Effects of two wintering practices on behavioral and physiological indicators of welfare of non-lactating, pregnant dairy cattle in a pasture-based system.

In countries with pasture-based dairy systems and relatively cold winters, such as New Zealand, it is common to manage pregnant, nonlactating cows on forage crop paddocks rather than pasture due to slow pasture growth rates. Wintering dairy cattle on grazed crops can compromise welfare if wet and muddy underfoot conditions occur, which can reduce lying. This study investigated behavioral and physiological indicators of welfare of cows under 2 wintering practices: cows managed on and grazed kale crop (Brassica oleracea), and cows managed on pasture with baled hay. Following dry-off (d 0), 80 cows were randomly assigned to one of the 2 wintering practices (40 cows/practice) and monitored between d 4 and d 32 (phase 1). During this period, lying and stepping behavior was continuously recorded using leg-based accelerometers. Blood samples were obtained at d 0 and 32 for measurements of thyroxine (T4), nonesterified fatty acids (NEFA), white blood cells (WBC), and red blood cells (RBC). All data for phase 1 were presented descriptively due to the lack of treatment replication. Daily mean air temperature during this period was 5.2°C (range: 0.0 to 10.7°C), and rainfall was 1.1mm/d (range: 0 to 5.6mm/d). Between d 4 and 32, cows in both groups spent similar amounts of time lying (pasture with hay cows: 8.9h/24h ± 2.57, kale crop cows: 8.7h/24h ± 3.06, mean ± SEM). Both groups reduced their lying on wet and cold days and there was evidence of rebound lying once unfavorable weather conditions stopped. Cows on kale crop had numerically higher NEFA and lower WBC compared with cows managed on pasture, although most physiological values were within normal ranges. In a second phase of the study (d 34 and 35), cows were managed under controlled, replicated conditions in the 2 wintering practices using typical on-farm stocking rates (2 or 4 cows per group in the pasture with hay and kale crop treatments, respectively; n = 10 groups/treatment). During this period, cow behavior, skin and surface temperatures, hygiene scores, feed intakes and ground conditions were measured. Weather conditions during the 48-h exposure were mostly cold and dry (mean air temperature: 7.8°C, range: -2.2 to 20.5°C). Cows managed on pasture with hay spent more time lying down on the first day of exposure, however, this was likely due to less space being available to kale cows on this day. Cows managed on pasture with hay ruminated more than cows on kale crop on both days of observations (Day 1: 37.9% vs 30.9% of observations, Day 2: 36.8% vs 28.7% of observations for pasture with hay and kale crop groups, respectively) and were lying more often in postures indicative of greater thermal comfort. Cows managed on pasture with hay had higher skin and surface temperatures compared with cows on kale crop, whereas cows on kale crop had dirtier coats. Results suggest that opportunities for thermal comfort were greater for cows managed on pasture with hay bales, which may be due to increased rumination activities and more insulated lying areas.

Effects of provision of drinking water on the behavior and growth rate of group-housed calves with different milk allowances.

Despite the clear importance of drinking water, calves are not always provided water on farm for the first few weeks of life. The main objective of this study was to investigate the effects of water provision (access or no access) and milk allowance (high or low) on the behavior and growth rate of calves. Fifty mixed-breed calves were each assigned to 1 of 4 treatments: (1) water and high (10 L/d) milk allowance (n = 13), (2) no water and high milk allowance (n = 12), (3) water and low (5 L/d) milk allowance (n = 12), or (4) no water and low milk allowance (n = 13). Visits to the water trough, water intake, milk drinking behavior (visits and drinking speed), proportion of observations eating hay and calf starter, and lying behavior were recorded from when the calves were, on average, 5 d of age (standard deviation: 2 d) for 4 consecutive weeks. Calves were weighed weekly. Some calves began to visit the water trough from the start of the recording period, as early as 4 d of age, and water intake increased with age for all calves that had access to it. This increase was greater for calves provided a high milk allowance. Water intake increased with ambient temperature, which highlights the importance of providing drinking water in warm conditions. Overall, calves spent a greater proportion of observations eating hay and calf starter with age. The provision of drinking water was associated with a greater proportion of observations eating hay but less eating calf starter. The increase in the proportion of observations eating calf starter with age was greater for calves on a low milk allowance than of those provided a high milk allowance; this is likely due to calves on a low milk allowance searching for nutrients and energy. Calves on a high milk allowance grew faster and spent more time lying compared with calves with a low milk allowance, thus suggesting greater satiety of well-fed calves. Our results suggest that calves should have free access to drinking water from birth and that access to drinking water may aid in hay (fiber) intake and possibly rumen development.

Physiological and behavioral responses as indicators for early disease detection in dairy calves.

This study investigated physiological and behavioral responses associated with the onset of neonatal calf diarrhea (NCD) in calves experimentally infected with rotavirus and assessed the suitability of these responses as early disease indicators. The suitability of infrared thermography (IRT) as a noninvasive, automated method for early disease detection was also assessed. Forty-three calves either (1) were experimentally infected with rotavirus (n = 20) or (2) acted as uninfected controls (n = 23). Health checks were conducted on a daily basis to identify when calves presented overt clinical signs of disease. In addition, fecal samples were collected to verify NCD as the cause of illness. Feeding behavior was recorded continuously as calves fed from an automated calf feeder, and IRT temperatures were recorded once per day across 5 anatomical locations using a hand-held IRT camera. Lying behavior was recorded continuously using accelerometers. Drinking behavior at the water trough was filmed continuously to determine the number and duration of visits. Respiration rate was recorded once per day by observing flank movements. The effectiveness of inoculating calves with rotavirus was limited because not all calves in the infected group contracted the virus; further, an unexpected outbreak of Salmonella during the trial led to all calves developing NCD, including those in the healthy control group. Therefore, treatment was ignored and instead each calf was analyzed as its own control, with data analyzed with respect to when each calf displayed clinical signs of disease regardless of the causative pathogen. Milk consumption decreased before clinical signs of disease appeared. The IRT temperatures were also found to change before clinical signs of disease appeared, with a decrease in shoulder temperature and an increase in side temperature. There were no changes in respiration rate or lying time before clinical signs of disease appeared. However, the number of lying bouts decreased and lying bout duration increased before and following clinical signs of disease. There was no change in the number of visits to the water trough, but visit duration increased before clinical signs of disease appeared. Results indicate that milk consumption, IRT temperatures of the side and shoulder, number and duration of lying bouts, and duration of time spent at the water trough show potential as suitable early indicators of disease.

Effects of 3 surface types on dairy cattle behavior, preference, and hygiene.

Muddy surfaces have negative effects on the health and welfare of dairy cattle, and if possible, cows will avoid this surface. However, it is unclear whether it is the moisture content or the contamination with manure that is aversive to the cows. This study aimed to assess the use and preference for different wood chip (0.4 m deep) surface types: (1) clean and dry (clean, dry matter content, DM: 44 ± 2.8%), (2) dirty (dirty, contaminated with manure, DM: 40 ± 3.7%), and (3) clean and wet (wet, wetted by water, DM: 23 ± 3.3%). Eighteen nonlactating, pregnant cows were tested individually (mean 24-h temperature: 9.9 ± 4.46°C, mean ± standard deviation for all preceding values). Cows were kept indoors in test pens for 18 h on wood chip without feed and 6 h on pasture to allow for daily feed intake. To ensure cows made informed choices and to measure changes in behavior and hygiene associated with each option, they were first exposed to each surface for 5 d (n = 12 cows per surface type when they were restricted on one surface; i.e., each cow was exposed to 2 treatment surfaces only). Cows on the wet surface spent the least amount of time lying when restricted to one surface for 18 h (wet: 21%, dirty: 57%, clean: 64%) and spent more time lying when on pasture for 6 h (wet: 13%, dirty: 4%, clean: 3%). The total lying times during the 5-d surface exposure were wet: 4.6 ± 1.04 h, dirty: 10.6 ± 0.25 h, and clean: 11.7 ± 0.25 h per 24 h. Cows restricted on the wet surface for 18 h had fewer bouts (no.) of lateral lying (wet: 0.9 ± 1.36, dirty: 6.3 ± 1.36, clean: 8.4 ± 1.38), spent less time lying with their heads supported (wet: 18.9 ± 7.17 min, dirty: 36.7 ± 7.17 min, clean: 39.1 ± 7.26 min), and spent less time with the front legs tucked (wet: 16 ± 4.3%, dirty: 41 ± 4.3%, clean: 50 ± 4.3% of time spent lying, mean ± standard error of the mean for all preceding values), than cows on the other surfaces. Cows on the dirty surface were less clean compared with the other treatment groups (0.6 of a score on a 5-point scale; standard error of the differences of means: 0.11 for both comparisons). They were then given a free choice between 2 known surfaces for 2 consecutive days (n = 6 per pairwise choice). Cows ranked the surfaces as clean > dirty > wet. In summary, there is compelling evidence that wet surfaces impair the welfare of dairy cattle by affecting the quantity and quality of rest. Rebound responses indicate that the motivation to rest is not fulfilled on wet surfaces. Finally, when given a choice, they show clearly that they will avoid wet and dirty surfaces. The combined results indicate that changes in affective state likely underlie these behavioral responses.

Widespread correction of central nervous system disease after intracranial gene therapy in a feline model of Sandhoff disease.

Sandhoff disease (SD) is caused by deficiency of N-acetyl-ß-hexosaminidase (Hex) resulting in pathological accumulation of GM2 ganglioside in lysosomes of the central nervous system (CNS) and progressive neurodegeneration. Currently, there is no treatment for SD, which often results in death by the age of five years. Adeno-associated virus (AAV) gene therapy achieved global CNS Hex restoration and widespread normalization of storage in the SD mouse model. Using a similar treatment approach, we sought to translate the outcome in mice to the feline SD model as an important step toward human clinical trials. Sixteen weeks after four intracranial injections of AAVrh8 vectors, Hex activity was restored to above normal levels throughout the entire CNS and in cerebrospinal fluid, despite a humoral immune response to the vector. In accordance with significant normalization of a secondary lysosomal biomarker, ganglioside storage was substantially improved, but not completely cleared. At the study endpoint, 5-month-old AAV-treated SD cats had preserved neurological function and gait compared with untreated animals (humane endpoint, 4.4±0.6 months) demonstrating clinical benefit from AAV treatment. Translation of widespread biochemical disease correction from the mouse to the feline SD model provides optimism for treatment of the larger human CNS with minimal modification of approach.

A field study of the behavioral and physiological effects of varying amounts of shade for lactating cows at pasture.

Shade reduces the negative effects of heat load, but little is known about how much is required for efficient cooling in commercial settings. The effect of the amount of shade on 8 Holstein-Friesian herds was studied for 2 consecutive summers (mean temperature: 23 °C) on 6 commercial, pasture-based dairy farms. Farms varied in the amount of natural shade provided (range: 0 to 15.6m(2) shade/cow). Time spent in shade, near water, eating, ruminating, lying, and standing were recorded between 1000 and 1530 h in 31 shaded and 11 unshaded paddocks using 20-min instantaneous scan observations of 15 focal cows/herd. Respiration rate and panting score (0 to 4.5) was recorded for focal animals once per hour. The total numbers of cows in shade, near water, and with panting scores ≥ 2 were recorded every 30 min. Cows without shade spent 4% more time lying than cows with shade (standard error of the difference, SED = 1.9%). A larger proportion of the herd had panting scores ≥ 2 when no shade was available (6 vs. 2% of the herd, SED = 1.2%), and respiration rates were higher by 8 breaths/min in cows without shade (SED = 4.7 breaths/min). Under the conditions tested, the maximum proportion of the herd that was observed using the shade increased by 3.1% for every 1-m(2) increase in shade size [standard error (SE) = 1.51%], and all cows were first seen simultaneously using shade when 2m(2)/cow was provided. For every 1-m(2) increase in shade, 0.3% fewer cows had panting score ≥ 2 (SE = 0.12%). We observed no significant relationships between the amount of shade available and any other variables. Although additional work is required to make specific recommendations, these results indicate that providing more shade allowed a higher proportion of animals to use this resource and reduced respiratory signs of heat load.

Effects of short-term repeated exposure to different flooring surfaces on the behavior and physiology of dairy cattle.

Dairy cattle managed in some pasture-based systems such as in New Zealand are predominantly kept outdoors all year around, but are often taken off pasture for periods of time in wet weather to avoid soil damage. It is common to keep cattle on concrete surfaces during such "stand-off" practices and we investigated whether the addition of rubber matting onto concrete areas improves the welfare of dairy cattle. Sixteen groups of 5 cows (4 groups/treatment, 5 cows/group) were allocated to 1 of 4 treatments (concrete, 12-mm-thick rubber mat, 24-mm-thick rubber mat, or deep-bedded wood chips) and kept on these surfaces for 18 h/24h for 4 consecutive days (6h on pasture/24h). Each 4-d stand-off period was repeated 4 times (with 7 d of recovery between periods) to study the accumulated effects of repeated stand-off. Lying behavior was recorded continuously during the experiment. Gait score, stride length, hygiene score, live weight, and blood samples for cortisol analysis were recorded immediately before and after each stand-off period. Cows on wood chips spent the most time lying, and cows on concrete spent the least time lying compared with those on other surfaces [wood chips: 10.8h, 24-mm rubber mat: 7.3h, 12-mm rubber mat: 6.0 h, and concrete: 2.8h/18 h, standard error of the difference (SED): 0.71 h]. Cows on concrete spent more time lying during the 6h on pasture, likely compensating for the reduced lying during the stand-off period. Similarly, cows on concrete spent more time lying on pasture between stand-off periods (concrete: 12.1h, 12-mm rubber mat: 11.1h, 24-mm rubber mat: 11.2h, and wood chips: 10.7h/24h, SED: 0.28 h). Cows on concrete had higher gait score and shorter stride length after the 4-d stand-off period compared with cows on the other surface types, suggesting a change in gait pattern caused by discomfort. Cows on rubber mats were almost 3 times dirtier than cows on concrete or wood chips. Cortisol and live weight decreased for all treatment groups during the stand-off period. We observed no major effect of the repeated stand-off exposure. In summary, adding rubber matting onto concrete surfaces for stand-off purposes is beneficial for animal welfare. A well-managed wood chip surface offered the best welfare for dairy cows removed from pasture, and the findings of this study confirm that a concrete surface decreases the welfare of cows removed from pasture.

Behavioral and physiological effects of a short-term feed restriction in lactating dairy cattle with different body condition scores at calving.

Body condition score (BCS) around calving, and the typical BCS loss for up to 100 d after parturition, is associated with both production and reproductive performance of dairy cattle. In addition, there is public concern that thin cows may have impaired welfare, particularly in early lactation where feed demand exceeds pasture growth, and a lag exists between peak milk energy requirements and intake. The aim of this experiment was to determine how BCS at calving influences behavioral and physiological responses to a short-term feed restriction at 47 DIM. Body condition score (on a 10-point scale) at calving was manipulated by modifying the diets in the previous lactation of healthy dairy cattle to generate 3 treatment groups: low BCS (3.4; n=17), medium BCS (4.6; n=18), or high BCS (5.4; n=20). Cows were tested in 4 groups for 8 consecutive days; testing consisted of different levels of feed allocation (d 1 and 2: 100%; d 3 and 4: 75%; d 5: 50%; d 6 to 8: 125%), where 100% was 15kg of DM/cow per day. All BCS groups had similar and marked behavioral and physiological responses to feed restriction. For example, they increased vocalization, time spent eating silage and grazing, aggressive behavior, and fat metabolism (as measured by concentrations of ß-hydroxybutyrate and nonesterified fatty acids), and reduced milk production. Body condition affected some of these responses. Fewer cows with low BCS engaged in aggressive interactions in a feed competition test (trough filled with silage that could be consumed in 15 min) on the first day of feed restriction (low: 32%; medium: 74%; high: 64%; standard error of difference=15.4%). High BCS cows had greater concentrations of ß-hydroxybutyrate and nonesterified fatty acids throughout the experimental period, which suggests more fat mobilization; however, plasma leptin and fecal glucocorticosteroid metabolite concentrations were unaffected by BCS. Whereas cows demonstrated marked responses to feed restriction, the results suggest that a BCS of 3.4, 4.6, or 5.4 in healthy cows at calving does not overwhelmingly influence this response at 47 DIM.

Dairy cattle prefer shade over sprinklers: effects on behavior and physiology.

Cattle will readily use shade in warm weather, but less is known about voluntary use of sprinklers. We examined preferences of 96 Holstein-Friesian dairy cows (milk yield: 12.7±3.48 kg per day; mean±SD) for sprinklers, shade, or ambient conditions after walking 2.0 km or 0.3 km before afternoon milking (n=48 cows/distance). Each cow was individually tested on 3 consecutive days with a different paired choice each day: 1) shade or sprinklers, 2) shade or ambient conditions, 3) sprinklers or ambient conditions. Average air temperature during testing was 22.3°C. Cows preferred shade over sprinklers (62 vs. 38% ± 5.0%; mean ± SE) and shade over ambient conditions (65 vs. 35% ± 5.1%; mean±SE). Cows showed no preference between sprinklers and ambient conditions (44% of the cows chose sprinklers, SE=5.3%). The preference for shade over sprinklers and ambient conditions increased with air temperature, solar radiation, and wind speed. Walking distance did not influence the preference for any treatment. Respiration rate was decreased most by sprinklers (38% decrease) but also decreased in shade and ambient conditions (17 and 13% decrease, respectively; standard error of the difference=4.7%). Similarly, surface temperature was decreased most by sprinklers (11.4% decrease), compared with that by shade (1.0% decrease), or that by ambient conditions (1.4% increase; standard error of the difference=1.82%). Furthermore, sprinklers reduced insect avoidance behaviors, including number of tail flicks and hoof stamps. In conclusion, dairy cattle preferred to use shade in summer despite sprinklers being more efficient in decreasing heat load and insect avoidance behavior.

The amount of shade influences the behavior and physiology of dairy cattle.

The objective was to understand how the amount of shade (shade cloth blocking 99% of solar radiation) influenced the behavior and physiology of Holstein-Friesian dairy cattle managed on pasture. We compared behavior, body temperature, and respiration rate of cattle provided with 1 of 3 treatments for 5 d: access to 2.4m(2) or 9.6m(2) shade/cow, or no shade (n=4 groups/treatment, 10 animals/group). Behavioral observations were carried out between 1000 and 1550h. Cows spent more than twice as much time in the larger shade (24 vs. 50% of observations for 2.4m(2) and 9.6m(2) shade/cow, respectively, SED: 1.7%) and engaged in fewer aggressive interactions when more shade was provided (10.7 vs. 3.2 aggressive interactions/m(2) during 5.8h of observation for 2.4m(2) and 9.6m(2) shade/cow, respectively, SED: 3.16 interactions/m(2)). Time around the water trough increased when little or no shade was provided (11, 5, and 2% of observations within 4.5m of water trough for no shade, 2.4m(2), and 9.6m(2) shade/cow, SED: 2.4%). Respiration rate was higher when cows had less shade available (62, 57, and 51 breaths/min for no shade, 2.4m(2), and 9.6m(2) shade/cow, respectively, SED: 2.1 breaths/min). All cows used the shade more when 9.6m(2) shade/cow was provided; simultaneous use was observed in 15 versus 0% of observations in the 9.6m(2) and 2.4m(2) treatments on the warmest day, respectively. Weather conditions influenced both the behavioral and physiological responses, and these changes were more pronounced when less or no shade was available. Cows spent more time in shade and less time lying with increasing heat load. In addition, aggressive interactions in the shade, time around the water trough, mean body temperature, and respiration rate increased with environmental heat load. Our findings highlight the importance of determining and providing an effective amount of shade to cattle.

Peptide phage display: opportunities for development of personalized anti-cancer strategies.

Personalized medicine is critical for cancer patients, because (1) cancer is a highly heterogeneous disease with major molecular differences in the expression and distribution of tumor cell surface markers among patients with the same type and grade of cancer, (2) cellular mutations tend to accumulate as cancer progresses, further increasing tumor heterogeneity, and (3) currently used cancer therapies often are toxic to normal cells, causing severe side effects rarely seen in other diseases. To prevent side effects and to improve effectiveness, cytotoxic therapies should be targeted and each patient should be profiled for the presence of cancer targets before the therapy is administered. Phage display technology utilizes combinatorial libraries of proteins expressed on phage particles that can be selected for specific binding to cancer cells. Such cancer-specific molecules can be used in a variety of applications, including identification of cell-specific targeting molecules; identification of cell surface biomarkers; profiling of specimens obtained from individual cancer patients, and the design of peptide-based anti-cancer therapeutics for personalized treatments. This review is focused on peptide phage display strategies that target cell surfaces because many biomarkers important in cancer are differentially expressed molecules located on the outside of the cell membranes.

Molecular markers of glial tumors: current targeting strategies.

Diagnosis and therapy for malignant gliomas represents one of the most challenging problems in clinical oncology. Current treatment of malignant glioma is multimodal, involving surgical resection, radiotherapy and chemotherapy. Even with these combined therapies, patients usually die within 1 to 2 years after onset of symptoms. Clearly, improved strategies for selective delivery of therapeutic agents to gliomas are needed to combat these devastating and usually fatal cancers. This review summarizes current knowledge concerning targetable molecular markers on the surface of glial tumor cells and tumor vasculature. Such markers are altered or up-regulated in gliomas compared to normal tissues, or they might be glioma-restricted. These markers include growth factor receptors, cell-surface adhesion molecules, and membrane-type matrix metalloproteinases. Current approaches that utilize growth factor peptides and peptide/antibodies identified via phage display technology as carrier ligands for targeting malignant gliomas are discussed.

Use of dual energy x-ray absorptiometry for noninvasive body composition measurements in clinically normal dogs.

OBJECTIVE: To determine quantitative values for components of body composition in clinically normal dogs of various breeds by use of dual energy x-ray absorptiometry (DEXA) and validate the precision and accuracy of DEXA technology in dogs. ANIMALS: 103 clinically normal sexually intact adult dogs. PROCEDURE: In a cross-sectional study, Beagles, Pembroke Welsh Corgis, Golden Retrievers, Great Danes, Pointers, Rottweilers, and nonpurebred dogs received total body DEXA scans. For the validation portion of the study, the results of DEXA scans of 6 dogs were compared with values obtained by chemical analyses of tissues from euthanatized dogs to determine the accuracy of this modality in dogs. RESULTS: Results (coefficient of variation) of the precision tests ranged from 0.10% for lean tissue to 5.19% for fat tissue, whereas accuracy tests revealed a difference between percentage bone mineral content and ash values. Body composition differed by sex, such as higher lean tissue and bone mineral content in males within some breeds, and among breeds. Regardless of body size or weight, the percentage of body weight that was bone mineral ranged from 3 to 4.0% [corrected]. CONCLUSIONS AND CLINICAL RELEVANCE: Results of this cross-sectional study provide valuable body composition data for clinically normal adult dogs, which may have research and clinical applications.

Body composition of growing and adult cats as measured by use of dual energy X-ray absorptiometry.

BACKGROUND AND PURPOSE: Total body scans were performed on 89 domestic cats of various ages, using dual energy x-ray absorptiometry (DEXA) to determine body composition, including fat, lean, and bone mineral content. Bone mineral density results from scans also are presented. METHODS: This cross sectional study included data for cats from a closed colony and from privately owned cats. Data were grouped by age and were analyzed by sex and reproductive status to provide information as to the rate of growth of the individual components of body composition. RESULTS: The results indicate that the rate of accretion of bone mineral, fat, and lean tissue differs throughout maturation and by sex. Regressions are provided to highlight age- and sex-related differences. CONCLUSIONS: The results of this study emphasize the benefits of examining the growth of each component of body composition when studying the effects of nutrition, disease processes, or therapeutic interventions.

Alterations in the growth hormone/insulin-like growth factor I pathways in feline GM1 gangliosidosis.

Cats affected with feline GM1 gangliosidosis, an autosomal, recessively inherited, lysosomal enzymopathy, have progressive neurological dysfunction, premature thymic involution, stunted growth, and premature death. Although increased membrane GM1 gangliosides can result in increased apoptosis of thymocytes, there is not a direct correlation between thymocyte surface GM1 and thymic apoptosis in vivo, suggesting that other factors may be important to the pathogenesis of thymic involution in affected cats. Because GH and insulin-like growth factor I (IGF-I) are important hormonal peptides supporting thymic function and affecting growth throughout the body, particularly in the prepubescent period, several components of the GH/IGF-I pathway were compared in GM1 mutant and normal age-matched cats. GM1 mutant cat serum IGF-I concentrations were reduced significantly compared with those in normal cats by 150 days of age, and GM1 mutant cats had no peripubertal increase in serum IGF-I. Additionally, IGF-binding protein-3 was reduced, and IGF-binding protein-2 was elevated significantly in GM1 mutant cats more than 200 days of age. Liver IGF-I messenger RNA and pituitary GH messenger RNA both were reduced significantly in GM1 mutant cats. After stimulation by exogenous recombinant canine GH, serum IGF-I levels increased significantly in GM1 mutant cats, indicating that GH/IGF-I signaling pathways within the liver remain intact and suggesting that alterations are external to the liver.

Evaluation of GM1 ganglioside-mediated apoptosis in feline thymocytes.

Cats with inherited GM1 gangliosidosis (GM1 mutant cats) have premature thymic involution characterized by decreased total thymocytes primarily affecting the CD4+ CD8+ subpopulation. While GM1 mutant cats have increased cell surface GM1 gangliosides, as determined by cholera toxin B binding, on both thymocytes and peripheral lymph node cells only thymocytes show increased apoptosis. To determine if GM1 gangliosides can increase the occurrence of apoptosis in feline thymocytes directly, we added exogenous GM1 ganglioside (GM1) to feline thymocyte primary cultures and compared the results to apoptotic changes seen in untreated cells or in cells treated with dexamethasone (Dex), a known inducer of thymocyte apoptosis in other species. Incorporation of exogenous GM1 into thymocyte cytoplasmic membranes was confirmed by flow cytometric analyses of cholera toxin B labelling. Apoptosis in feline thymocytes was analyzed by electron microscopy, spectrophotometric evaluation of DNA fragmentation, flow cytometric enumeration of apoptotic nuclei, and gel electrophoretic analysis of degraded DNA. Alterations in percentages of thymocyte immunophenotype following GM1 incorporation were determined by flow cytometric analyses of labelled cell surface markers for feline CD4 and CD8. Because in vitro addition of GM1 gangliosides has been reported in other species to decrease surface expression of CD4 on both thymocytes and peripheral lymphocytes, we evaluated GM1-associated down-regulation of CD4 on the surface of feline thymocytes and peripheral lymph node cells by flow cytometry. Additionally, we compared the apoptotic response of the more mature peripheral lymph node cells to the less mature thymocytes. Our results indicate that incorporation of exogenous GM1 into feline thymocyte cell membranes produces a dose-dependent increase of apoptotic cell death. Although, CD4 expression on both feline thymocyte and lymph node cell membranes was abruptly decreased after introducing exogenous GM1, enhanced apoptotic death was observed only in thymocytes, not in lymph node cells at the same GM1 concentration. Enhancement of thymocyte apoptosis appears to be age-related since cells derived from cats <3 months of age were more vulnerable than those from cats >3 months of age.

Thymic alterations in feline GM1 gangliosidosis.

GM1 gangliosidosis is an inherited metabolic disease characterized by progressive neurological deterioration with premature death seen in children and numerous animals, including cats. We have observed that thymuses from affected cats greater than seven months of age (GM1 mutant cats) show marked thymic reduction compared to age-matched normal cats. The studies reported here were done to describe alterations in the thymus prior to (less then 90 days of age) and during the development of mild (90 to 210 days of age) to severe (greater than 210 days of age) progressive neurologic disease and to explore the pathogenesis of the thymic abnormality. Although histologic examination of the thymus from GM1 affected cats less than 210 days of age showed no significant differences from age-matched control cats, thymuses from GM1 mutant cats greater than 210 days of age were significantly reduced in size (approximately 3-fold). Histologic sections of lymph nodes, adrenal glands, and spleens from GM1 gangliosidosis-affected cats showed no significant differences. Flow cytometric analyses showed a marked decrease in the percentage of immature CD4+CD8+ thymocytes (p < 0.001) and significantly increased CD4-CD8+ cells (p < 0.01) in GM1 mutant cats greater than 210 days of age when compared to normal age matched cats. Co-labelling with CD4, CD8, and CD5 indicated an increase in the percentage of GM1 mutant cat thymocytes at this age which were CD5high, suggesting the presence of more mature cells. Cytometric analyses of subpopulations of peripheral lymphocytes indicated an increase in CD4-CD8+ cells (p < 0.05) with concurrent decreases in CD4+CD8- and CD4-CD8- cells (which were not significant). Similar analyses of thymocyte and lymphocyte subpopulations from cats < 210 days of age showed no significant differences between GM1 mutant and normal cells. GM1 mutant cats at all ages had increased surface binding of Cholera toxin B on thymocytes, indicating increased surface GM1 ganglioside expression. Increases were highly significant in GM1 mutant cats greater than 210 days of age. In situ labelling for apoptosis was increased in GM1 mutant cats between 90 to 200 days of age when thymic masses were within normal limits. In GM1 mutant cats over 200 days of age, decreased labelling was observed when thymic mass was reduced and the CD4+CD8+ subpopulation, known to be very susceptible to apoptosis, was significantly decreased. These data describe premature thymic involution in feline GM1 gangliosidosis and suggest that increased surface GM1 gangliosides alters thymocyte development in these cats.

Gangliosides enhance apoptosis of thymocytes.

Monosialogangliosides, normal components of cell membranes, regulate cell development and differentiation in several organs. Our previous observation of dramatic premature thymic involution in cats with feline GM1 gangliosidosis, whose thymocytes have abnormally high cell surface gangliosides, suggested that excess GM1 ganglioside (GM1) could modulate thymocyte apoptosis in this disease (Cox et al., "Thymic Alterations in Feline GM1 Gangliosidosis," submitted). In these studies, we added exogenous GM1 to murine primary thymocyte cultures and demonstrated enhanced apoptosis in treated cells by DNA fragmentation, apoptotic body, and electrophoretic analyses. GM1-enhanced apoptosis was blocked by common apoptotic pathway inhibitors including aurintricarboxylic acid (inhibitor of endonuclease activity), actinomycin D (inhibitor of RNA transcription), and cycloheximide (inhibitor of protein synthesis). GM1 treatment primarily affected the immature CD4+ CD8+ subset, as shown by flow cytometric evaluation of fetal thymic organ culture and primary thymocyte cultures. Apoptosis also could be induced by GM2, GM3, and GT1b, whereas asialo-GM1 failed to do so, suggesting that the sialic acid moiety may play an important role in the induction of thymocyte apoptosis.

The effect of prepubertal immunization against gonadotropin-releasing hormone on the development of sexual and social behavior of bulls.

To determine the effect of prepubertal immunization against GnRH on the development of sexual and social behavior of Friesian bulls, 90 calves were randomly assigned to five treatments: 1) I2, immunized against GnRH at 2 and boosted at 2.5, 4, and 7.5 mo of age, n = 2 x 10; 2) I4, immunized against GnRH at 4 and boosted at 4.5 and 7.5 mo of age, n = 2 x 10; 3) I7.5, immunized against GnRH at 7.5 and boosted at 8 mo of age n = 2 x 10; 4) S, steers castrated at 2 mo of age, n = 10; and 5) B, intact bulls, n = 2 x 10. Blood samples were collected initially every 2, then every 3 wk. Plasma was analyzed for anti-GnRH titers and plasma testosterone concentration. Sexual and agonistic behavior, male-male mounting, and damage to paddocks was assessed throughout the experiment. All immunized calves developed antibodies against GnRH (32.3 +/- 2.0% bound at a 1:10 plasma:PBS-BSA dilution, 14 d after first boost). Plasma testosterone concentrations were < 1 ng/mL for all immunized animals until 11 mo of age, when they increased to levels found in intact bulls at 14 mo of age. At slaughter, testes and seminal vesicle weights were 38.3 and 31.6% lighter, respectively, for all immunized treatments compared to B. There were no significant differences between I2, I4, and I7.5 in any of the sexual or agonistic behavior tests. Bulls scored higher than steers in all sexual behavior tests. Immunized bulls scored lower than bulls in sexual behavior tests from 10 to 17 mo of age. The proportion of immunized animals that serviced an estrous cow was lower than the proportion of intact bulls at 10, 12.5, 14, and 17 mo of age. Immunized animals scored lower than bulls in bull challenge tests at 8.5, 11.5, 13, 14.5, and 17 mo of age. Paddock damage by animals on the three immunization treatments was lower than that by bulls from 7 to 14.5 mo of age, as were leg were scores (an indicator of male-male mounting behavior) from 9 to 14 mo of age. There was no difference in sexual behavior between immunized bulls (I2, I4, and I7.5) and bulls while held in lairage pens for 16 h before slaughter, but all treatment groups scored higher than steers. There was a similar trend for agonistic behavior, although I4 bulls were no different from steers. Prepubertal immunization against GnRH at 2, 4, and 7.5 mo of age impaired testes function and affected the development of social and sexual behavior of young bulls.