An integrative insight into the synsacral canal of fossil and extant Antarctic penguins

The lumbosacral-canal system in birds most likely operates as a sense organ involved in the control of balanced walking and perching, but our knowledge of it is superficial. Penguins constitute interesting objects for the study of this system due to their upright walking, but only the Humboldt penguin, Spheniscus humboldti, and some incomplete fossil penguin synsacra have been studied in this respect. Here, we give an integrative comparative insight into the synsacral canal of extant Emperor penguin, Aptenodytes forsteri, Adelie penguin, Pygoscelis adeliae, and Eocene giant Anthropornis and/or Palaeeudyptes Antarctic penguins, using computed tomography imaging and associated data-extraction methodologies, complemented by analytical approaches ranging from geometric morphometrics to modularity, curvature, and wavelet analyses. We document that the variability in the number of synsacro-lumbar vertebrae is evolutionarily conserved, and all studied synsacra possess osteological correlates of the lumbosacral-canal system. We also found that Eocene and extant Antarctic penguins were separable on the basis of the main direction of the shape-related (size-independent) variability within said system, and A. forsteri was unique in the entire studied set in terms of the relative cranial shift of this compound structure. Moreover, we suggest that the evolutionary processes, shaping both the terrestrial posture and gait, were responsible, in extant penguins, for the increased simplicity and stability of the synsacral canal cross-sectional periodic patterns, as well as pave the way for the lumbosacral-canal system modularity characterized by reduced atomization/complexity.     

Profiling the New Zealand Thoroughbred racing industry. 2. Conditions interfering with training and racing

AIM: To describe and enumerate conditions that interrupted training and racing in a population of Thoroughbred racehorses in New Zealand.

METHODS: A longitudinal study design was used to collect data on horses training under the care of 20 licensed racehorse trainers from venues in the mid to lower regions of the North Island between October 1997 and July 2000. Incidence rates were reported for first and second occurrences for different categories of musculoskeletal injury (MSI), and first occurrences of upper and lower respiratory tract disease, using training days as time-at-risk. The proportion of horses that retired or died due to MSI, respiratory tract or miscellaneous conditions was used to estimate risk of exit for each type of event. Duration of training preparation, starts per 100 training days, and proportion of starts that ended in first, second or third place, were calculated for horses at risk for first MSI, and all subsequent MSIs. In training preparations that had at least one start and that ended in MSI, the cumulative percentage of MSIs by day of diagnosis was reported for 0–21 days after the last start in the preparation.

RESULTS: Horses (n=1,571) were followed during 3,333 training preparations and 392,290 training days. Events associated with the end of a training preparation or spell period included MSI (n=834), respiratory event (RE; n=165), miscellaneous event (ME; n=58), and voluntary retirements (n=360). Causes of MSI included lameness (n=400), shin soreness (n=207), tendon and ligament conditions (n=98), injury or laceration (n=56), fractures (n=55), and back disorders (n=18). MSIs involved the limbs in 97% of cases, and the lower limbs up to the carpus or hock in the fore- and hindlimbs, respectively, in 81% of cases. Most (93%) lower limb conditions involved a forelimb while 70% of MSIs that involved structures above the carpus or hock involved a hindlimb. Incidence rates (IRs) are reported for each age group for first and second occurrences of MSI, and first occurrence of upper and lower respiratory tract disease. The risk of MSI was higher in horses that had incurred one previous MSI (RR 1.4, 95% CI=1.2–1.7; p>0.001) than in horses without any previous MSI. The proportion of horses that exited due to death or retirement varied with the type of injury, and the highest proportion was associated with recurrent fractures, and tendon and ligament injuries (46.2 and 44.4%, respectively). The overall IR of horses exiting the study due to retirement or death increased with increasing age, and was higher in females than males for horses aged 2, 3, 4, and ≥5 years. A reduction in the number of starts per 100 training days was observed in horses aged ≥5 years when returning to training after an initial MSI (p=0.004). Male horses of all age groups and females younger than 4 years had shorter median training preparations (p>0.05) when returning to training after an initial MSI compared with preparations at risk for a first occurrence of MSI. Between 27 and 62% of cases of MSI that occurred in training preparations after at least one start were reported on the day of the last start, and the remainder were reported in the days to weeks following the last start of that preparation.

CONCLUSION: Incidence rates, and proportions of affected horses that retired or died as a result of injury or disorder varied with type of injury and age of horse. Horses returning to training after an initial MSI were at higher risk of subsequent MSIs and showed changes in duration of training preparations, but little change in starts per 100 training days or probability of placing in each start. MSIs in racing horses were less likely to be reported on the day of a race than at other times in the training preparation for all ages except 2-year-olds.     

Effect of Physiologically Relevant Heat Shock on Development,Apoptosis and Expression of Some Genes in Buffalo (Bubalus bubalis) Embryos Produced In Vitro

For investigating the effects of physiologically relevant heat shock, buffalo oocytes/embryos were cultured at 38.5°C (control) or were exposed to 39.5°C (Group II) or 40.5°C (Group III) for 2 h once every day throughout in vitro maturation (IVM), fertilization (IVF) and culture (IVC). Percentage of oocytes that developed to 8‐cell, 16‐cell or blastocyst stage was lower (p < 0.05) and the number of apoptotic nuclei was higher (p < 0.05) for Group III > Group II > controls. At both 8–16‐cell and blastocyst stages, relative mRNA abundance of stress‐related genes HSP 70.1 and HSP 70.2 and pro‐apoptotic genes CASPASE‐3, BID and BAX was higher (p < 0.05) in Groups III and II than that in controls with the exception of stress‐related gene HSF1. Expression level of anti‐apoptotic genes BCL‐XL and MCL‐1 was also higher (p < 0.05) in Groups III and II than that in controls at both 8–16‐cell and blastocyst stages. Among the genes related to embryonic development, at 8–16‐cell stage, the expression level of GDF9 was higher (p < 0.05) in Group III than that in controls, whereas that of GLUT1, ZAR1 and BMP15 was not significantly different among the three groups. At the blastocyst stage, relative mRNA abundance of GLUT1 and GDF9 was higher (p < 0.05) in Group II than that in controls, whereas that of ZAR‐1 and BMP15 was not affected. The results of this study demonstrate that exposure of buffalo oocytes and embryos to elevated temperatures for duration of time that is physiologically relevant severely compromises their developmental competence, increases apoptosis and affects stress‐, apoptosis‐ and development‐related genes.     

Concurrent gastro-oesophageal intussusception,trichobezoar and hiatal hernia in a cat

CASE HISTORY: An adult male Birman cat was evaluated for recurrent, intermittent vomiting or regurgitation, occasionally associated with abdominal discomfort.

CLINICAL FINDINGS AND DIAGNOSIS: Radiographs, including an oesophogram, indicated an oesophageal obstruction. Prior to treatment, the cat's condition deteriorated and it was euthanised at the owner's request. Post-mortem examination revealed a gastro-oesophageal intussusception, a trichobezoar impacted into the intussusceptum, and a dilated oesophageal hiatus consistent with a chronic hiatal hernia.

CLINICAL RELEVANCE: Gastro-oesophageal intussusception is a rare condition in cats. Its aetiology in relation to a pre-existing hiatal hernia and a trichobezoar is discussed.     

Buffalo (Bubalus bubalis) ES Cell–Like Cells are Capable of In Vitro Skeletal Myogenic Differentiation

When buffalo embryonic stem (ES) cell–like cells that expressed surface markers SSEA‐4, TRA‐1‐60, TRA‐1‐81, CD9 and CD90 and intracellular markers OCT4, SOX2 and FOXD3, as shown by immunofluorescence, and that expressed REX‐1 and NUCLEOSTEMIN as confirmed by RT‐PCR, were subjected to suspension culture in hanging drops in absence of LIF and buffalo foetal fibroblast feeder layer support, they differentiated to form three‐dimensional embryoid bodies (EBs). Of 231 EBs examined on Day 3 of suspension culture, 141 (61.3 ± 3.09%) were of compact type, whereas 90 (38.4 ± 3.12%) were of cystic type. The cells obtained from EBs were found to express NF‐68 and NESTIN (ectodermal lineage), BMP‐4 and α‐skeletal actin (mesodermal lineage), and α‐fetoprotein, GATA‐4 and HNF‐4 (endodermal lineage). When these EBs were cultured on gelatin‐coated dishes, they spontaneously differentiated to several cell types such as epithelial‐ and neuron‐like cells. When EBs were cultured in the presence of 1 or 2% DMSO or 10^?8 m or 10^?7 m retinoic acid for 25 days, ES cells could be directed to form muscle cell–like cells, the identity of which was confirmed by expression of α‐actinin by immunofluorescence and of MYF‐5, MYOD and MYOGENIN genes by RT‐PCR. MYOD was first detected on Day 10 in both treatment groups and on Day 15 in controls, whereas MYOGENIN was first detected on Day 10, Day 15 and Day 25 in the presence of retinoic acid, in the presence of DMSO and in controls, respectively. The present study demonstrates the ability of buffalo ES cell–like cells to undergo directed differentiation to cells of skeletal myogenic lineage.     

Effect of training location and time period on racehorse performance in New Zealand. 2. Multivariable analysis

AIM: To investigate training location (horses trained in Matamata vs those trained at all other venues in New Zealand), and time period (1996–1997 and 1998–1999), while controlling for other horse- and race- or trial-related factors, as a means of assessing the possible impact of construction of a new training surface at the Matamata Racing Club on indirect measures of racehorse performance (number of starts, and failure to race within 6 months of any start).

METHODS: Multivariable logistic regression and poisson analysis were used to analyse data derived using a retrospective cohort approach. Multivariable logistic regression was also used to analyse a case-control study. All data were derived from New Zealand Thoroughbred Racing (NZTR), records of race and trial results for racehorses trained in Matamata and other venues in New Zealand, covering two 19-month time periods (1996–1997 and 1998–1999). Outcome variables included whether a horse started again in the 6 months following any start that occurred in the first 13 months of either time period, and a count of the total starts for every horse.

RESULTS: Factors associated with increased risk of a start being followed by a 6-month no-race period included training location other than Matamata in comparison to horses trained in Matamata in the 1996–1997 time period, increasing age, 1998–1999 over 1996–1997, starting in a trial rather than a race, placing fourth or worse in a start, softer track conditions, summer vs autumn, increasing cumulative exercise intensity in the 60 days prior to a start, and increasing race distance. Factors associated with an increase in the total number of starts included horses trained at Matamata in 1996–1997 compared with other time period-location combinations, younger age of horses at the time of a start, longer race distance, and an increasing proportion of starts in stakes races.

CONCLUSIONS: Official race and trial results data provided a valuable resource for epidemiological studies of factors influencing racehorse performance. Results of analyses performed here provided little evidence of any adverse impact of a new training surface at the Matamata Racing Club on indirect measures of racehorse performance.     

Assessment of DNA Damage during In Vitro Development of Buffalo (Bubalus bubalis) Embryos: Effect of Cysteamine

Comet assay was used in the present study to examine DNA damage to buffalo oocytes and embryos during in vitro culture. Embryos were produced in vitro from oocytes obtained from slaughterhouse ovaries in presence of cysteamine (IVM and IVC media supplemented with 50 and 100 μm , respectively) or in its absence (controls). Compared to controls, cysteamine supplementation increased (p < 0.01) cleavage rate and proportion of oocytes that developed to 8‐ to 16‐cell stage. The incidence of DNA damage was lower (p < 0.01) in cysteamine group than that in controls at 8‐ to 16‐ (19.3 ± 4.24 vs 72.0 ± 5.22%) but not in 2‐cell stage embryos (11.7 ± 5.63 vs 20.8 ± 5.49%) or in mature oocytes (5.3 ± 3.43 vs 10.3 ± 4.73%). The tail length, which indicates magnitude of DNA damage, was shorter (p < 0.01) in cysteamine group than in controls in mature oocytes (25.5 ± 0.5 vs 36.0 ± 0.71 pixels) and 8‐ to 16‐cell stage (49.2 ± 1.64 vs 152.7 ± 1.28 pixels) but not in 2‐cell stage embryos (36.3 ± 1.54 vs 36.4 ± 0.75 pixels). Also, exposure of oocytes/embryos to UV radiation or H₂O₂ caused extensive DNA damage. In conclusion, these results suggest that oocytes/embryos suffer from DNA damage during progress of in vitro culture, which can be partly ameliorated by cysteamine supplementation of culture media.     

Short‐Term Antiandrogen Flutamide Treatment Causes Structural Alterations in Somatic Cells Associated with Premature Detachment of Spermatids in the Testis of Pubertal and Adult Guinea Pigs

In spite of widespread application of flutamide in the endocrine therapies of young and adult patients, the side effects of this antiandrogen on spermatogenesis and germ‐cell morphology remain unclear. This study evaluates the short‐term androgen blockage effect induced by the administration of flutamide to the testes of pubertal (30‐day old) and adult (65‐ and 135‐day old) guinea pigs, with an emphasis on ultrastructural alterations of main cell types. The testes removed after 10 days of treatment with either a non‐steroidal antiandrogen, flutamide (10 mg/kg of body weight) or a pharmacological vehicle alone were processed for histological, quantitative and ultrastructural analysis. In pubertal animals, flutamide androgenic blockage induces spermatogonial differentiation and accelerates testes maturation, causing degeneration and detachment of primary spermatocytes and round spermatids, which are subsequently found in great quantities in the epididymis caput. In post‐pubertal and adult guinea pigs, in addition to causing germ‐cell degeneration, especially in primary spermatocytes, and leading to the premature detachment of spherical spermatids, the antiandrogen treatment increased the relative volume of Leydig cells. In addition, ultrastructural evaluation indicated that irrespective of age antiandrogen treatment causes an increase in frequency of organelles involved with steroid hormone synthesis in the Leydig cells and a dramatic accumulation of myelin figures in their cytoplasm and, to a larger degree, in Sertoli cells. In conclusion, the transient exposition of the guinea pigs to flutamide, at all postnatal ages causes some degenerative lesions including severe premature detachment of spermatids and accumulation of myelin bodies in Leydig and Sertoli cells, compromising, at least temporarily, the spermatogenesis.     

Cryopreservation of Zona‐Free Cloned Buffalo (Bubalus Bubalis) Embryos: Slow Freezing vs Open‐Pulled Straw Vitrification

This study was carried out to compare the post‐thaw cryosurvival rate and the level of apoptosis in vitro produced zona‐free cloned buffalo blastocysts subjected to slow freezing or vitrification in open‐pulled straws (OPS). Zona‐free cloned embryos produced by handmade cloning were divided into two groups and were cryopreserved either by slow freezing or by vitrification in OPS. Cryosurvival of blastocysts was determined by their re‐expansion rate following post‐thaw culture for 22–24 h. The post‐thaw re‐expansion rate was significantly (p < 0.05) higher following vitrification in OPS (71.2 ± 2.3%) compared with that after slow freezing (41.6 ± 4.8%). For examining embryo quality, the level of apoptosis in day 8 frozen‐thawed blastocysts was determined by TUNEL staining. The total cell number was not significantly different among the control non‐cryopreserved cloned embryos (422.6 ± 67.8) and those cryopreserved by slow freezing (376.4 ± 29.3) or vitrification in OPS (422.8 ± 36.2). However, the apoptotic index, which was similar for embryos subjected to slow freezing (14.8 ± 2.0) or OPS vitrification (13.3 ± 1.8), was significantly (p < 0.05) higher than that for the control non‐cryopreserved cloned embryos (3.4 ± 0.6). In conclusion, the results of this study demonstrate that vitrification in OPS is better than slow freezing for the cryopreservation of zona‐free cloned buffalo blastocysts because it offers a much higher cryosurvival rate.