Quantitative analysis of long-bone growth in the horse

Long-bone growth at the distal ends of the radial bones, the distal ends of the 3rd metacarpal bones, the distal ends of the 3rd metatarsal bones, and the proximal ends of the proximal phalangeal bones (of thoracic and pelvic limbs) was quantitatively analyzed in 9 Thoroughbred-Quarter Horse foals from birth to 2 years of age. Metal growth markers were surgically implanted in the bones of the animals at 2 to 4 days of age. Radiographs of the bones were made on the day of surgical manipulation, the next day, and then once a week for 8 months, and once a month thereafter for an additional 18 months. On each radiograph, the intervals between the growth markers were measured and plotted, and these accumulated growth data were subjected to statistical analyses. Growth curves, cumulative growth, and relative growth rates were determined. The ages of radiographic growth plate closure also were determined. Total bone growth observed in these foals was proportionately greater than that observed in a similar study of Shetland-Welsh ponies, probably indicating a breed difference. The most rapid growth rate at the 5 anatomic sites in the foals occurred from birth to 10 weeks of age; however, in the distal part of the radius, there was a continuous, though declining growth rate until 60 weeks of age, whereas in the distal ends of the 3rd metacarpal and metatarsal bones and the proximal end of the proximal phalanges growth ceased abruptly and nearly plateaued after 10 weeks of age.(ABSTRACT TRUNCATED AT 250 WORDS)     

Quantitative studies of the optic nerve fiber layer in the chicken retina.

The optic nerve fiber layer (NFL) of the chicken retina was studied quantitatively and morphologically at 17 positions along seven radially arranged bands from the dorsal tip of the pecten oculi using electron microscopy. The number of nerve fibers was counted in areas 6 microm in width x the full thickness of the NFL. Myelinated nerve fibers in the NFL were also identified immunohistochemically using anti-myelin basic protein serum. The dorsal area (dorsal, dorso-temporal and dorso-nasal bands) in the retina had thin NFL and contained the largest number of nerve fibers, which were mainly thin and unmyelinated. The ventral area (ventral and ventro-temporal bands) had a thick NFL and contained a relatively small number of nerve fibers, many of which were myelinated. The nasal band had the thickest NFL and contained as many nerve fibers as the dorsal area, with the temporal band showing a high ratio of myelinated fibers. The band had a thick NFL and contained many nerve fibers with a relatively low ratio of myelinated fibers. The relationship between the number and composition of nerve fibers in the NFL to the chicken visual characteristics was discussed. Although the myelin in the chicken retina was loose type, the myelin-forming cells were similar in appearance to dense oligodendrocytes. retina, morphometry, myelinated fiber, nerve fiber layer.     

Quantitative analysis of the optic nerve of the horse (Thoroughbred)

Three optic nerves (L1, R2, R3) 12-18 mm behind the eyeball of the horse (Thoroughbred) were investigated quantitatively under light and electron microscopes. Thin sections at the thickness of 0.35 microm were cut, stained by toluidine blue and observed under the light microscope. The areas of the optic nerve and the axon bundles were 20.03 +/- 1.04 and 16.59 +/- 0.79 mm2 (mean +/- SD, n=3), respectively. The axon numbers for optic nerve L1, R2 and R3, estimated from light micrographs, were about 481 x 10(3), 543 x 10(3), and 494 x 10(3), respectively. Axons of optic nerve L1 were also counted from electron micrographs and the total number of 488 x 10(3) was received. Furthemore, axon diameters of optic nerve L1 were also measured from electron micrographs. The diameter of a circle with the same peripheral length as an axon, was regarded as its diameter. The medullary sheath of the axon was not included during measuring. Altogether 5,744 axons were measured and axon diameters were in a range of 0.23-12.69 microm, with a mean of 2.56 +/- 1.45 microm (mean +/- SD). A regional difference of axonal diameters was found across the optic nerve: the mean diameter of axons in the centrodorsal region (2.28 microm) was the smallest, and had significant difference with those in several peripheral regions (P<0.05).     

Quantitative analysis of cells in the ganglion cell layer of the chick retina: developmental changes in cell density and cell size

Changes in cell density and size in the ganglion cell layer (GCL) of the retina were studied in chick embryos and post-hatching chicks. The total number of cells in the GCL increased from 3.64 million at embryonic day 8 (E8) to the maximal 7.85 million at E14. After E14, the number of cells decreased to 6.08 million at post-hatching day 1 (P1) and 4.87 million at P8. Cell density in the GCL decreased unevenly according to retinal regions; cell density in the presumptive central area (pCA) of P8-chicks decreased to approximately 45% of that in E8-embryos. Densities of the nasal peripheral retina (NP) and temporal peripheral retina (TP) of P8-chicks decreased to 23 and 18% of E8-embryos, respectively. Differentiation of the central (44,000 cells/mm(2) in pCA) - peripheral (28,000 cells/mm(2) in TP) gradient in cell density was formed by E8. The presumptive dorsal area (pDA) was shaped by E11, but became obscure with age. Although ganglion cell sizes were basically uniform at E8, differentiation occurred with the appearance of larger ganglion cells after E14. Mean size of retinal ganglion cells increased 2.8-fold in the pCA and 3.8-fold in the TP between E8 and P8, accompanying a similar scale of decreases in cell densities.     

Topography of ganglion cells in the retina of the horse

Topography of ganglion cells in the retina of the horse (Thoroughbred) was analyzed in the wholemount retina stained with cresyl violet, and a total number of ganglion cells were estimated. Sizes of ganglion cells were also measured and size spectra were analysed. The main results showed that: (1) a common point in 4 wholemount retinae, based on cell densities and retinal locations, was that a retina could be divided into 5 regions, namely visual streak, nasal, temporal, dorsal and ventral region to the visual streak. A maximum cell density of 4,000 cells/mm2 was found in the visual streak. And a total number of ganglion cells was estimated in a range of 398 x 10(3) - 469 x 10(3), with a mean of 441 x 10(3) +/- 31 x 10(3) (n=4). (2) cell sizes were measured as the mean lengths of the major and minor axes of the somas, and were in a range of 5-53.8 microm. The lowest mean diameter was 14.0 microm (+/- 3.7) in the visual streak and the highest was 25.9 microm (+/- 7.6) in the ventral region. Cell size spectra were unimodal and positively skewed. It is expected that these analyses will provide an anatomical and physiological background for further study of the visual system in the horse.     

Quantitative motor unit action potential analysis of skeletal muscles in the Warmblood horse

Motor unit action potential (MUP) analysis in human medicine is a valuable and important diagnostic technique enabling discrimination between myogenic and neurogenic problems. This study establishes normative data in subclavian, triceps and lateral vastus muscles for clinical application of MUP analysis in the Warmblood horse, and examines whether muscle differences are present. Electromyographic (EMG) needle examination and MUP analysis were performed of the triceps, lateral vastus and subclavian muscles in 7 awake, nonsedated, Warmblood horses age 4-10 years. The amplitude, duration, number of phases and turns were calculated from the recorded superimposed MUPs together with intramuscular and rectal temperatures. No significant differences were found in duration of insertional activity between the 3 muscles. The mean +/- s.d. duration of the insertional activity was 526 +/- 1483 ms. The MUP amplitude of all 3 muscles differed significantly, with the highest amplitude (427 +/- 3.20 microV) in the triceps and the lowest (220 +/- 2.08 microV) in the subclavian muscle. The number of turns of the lateral vastus (3.0 +/- 1.22) was significantly higher than that of the triceps muscle (2.7 +/- 1.51). No differences were found in MUP duration (5.9-6.4 ms).     

Postnatal development of the photoreceptor inner segment of the retina in dogs

In dogs, the retina develops during the postnatal period in a manner similar to that in other animals born with closed eyelids. Photoreceptor inner segments are initially observed as a cytoplasmic bulge protruding sclerad through the external limiting membrane. Outer segment formation begins when a centriole within the inner segment attaches to the distal inner segment cell membrane. A few round mitochondria are observed within the early inner segments. As maturation proceeds, the number of mitochondria within the inner segments increases and the mitochondria elongate, orienting parallel to the long axis of the inner segment.     

Regional specialization of ganglion cell layer of the chick retina

Specialization of the ganglion cell layer (GCL) was studied by Nissl-staining and axonal tract-tracing methods in chicks and chick embryos. The changes in the retinal area and the cell number in the GCL produced a disparity in the cell density that occurred through the two different processes, cell generation (before embryonic days 10-14, E10-14) and cell loss (after E10-14). One high-density area was found in the retinal fundus on E8 (presumptive central area, pCA) and its density decreased toward the peripheral retina. Another high-density area was found in the dorsal retina on E11 (presumptive dorsal area, pDA). Cell densities of the pCA and the pDA on E11 decreased gradually to 25-30% by P1, and after that they further decreased to 40-60% by P30. The pCA was still identified on P30, but the pDA became very obscure by this age. In contrast, ganglion cell sizes increased 5-7 times in the pCA and pDA from E8 to P30, and increased 12 times in the temporal periphery. The present study suggests that the center-peripheral gradient of cell density results from lager scale of cell genesis in the pCA, but not from lager scale of cell loss in the peripheral retina. However, obscuration of the pDA results from equalization of cell density in cellular degeneration processes.     

The presence of megamitochondria in the ellipsoid of photoreceptor inner segment of the zebrafish retina

Although the megamitochondria (MM) were localized in various pathological conditions, normal retina of some mammalian species was reported to include MM for various physiological roles. However, it was not clearly confirmed whether the MM is present in the retina of lower vertebrate as well. In this study, we tried to show the presence of the MM in the zebrafish retina using electron microscopic technique. In all the photoreceptors including rods, cones and double cones of the zebrafish retina, MM were observed in the ellipsoid of inner segment. In the photoreceptor epllipsoid of the zebrafish retina, the mitochondria located in the central portion of the ellipsoid had a highly electron-dense matrix, which were accompanied by the mitochondria with electron-lucent matrix in the apical portion of the ellipsoid. The presence of MM was more clearly discernable in the rods, which were localized under the double cones. This finding is somewhat different from those observed in the previous studies because MM were localized in the inner segment of cones, but were not in those of rods in the case of mammalian retina. Although the exact physiological meaning for the presence of MM in some vertebrate species should be further studied, the present study could show that the MM in the ellipsoid of the retinal photoreceptors was not only restricted in some mammalian species.     

Genetic characterisation of the Uruguayan Creole horse and analysis of relationships among horse breeds

The genetic variability within the Uruguayan Creole horse and its relationship to a group of geographically or historically related breeds (Spanish Pure-bred, Barb, Quarter horse, Paso Fino, Peruvian Paso, Arabian and Thoroughbred horse), was evaluated using 25 loci (seven of blood groups, nine of protein polymorphisms and nine microsatellites) analyzed on a total of 145 Uruguayan Creole horses. In this study, blood group and protein polymorphism variants that are considered to be breed markers of Spanish Pure-bred and Barb horses were detected in the Creole breed. Conversely, some microsatellites and protein polymorphisms alleles were found uniquely in the Creole horse. American horse breeds together with Barb and Arabian horses clearly formed a separate cluster from the Spanish pure-bred and Thoroughbred breeds, as shown by an UPGMA dendrogram based on Nei's standard genetic distance. Data in this study provided evidence for considerable genetic variation within Uruguayan Creole horses and of a distinctive breed profile. Both traits were most likely inherited from the XVIth century Spanish horses, more closely related to Barb than to Spanish Pure-bred.     

Immunohistochemical studies of cone photoreceptors and cells of the inner retina in feline rod-cone degeneration

Previous studies using electroretinography and immunohistochemistry have shown normal cone function and structure in early stages of hereditary rod‐cone degeneration of Abyssinian cats. To further investigate the cone photoreceptors and the inner retina of dystrophic cats, antibodies against green‐ and blue‐sensitive cones and specific cell types of inner retina were used in seven cats with the recessively inherited rod‐cone degeneration, and three normal European short‐haired cats. There was a reduction in number of both types of cones early in the disease. Changes at early stages of disease also occurred among horizontal cells in which there was an extension and a thickening of their lateral processes. The regular configuration of bipolar cells was changed in the more advanced stages of disease and their apical dendrites were lost. Abnormalities were not observed in the amacrine cells and in the ganglion cell layer in any of the present cases. This study shows that the cone system is morphologically abnormal in young cats at an earlier stage of disease than previously shown. The present findings also support the assumption that the inner retina is largely preserved throughout the disease process.     

The development of the inner layer of backfat in fetal and young pigs

The development of the innermost layer of backfat was studied histologically and histochemically in fetuses from Yorkshire sows at 45, 60, 65, 75, 85, 90, 105, 110, and 112 d of gestation. Samples were also obtained from fetuses at 110 d of gestation from Ossabaw sows and from sows selected for high (obese) or low (lead) backfat thickness. Fetuses from some sows were decapitated at 45 d of gestation and samples were obtained from these fetuses at 110 d of gestation. The inner layer develops before 75 d of gestation and has stromal-vascular characteristics that clearly distinguish it from the middle layer of backfat. For instance, a well organized and dense collagen matrix immediately surrounds small fat cell clusters in the inner layer. Most fat cell cluster capillaries (in the inner layer) reacted strongly for ATPase histochemically and are histologically immature. The inner layer morphology and cell size was similar in Ossabaw, obese and lean fetuses. The number of PAS (Periodic Acid Schiff) positive cells were dependent on fetal strain. The number was highest in obese fetuses and lowest in Ossabaws. Comparison of decapitated fetuses and weight-matched controls indicated that the inner layer was most affected by decapitation. A decreased organization and amount of collagen was associated with larger fat cell clusters in decapitated fetuses, and most fat cell cluster capillaries reacted little for ATPase histochemically and were histologically mature. The range in inner-layer fat cell diameter was wider in decapitated fetuses (11 to 55 micron) than in control fetuses (11 to 30 micron). These results demonstrate that the characteristics of the inner layer are clearly different from the other layers and that the amount and organization of collagen was inversely associated with the size of fat cell clusters and fat cells and with the maturity of the adipocyte-associated capillaries.     

Quantitative analysis of animal model lens anatomy: accommodative range is related to fiber structure and organization

Purpose The results of a recent study on accommodation in humans and baboons has revealed that lens fiber structure and organization are key components of the mechanism of accommodation. Dynamic focusing involves the controlled displacement and replacement, or realignment, of cortical fiber-ends at sutures as the mechanism of accommodation at the fiber level. This emended explanation of the mechanism of accommodation raises the following question: as the structure of crystalline lenses are only similar, not identical between species, is accommodative amplitude related to differences in the structure and organization of fibers between species? To address this question, we have quantitatively examined the structure and organization of fibers in a number of the more commonly used animal models (mice, cattle, frogs, rabbits and chickens) for lens research. Materials and methods Lenses (a minimum of 12–18 lenses/species) from mice, cattle, frogs and rabbits were used for this study. Prior to fixation for structural analysis, measurements of the gross shape of the lenses (equatorial diameter, anterior and posterior minor radii [anterior + posterior minor radius = polar axis]) were taken directly through a stereo surgical dissecting microscope equipped with an ocular reticle. Lenses were then prepared for and examined by light (LM), transmission (TEM) and scanning electron microscopy (SEM). Scale computer-assisted drawings (CADs) of lenses and lens fibers were then constructed from quantitative data as described above and from quantitative data contained in micrographs. Results The differences in fiber structure and organization that effect accommodative range arise early in development and are continued throughout lifelong lens growth. In umbilical suture lenses (avian) secondary fibers develop with almost completely tapered anterior ends (85–90% reduction of their measures of width and thickness at the equator). By comparison, in lenses with line sutures (e.g. frogs and rabbits) secondary fibers develop with just a 50–60% reduction in anterior fiber taper. In lenses with Y sutures (mice and cattle), fiber width taper is only 25–40%. However, in all cases, while the taper of the posterior end width of fibers is just slightly less (approx. 15–20%) than that of anterior ends, posterior end thickness is only reduced by one half that of anterior thickness. Conclusions In humans, the mechanism of accommodation at the fiber level involves the controlled realignment of very flattened and flared, rather than tapered fiber-ends at sutures. In this manner, the simultaneous increase in lens thickness and surface curvature in the accommodated state is the result of fiber-ends being overlapped along multiple (9–12) suture branches covering the majority of the anterior and posterior surfaces. The results of this animal study strongly suggest that accommodative range is directly related to quantitative differences in fiber structure and organization in the different suture types. The very broad accommodative range in birds is made possible, at least in part, by the almost complete tapering of fiber-ends at umbilical sutures. In contrast, the essentially negligible accommodative range of animals that have line- and Y-suture lenses is at least partially the result of the fact that these lenses have fibers with very little end taper. Thus, the blunt ends of fibers in line- and Y-suture lenses precludes any significant overlap of end segments to effect accommodation.     

Quantitative detection of atropine-delayed gastric emptying in the horse by the 13C-octanoic acid breath test

The 13C-octanoic acid breath test has been correlated significantly to radioscintigraphy for measurement of gastric emptying indices in healthy horses. The objective of this study was to investigate the validity of the test for measurement of equine delayed gastric emptying, prior to its potential clinical application for this purpose. A model of atropine-induced gastroparesis was used. Gastric emptying rate was measured twice in 8 horses using concurrent radioscintigraphy and/or breath test after treatment i.v. with either atropine (0.035 mg/kg bwt) or saline in randomised order. Analysis of both data sets demonstrated that the atropine treatment had caused a significant delay in gastric emptying rate. Paired breath test data showed an atropine-induced delay in gastric half-emptying time (t 1/2), with no overlap in the 99% CI range (P < 0.001). Significant correlations were found between scintigraphy and 13C-octanoic acid breath test for calculation of both t 1/2 (P < 0.01) and lag phase duration (P < 0.05) in the atropine-delayed emptying results. The mean (s.d.) bias in breath test t 1/2 when compared with scintigraphy was 1.78 (0.58) h. The results demonstrated that the 13C-octanoic acid breath test was an effective diagnostic modality for the measurement of equine delayed gastric emptying. The technique offers advantages to existing methods for clinical investigation, as it is noninvasive, not radioactive, quantitative and requires minimal equipment or training to perform.     

Genetic analysis of three South African horse breeds

Genetic variability at 7 blood-group and 10 biochemical genetic loci was examined in 3 South African horse breeds, the Nooitgedacht, Boerperd and Basuto Pony. Observed heterozygosity for these breeds was intermediate for domestic horses, with the highest heterozygosity in the Boerperd and the lowest in the Basuto Pony. The 3 breeds show greater genetic similarity to each other than to other domestic horse breeds. Compared to other breeds, the South African breeds show greater genetic similarity to breeds such as the Thoroughbred, Holstein, Trakehner and Hanovarian and also to North American breeds such as the Saddlebred, Standardbred and Morgan Horse.     

Histochemical detection of glycoconjugates in the inner perivitelline layer of Japanese quail (Coturnix japonica)

The avian inner perivitelline layer (IPVL), a homologous structure to the mammalian zona pellucida, is deposited between the granulosa cells and the oocyte cell membrane during folliculogenesis. In this glycohistochemical study, a panel of fluorescein isothiocyanate (FITC)-labelled lectins was used to characterise and localise the oligosaccharide sequences of the IPVL glycoproteins at different stages of follicular development in the quail ovary. Deacetylation and sialidase digestion were also performed prior to lectin cytochemistry. Contrary to mammals, where the topographical distribution of these carbohydrates is not uniformly distributed throughout the zona pellucida, indicating the regionalisation of oligosaccharide chains, our results demonstrated a homogenous lectin staining of the comparatively thin IPVL. We also found variations in the presence and distribution of the carbohydrate residues in the IPVL during different stages of follicular growth. The IPVL of pre-vitelline follicles distinctly stains with WGA, sWGA and SBA, demonstrating the presence of D-GlcNAc, Neu5Ac and α-D-GalNac in the glycoproteins of the forming IPVL. No staining was found with ConA (specific for α-D-Man, α-D-Glc), LCA (α-D-Man, α-D-Glc), PNA (β-D-Gal-(1-3)-D-GalNAc, VAA (Gal), DBA (α-D-GalNAc(1-3)-GalNAc and UEA-I (α-L-Fuc). With continuing follicular growth of the oocyte and the follicle, this staining pattern changed. LCA and PNA-staining in the IPVL became distinctly positive. As the IPVL of immature oocytes distinctly stains with WGA/sWGA-FITC, but spermatozoa do not bind to immature zona, it appears questionable that carbohydrate residues detected by WGA/sWGA play a major role in sperm-IPVL binding, as suggested in previous investigations.     

In vitro formation of holes on the inner perivitelline layer of quail ovum by chicken spermatozoa

The aim of the present study was to examine whether chicken semen can be substituted for quail semen to conduct in vitro experiments on fertilization. Chicken spermatozoa was incubated with the inner perivitelline layer (IPL) isolated from the largest follicle in the quail ovary under in vitro conditions. The perforation of chicken and quail spermatozoa were assessed by counting the number of all visible holes in the pieces of IPL. No difference was found in the number of holes formed by the chicken sperm and quail IPL interaction compared with that between intraspecies. In addition, the number of holes in the IPL was significantly increased with the increase in sperm concentration from 1 × 10⁶ to 8 × 10⁶ sperm/mL (P < 0.05). Interestingly, after treatment of chicken spermatozoa with 2.5 mg/mL of the solubilized quail IPL followed by incubation with the intact chicken IPL, the number of holes in the intact chicken IPL was significantly decreased as compared with that of spermatozoa treated without the solubilized IPL (P < 0.05). This indicates that sperm receptors in the solubilized quail IPL and binding ligands on the chicken spermatozoa would find each other and bind, forming complexes and these complexes blocked the interaction between chicken spermatozoa and intact chicken IPL. These results show that: (i) chicken spermatozoa possess the penetrability into quail IPL; and (ii) a high degree of affinity via the receptor interactions exists between chicken spermatozoa and quail IPL. Therefore, it appears that the substitution of chicken semen for quail semen is possible to use as an in vitro technique to examine the sperm‐IPL interaction during fertilization in quail.     

Rectal tears in the horse: an analysis of 35 cases

The records of 35 horses with Grade 3 or 4 rectal tears, presented to the Veterinary Medical Center at Texas A & M University over a five year period, were reviewed. Grade 3 tears were sub-classified according to whether the remaining tissue was serosa (Grade 3a) or mesorectum (Grade 3b). Five horses were destroyed on presentation and 30 were treated by primary suture closure (8 horses), faecal diversion alone (9 horses) or in combination with suture closure (11 horses) and packing of the tear with medicated gauze sponges (two horses). Faecal diversion was achieved with a temporary indwelling rectal liner (TIRL) in 19 horses and colostomy in one. Survival was related to classification of the tear, efficacy of first aid measures administered at time of injury and method of treatment. Seventy-four per cent of horses with Grade 3a tears and 44 per cent of those with Grade 3b tears survived. Grade 4 tears had a grave prognosis. Horses given adequate first aid before admission had a better survival rate. With proper patient selection, primary closure of the tear with sutures yielded excellent results. In horses which were not candidates for suture closure alone, a combination of faecal diversion and suturing gave better results than faecal diversion only. In addition, selected horses were treated successfully by packing the rectal tear with gauze sponges. The results demonstrate the value of a TIRL to divert faeces and appropriate first aid measures in treating rectal tears.