral detergent fiber digestibility was not affected by supplementation or amount of supplement provided (P ≥ 0.10). In contrast, N digestibility increased with supplementation and for F vs. H (P < 0.01). Digestibility of DM, OM, and N increased linearly as SF decreased (P ≤ 0.03). When all supplements were provided, ruminal NH₃, total VFA, and molar proportions of all individual VFA increased with supplementation (P ≤ 0.04), whereas acetate:propionate ratio decreased (P < 0.01). When only daily supplements were provided, none of the aforementioned fermentation parameters were affected (P ≥ 0.09). In summary, reducing the amount of supplemental CP provided to ruminants consuming low-quality forages, when supplementation intervals are >5 d, can be a management tool to maintain acceptable levels of DMI, nutrient digestibility, and ruminal fermentation while reducing supplementation cost.