Host-parasite-environment relationship, morphology and molecular analyses of Henneguya eirasi n. sp. parasite of two wild Pseudoplatystoma spp. in Pantanal Wetland, Brazil

A new myxosporean species, Henneguya eirasi n. sp., is described parasitizing the gill filaments of Pseudoplatystoma corruscans and Pseudoplatystoma fasciatum (Siluriformes: Pimelodidae) caught in the Patanal Wetland of the state of Mato Grosso, Brazil. The parasite formed white, elongated plasmodia measuring up to 3mm. Mature spores were ellipsoidal in the frontal view, measuring 37.1 ± 1.8 μm in total length, 12.9 ± 0.8 μm in body length, 3.4 ± 0.3 μm in width, 3.1 ± 0.1 μm in thickness and 24.6 ± 2.2 μm in the caudal process. Polar capsules were elongated and equal in size, measuring 5.4 ± 0.5 μm in length and 0.7 ± 0.1 μm in width. Polar filaments had 12-13 coils. Histopathological analysis revealed that the parasite developed in the sub-epithelial connective tissue of the gill filaments and the plasmodia were surrounded by a capsule of host connective tissue. The plasmodia caused slight compression of the adjacent tissues, but no inflammatory response was observed in the infection site. Ultrastructure analysis revealed a single plasmodial wall connected to the ectoplasmic zone through numerous pinocytotic canals. The plasmodial wall exhibited numerous projections and slightly electron-dense material was found in the ectoplasm next to the plasmodial wall, forming a line just below the wall. Partial sequencing of the 18S rDNA gene of H. eirasi n. sp. obtained from P. fasciatum resulted in a total of 1066 bp and this sequence did not match any of the Myxozoa available in the GenBank. Phylogenetic analysis revealed the Henneguya species clustering into clades following the order and family of the host fishes. H. eirasi n. sp. clustered alone in one clade, which was the basal unit for the clade composed of Henneguya species parasites of siluriform ictalurids. The prevalence of the parasite was 17.1% in both fish species examined. Parasite prevalence was not influenced by season, host sex or host size.     

Light and scanning electron microscopy of Henneguya arapaima n. sp. (Myxozoa: Myxobolidae) and histology of infected sites in pirarucu (Arapaima gigas: Pisces: Arapaimidae) from the Araguaia River, Brazil

In this report, we describe Henneguya arapaima n. sp., a parasite of the gill arch and gall bladder of Arapaima gigas (pirarucu) collected in the Araguaia River, in the municipality of Nova Crixás, Goiás State, central Brazil. The plasmodia were white, round or ellipsoidal and measured 200-600mum. Parasite development was asynchronous and the mature spores were fusiform and had smooth wall. The spores measurements were (range, with means+/-S.D. in parentheses): total length-48.4-53.1mum (51.6+/-3.4mum), body length-13.5-15.2mum (14.2+/-0.8mum), body width-5.1-6.1mum (5.7+/-0.5mum), body thickness-4.7-5.3mum (4.9+/-0.2mum) and caudal process length-38.0-41.2mum (38.3+/-2.9mum). The polar capsules were elongated and of unequal size, with lengths of 6.3-6.8mum (6.5+/-0.2) and 6.2-6.6mum (6.3+/-0.1) for the longest and shortest axes, respectively. Capsule width was 1.4-1.6mum (1.5+/-0.1). Histological analysis showed that the plasmodia occurred in the tunica adventitia of the gall bladder and were delimited by a thin capsule of connective tissue. In the gill arch, the plasmodia were also surrounded by connective tissue similar to the endomesium of striated skeletal muscle cells. Sixty-five juvenile specimens of A. gigas weighing 1.0-25.0kg were examined, 17 (26.1%) of which were infected. Of these, 14 (82.3%) had cysts in the gall bladder, two (11.7%) had cysts in the gill arch and only one (5.9%) had cysts in both organs. When the fish were grouped by weight, the prevalence of infection in fish weighing up to 10.0kg (20.7%) was significantly lower than in fish weighing 10.1-25.0kg (50%) (G=3.93; d.f.=1; p<0.05).     

Effects of fasting on circulating IGF-binding proteins, glucose, and cortisol in channel catfish (Ictalurus punctatus)

The effects of fasting on IGF-binding proteins (IGFBPs), glucose, and cortisol in channel catfish were examined. Fed fish (controls) were compared to 14-, 30-, and 45-day fasted fish and 45-day fasted fish refed for 15 additional days. Body length and weight changes, condition factor (CF), hepatosomatic index (HSI), and plasma glucose and cortisol were assessed to determine growth and metabolic status. Body length and growth rates were inhibited (P<0.05) after 14, 30, and 45 days of fasting. The 14-, 30-, and 45-day fasted fish exhibited hypoglycemia and reduced CF and HSI. Cortisol levels were increased (22.8 +/- 15.2 ng/ml versus 4.7 +/- 3.9 ng/ml) in 30-day fasted fish compared to fed controls (P<0.05). Associated with the increase in cortisol in fasted fish was a concomitant increase in plasma levels of a 20-kDa IGFBP through day 45. A 35- and a 45-kDa IGFBP were also identified but were similar between fed and unfed fish throughout the experiment. At the end of 15 days of refeeding, 20-kDa IGFBP, glucose, and cortisol levels were similar to fed controls. Refeeding also caused an increase in growth rates. These results suggest the existence of a catfish counter part to mammalian IGFBP-1, similar to lower molecular mass IGFBPs reported in other species of fish. These results also suggest that a 20-kDa IGFBP is upregulated during fasting-induced growth inhibition of channel catfish and provide additional evidence of the conserved nature of the IGF-IGFBP-growth axis in fish.     

Longevity of Bolbophorus damnificus infections in channel catfish

The digenean Bolbophorus damnificus infects commercial channel catfish Ictalurus punctatus, causing mortality, lower feed consumption, and reduced growth in surviving fish. The purpose of this study was to determine the length of time for which B. damnificus prodiplostomulum metacercariae (juvenile trematode stage that infects fish) would remain viable (parasite appearing to be intact or exhibiting movement) in channel catfish. Fish (n = 210) were infected with molecularly confirmed B. damnificus cercariae harvested from naturally infected marsh rams-horn snails Planorbella trivolvis. During the first sampling (at 20 d postinfection), 8.3 +/- 3.6 metacercariae/fish (mean +/- SD) were found in the host muscle and visceral organs. The channel catfish were then acclimated to a water temperature of either 18 degrees C or 28 degrees C. After 11 months, 6.8 +/- 3.5 and 5.9 +/- 3.0 metacercariae/fish were found in groups held at 18 degrees C and 28 degrees C, respectively. The mean number of parasites per fish did not significantly differ between fish held at the two temperatures and did not significantly decline over time at either temperature. Fish examined from 13 to 30 months postinfection all contained viable metacercariae that were morphologically and molecularly identified as B. damnificus. At 18 months, 12 metacercariae (of which 11 were intact and 10 displayed movement) were found in the one fish sampled; at 30 months, the last fish sampled contained three intact metacercariae (one displayed slight movement). Our results indicate that B. damnificus metacercariae can remain viable in channel catfish for at least an 18-30-month production cycle during which they have the potential to affect fish growth; in addition, infected fish may serve as intermediate hosts for these metacercariae for at least 2.5 years postinfection.     

Fatal coccidiosis by Isospora icterus (Upton & Whitaker, 2000) in captive Campo Troupial (Icterus jamacaii) in Brazil

An outbreak of coccidiosis by Isospora icterus (I. icterus, Upton & Whitaker, 2000) in captive Campo Troupial (Icterus jamacaii) (Gmelin, 1788) at the Wild Animals Triage Center (IBAMA, Belo Horizonte, Brazil) is described. Clinical history and the necropsy findings documented diarrhea with diffuse necrotic enteritis. Sporulated oocysts (n = 100) had a bilayered wall, were subspherical, and measured 30.1 (27.5-32.5) microm in length and 28.5 (26.2-30.0) microm in width. A polar body but no micropyle was present and the length/width ratio was 1.1 (1.00-1.2). Each oocyst contained two ellipsoidal sporocysts measuring 17.6 (15.0-20.0) microm in length and 12.9 (12.5-15.0) microm in width, with a length/width ratio of 1.4 (1.2-1.5), and with Stieda and sub-Stieda bodies. Each sporocyst contained four sporozoites with granular sporocyst residuum. Oocysts were compatible with those from I. icterus, previously described in Campo Troupial.     

Effects of recombinant bovine somatotropin on growth and abundance of mRNA for IGF-I and IGF-II in channel catfish (Ictalurus punctatus)

Research was conducted to examine growth rates, circulating concentrations of IGF-I, and mRNA abundance levels of IGF-I and IGF-II in channel catfish (Ictalurus punctatus) given recombinant bovine ST (rbST; Posilac, Monsanto Co., St. Louis MO). In the first study, juvenile catfish (5.5 +/- 0.5 g) were randomly assigned to one of three treatments: 1) sham-injected control (one needle puncture per week); 2) rbST (30 microg x g BW(-1) x wk(-1); Posilac); and 3) nonhandled control (control). At the end of the 6-wk study, the fish were weighed, measured for length, and G:F was determined. Compared with sham and control treatments, rbST-treated fish had 48% greater final BW, 14% greater total length, and 52% greater G:F (P < 0.001). In the second study, juvenile catfish (41.1 +/- 1.5 g) were assigned randomly to one of two treatments: 1) sham or 2) rbST. Eight fish per treatment were sampled on d 0, 1, 2, 7, 14, and 21 for blood, muscle, and liver. Relative expression of IGF-I and IGF-II mRNA was determined by real-time PCR and plasma concentrations of IGF-I were measured using a validated fluoroimmunoassay. Circulating concentrations of IGF-I were increased (37.9 +/- 5.5 vs. 22.0 +/- 6.6 ng/mL; P < 0.05) in rbST-injected fish compared with sham-injected controls by d 14. Liver IGF-I and IGF-II mRNA was increased 4.3-and 14.4-fold, respectively, by d 1 in rbST-injected fish compared with controls (P < 0.05); however, abundance of liver IGF-I and IGF-II mRNA did not differ from controls on d 0, 2, 7, 14, and 21. Abundance of muscle IGF-I and IGF-II mRNA did not differ in rbST-injected fish compared with controls throughout the study. Results of the first study demonstrated that rbST improves growth performance of channel catfish. Results of the second study showed that the growth-promoting effects of rbST were not mediated by the expression of IGF-I or IGF-II mRNA in the muscle. Instead, the results suggest that rbST promotes growth by stimulating plasma IGF-I release, possibly through its direct effect on the liver or on local tissues to synthesize IGF-I. The changes in mRNA abundance and plasma concentrations of IGF-I support the role of IGF-I in growth regulation of channel catfish.     

Flavobacterium columnare genomovar influences mortality in channel catfish (Ictalurus punctatus)

Flavobacterium columnare, causal agent of columnaris disease, is pathogenic to many species of freshwater fish throughout the world. The United States channel catfish (Ictalurus punctatus) aquaculture industry is severely impacted by columnaris disease. The majority of the F. columnare isolates recovered from diseased channel catfish belonged to either genomovars I or II. The objective of the present study was to determine if differences existed in the ability of these genomovars to induce mortality in channel catfish. Single strand conformation polymorphism analysis (SSCP) was used to ascribe the isolates used in this study to the appropriate genomovar. Immersion challenge experiments (15min immersion exposure to approximately 5x10(5) to 1x10(6) CFU/mL) were carried out to assess virulence of genomovar I and II isolates to channel catfish. The results demonstrated that genomovar II (n=4) isolates were significantly (P<0.05) more virulent to channel catfish fry (92-100% mortality) than genomovar I (n=3) isolates (0-46% mortality). In vivo adhesion of the genetically characterized F. columnare also correlated (r2=0.73) to increased mortality in the challenged fry. In fingerling channel catfish, significantly higher mortality (P<0.05) resulted with genomovar II isolates ALM-05-182 and ALG-00-530 as compared to all the genomovar I isolates (n=3). Mortality of genomovar II isolate BGFS-27 with similar to genomovar II isolate (ALG-00-530) and two genomovar I isolates (ALM-05-53 and 140). The results suggest that although both genomovars are present in the aquatic environment, genomovar II appears to be more pathogenic for channel catfish.     

Spermatozoa morphology during the breeding season in Thoroughbred stallions in Japan

The morphology of spermatozoa of modern Thoroughbred stallions in Japan was investigated during the breeding season. A total of 299 semen samples were collected from the penises of 16 stallions immediately after service. The rate of abnormalities in sperm heads and tails, spermatozoa with cytoplasmic droplets and slides with medusa cells to total observed slides in each stallion were 3.9 +/- 2.1%, 11.5 +/- 5.9%, 2.4 +/- 2.6% and 20.1%, respectively. The values for the area, length, width and aspect ratio of the stallion sperm head were 12.54 +/- 1.34 microm(2), 5.93 +/- 0.40 microm, 2.69 +/- 0.21 microm and 0.46 +/- 0.05, respectively. With the exception of medusa cells, the features were significantly different among the stallions (P<0.05).     

Sporocyst size of isolates of Sarcocystis shed by the Virginia opossum (Didelphis virginiana)

The Virginia opossum (Didelphis virginiana) is a definitive host for multiple Sarcocystis species including Sarcocystis neurona, one of the causative agents of equine protozoal myeloencephalitis (EPM), a severe, neuromuscular disease of horses. Size and morphologic characteristics of isolates of Sarcocystis shed by the opossum were examined to determine if differences were useful in discriminating between the isolates and/or species. Collections of sporocysts from 17 opossums were molecularly characterized and measured using an ocular micrometer. The mean sporocyst size of isolates of S. neurona was 10.7 microm x 7.0 microm, Sarcocystis falcatula 11.0 microm x 7.1 microm, Sarcocystis speeri 12.2 microm x 8.8 microm, 1085-like isolate 10.9 microm x 6.8 microm, and 3344-like isolate 19.4 microm x 10.5 microm. The length and width of S. speeri were statistically different (p < 0.05) from the sporocysts of other types. The length of S. neurona and S. falcatula sporocysts were statistically different (p < 0.05) from each other and the width of S. falcatula and 1085 differed (p < 0.05). The fifth sporocyst type (3344) was observed, but due to pronounced morphological characteristics, statistical analysis was not performed. There was no consistent difference between the taxa based on internal structure of the sporocyst.     

Pedicle skin flaps in ponies: viable length is related to flap width.

The relationship between pedicle flap width and viable length was characterised for skin flaps of the flank in ponies. Four dorsally based, pedicle type skin flaps of 20 cm in length and 3, 6, 9 or 12 cm in width were created in a random sequence on one flank in each of 10 ponies. Flap survival length was assessed by skin texture and appearance, depilation of hair and wound healing at 14 days after surgery. There was considerable variation between animals in the viable length of flaps of the same width; however, a significant difference in the viable length of flaps of different widths was detected (P = 0.002). The viable length of the 3 cm flaps was significantly different from that of the 12 cm flaps (9.69 +/- 1.1 cm vs 12.97 +/- 1.0 cm, mean +/- se P less than 0.05). There was a positive correlation between flap width and viable length (r2 = 0.141, P = 0.017). There was no effect of flap order (cranial to caudal positioning relative to the other flaps) on viable length (P = 0.286). The results of this study demonstrate a significant relationship between flap width and viable length, confirming a previously unsupported assumption that flap width and viable length are related. These findings suggest that the clinician should employ broad pedicles when using local flaps to reconstruct skin defects on the flank of the horse.     

Comparative pharmacokinetics of oxytetracycline in rainbow trout (Salmo gairdneri) and African catfish (Clarias gariepinus)

A comparative pharmacokinetic study was conducted in rainbow trout (Salmo gairdneri) and African catfish (Clarias gariepinus) following intravenous (i.v.) and intramuscular (i.m.) administration of oxytetracycline (OTC) at a dose rate of 60 mg/kg body weight. Trout and catfish were kept in aerated tap water in tanks at constant temperatures of 12 degrees C and 25 degrees C, respectively. The two- and three-compartment open models adequately described plasma drug disposition in African catfish and rainbow trout respectively, following i.v. OTC administration. Compared to catfish (COP = 86 +/- 10 micrograms/ml) an eightfold higher extrapolated zero time concentration was obtained in trout (COP = 753 +/- 290 micrograms/ml). A significant difference was observed with respect to the relatively large apparent distribution volumes (Vd(area] after i.v. OTC administration (trout, mean value: 2.1 l/kg; catfish, mean value: 1.3 l/kg). The mean final elimination half-lives of both fish species were greater than previously reported in mammals (trout, 89.5 h; catfish, 80.3 h). A mean maximum plasma concentration (Cmax = 56.9 micrograms/ml) was obtained in trout at 4 h after i.m. administration of OTC. In catfish a lower Cmax of 43.4 micrograms/ml was determined at about 7 h. No significant difference was observed with respect to bioavailability following i.m. administration of OTC (trout, 85%; catfish, 86%).     

The effect of high total ammonia concentration on the survival of channel catfish experimentally infected with Flavobacterium columnare

Ammonia concentrations in water can affect the severity of Flavobacterium columnare infections in fish. Two trials lasting 7 d each were conducted to determine the effect of a single immersion flush treatment of total ammonia nitrogen (TAN; 15 mg/L) on the survival of channel catfish Ictalurus punctatus infected with E columnare; the chemical was added while the water flowed continuously through the tanks. Both trials consisted of four treatments: (1) no ammonia exposure and no bacterial challenge (control), (2) ammonia exposure only, (3) bacterial challenge only, and (4) both ammonia exposure and bacterial challenge. Two hours after exposure to ammonia, the highest un-ionized ammonia level was 0.43 mg/L. The percent un-ionized ammonia is based on TAN, temperature, and pH. Caudal fins from three fish in each treatment were sampled at 24 h posttreatment to be analyzed by quantitative real-time polymerase chain reaction (qPCR). No significant difference in survival (mean +/- SE) was noted between the channel catfish in treatment 1 (95.2 +/- 1.2%) and those in treatment 2 (95.6 +/- 1.0%); however, survival in both treatments 1 and 2 differed significantly from that in treatments 3 (8.5 + 4.5%) and 4 (41.8 +/- 12.7%). Treatment 4 catfish had significantly higher survival than treatment 3 catfish. Quantitative PCR data showed that treatment 4 fish had significantly less F. columnare (7.6 x 10(5)) than did treatment 3 fish (1.2 x 10(7)), and treatment 2 fish (8.5 x 10(3)) had significantly less bacteria than did treatment 1 fish (6.9 x 10(4)), indicating that ammonia limited the F. columnare infection. The highest mean concentration of the bacteria (3.9 x 10(7)) was found on moribund fish. The ammonia concentrations tested did not negatively influence fish survival but interfered with the infection process. An in vitro assay was also conducted to evaluate the direct effects of ammonia on F columnare.     

Sample prevalence and molecular characterisation of Cryptosporidium andersoni within a dairy herd in the United Kingdom

Bovine cryptosporidiosis is usually an acute diarrhoeal disease of young calves caused by Cryptosporidium parvum. However, chronic infection with Cryptosporidium andersoni has been associated with gastritis, reduced milk yield and poor weight gain in adult cattle. Here we describe the first genetic confirmation and characterisation of C. andersoni from cattle in the United Kingdom and its sample prevalence within a dairy herd. Oocysts measured 7.5+/-0.4 microm x 5.5+/-0.4 microm (7.0-8.5 microm x 4.5-6.5 microm) with a length-to-width ratio of 1.37 (1.08-1.60). The within-herd sample prevalence was 16% (95% confidence intervals=10.4-21.6%). Nested polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) and sequence analysis of the small subunit rDNA was used to confirm the species and characterise the isolates. Due to the lack of overt, acute, clinical symptoms, the incidence, prevalence and importance of this parasite is probably currently underestimated in cattle in the UK. The potential for zoonotic transmission is unknown.     

Effect of size and temperature on the quantity of immunoglobulin in channel catfish, Ictalurus punctatus

Concentrations of serum immunoglobulin (Ig) were determined for 30 channel catfish from pond water at 10 degrees C. These values were compared to measurements of 15 channel catfish from pond water at 30 degrees C. Channel catfish from 10 degrees C pond water had no significant (P greater than 0.05) different Ig concentrations (mean, 398 mg/dl) than catfish from 30 degrees C pond water (mean, 367 mg/dl). Serum Ig concentrations appear not to be different in cold (10 degrees C) vs warm (30 degrees C) pond water for 37.5-45 cm catfish. Channel catfish, 7.5-15 cm (n = 24) had significantly (P less than 0.05) lower Ig levels (mean, 104 mg/dl) than catfish either 7.5-25.5 cm (n = 57, mean, 232 mg/dl) or 37.5-45 cm (n = 45, mean, 388 mg/dl). Also, catfish 17.5-25.5 cm had a significantly (P less than 0.05) less Ig than catfish 37.5-45 cm. The concentrations of serum Ig increase with size (P = 0.0001) of catfish. The mean Ig concentration for 7.5-45 cm catfish (n = 126) was 263 mg/dl. The Ig concentration range was 44 to 650 mg/dl of serum.     

Relation between Physical Properties of the Zona Pellucida and Viability of Bovine Embryos after Slow-freezing and Vitrification

In vitro-produced bovine morulae/blastocyst embryos (n = 119) were slow-frozen and vitrified and the physical alterations of the zona pellucida (ZP) was observed by scanning electron microscopy (SEM) to find an explanation for the loss of developmental capacity of the embryos after freezing/thawing. A control group was provided, in which embryos (n = 38) were neither frozen nor vitrified. Embryos were in vitro-cultured in a standard CO2 Heraeus incubator and their viability was assessed 24 and 48 h after the start of culture, evaluating their morphological aspect. After 24 h of culture, embryo survival rate for slow-freezing/thawed (n = 23), vitrified/thawed (n = 20) and control embryos (n = 20) was 39, 27 and 90%, and 35, 14 and 65% after 48 h of culture, respectively. For evaluation of physical changes occurring in ZP, 20 embryos were slow-frozen, 18 were vitrified and 18 were used as control. All embryos were fixed, dried and examined under an SEM. Embryo's diameter, as well as the number of pores and their diameter was measured in squares of 6.4 microm width. We observed that, on average, the diameter of the embryos (92.26 +/- 10.15 microm) did not differ significantly among all embryos. As far as the diameter of the pores in the outer surface of the ZP is concerned, the results revealed a significant difference (P < 0.05) between control (0.48 +/- 0.0025 microm), slow-frozen (0.34 +/- 0.0007 microm) and vitrified (0.27 +/- 0.0006 microm) embryos. For the number of pores, statistical differences (p < 0.05) were observed between control and vitrified embryos (45.4 +/- 7.3 vs 38.2 +/- 8.2). It is possible that ZP functions as a barrier which is positive when dealing with pathogens, but is harmful when nutrients were supplied from the outside, especially at 48 h of culture. Results indicate that the steps of cryopreservation cause alterations in ZP, with irreversible damage on the further developmental competence of bovine embryos.     

Morphological diagnosis of infective larvae of Libyostrongylus douglassii (Cobbold, 1882) Lane, 1923 and L. dentatus Hoberg, Lloyd and Omar, 1995 (Nematoda: Trichostrongylidae) of ostriches

The differentiation of the species of the Libyostrongylus genus is only possible with the obtainment of the adult parasites in the ostriches proventriculus and gizzard. The present work confirms that it is possible to differentiate the infective larvae of L. douglassii and L. dentatus allowing the differential diagnosis of these species by fecal culture. To show this, adult females from both species were collected from ten proventriculus from adult ostriches and separated by species. Both groups were macerated individually added to sterilized feces for standard fecal cultures. The infective larvae were recovered, identified, quantified and measured. All proventriculus analyzed were parasitized by Libyostrongylus spp. and a clear heterogeneous location for each species was observed. The infective larvae from the fecal cultures of macerated L. douglassii presented a mean total length of 874.3+/-33.80 microm, and a short sheath tail (29.5+/-4.11 microm) with acute termination. The infective larvae from the macerated L. dentatus presented mean total length of 856.0+/-43.63 microm, long sheath tail (61.2+/-9.52 microm) with filamentous termination. The mean measures of the tails of both species had a significant difference. The differentiation of the infective larvae of L. douglassii and L. dentatus by fecal cultures will facilitate the diagnosis of both species for further understanding the Libyostrongylus biology.     

紫花苜蓿根腐病原菌--拟枝孢镰刀菌的鉴定及其生物学特性研究

本文对从内蒙古赤峰巴雅尔草业基地紫花苜蓿根部分离到的菌株 BYE27-2-5进行了致病性测定、形态学和rDNA-ITS 序列分析鉴定及生物学特性测定。结果表明,该菌菌丝生长、产孢和孢子萌发的最适温度分别为25,30和28℃;pH 值5．0～11．0均宜于菌丝生长,产孢最适 pH 8．0,孢子萌发最适 pH 7．0;光暗交替利于菌丝生长,光照利于产孢和孢子的萌发;孢子在相对湿度低于75％不萌发,高于95％萌发较快;葡萄糖蛋白胨培养基利于菌丝生长和孢子萌发,马铃薯蔗糖琼脂培养基利于产孢;碳源中葡萄糖利于菌丝生长和孢子萌发,蔗糖利于产孢;氮源中蛋白胨利于菌丝生长,酵母膏利于产孢和孢子萌发;菌丝致死温度为54℃（10 min）,分生孢子的致死温度为48℃（10 min）;根据其形态特征和 rDNA-ITS 序列分析结果,鉴定其为拟枝孢镰刀菌（Fusarium sporotrichioide ）。     

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).     

The Effects of Cryopreservation on the Morphometric Dimensions of Iberian Red Deer (Cervus elaphus hispanicus) Epididymal Sperm Heads

Computer-automated sperm-head morphometry was used in this study to determine the effects of cryopreservation on red deer sperm-head morphometry. Epididymal sperm samples were collected from 40 mature stags and were divided. One portion was diluted at room temperature in a Tris-citrate egg yolk medium, containing 6% glycerol. A microscope slide was prepared from single extended sperm samples prior to freezing. The remainder of each sample was frozen in nitrogen vapours. After thawing, sperm smears were prepared as described above. All slides were air dried and stained with Hemacolor. The sperm-head dimensions for length, width, area, perimeter and shape factor (length/width), for a minimum of 135 spermatozoa were determined for each slide by means of the Sperm-Class Analyser (SCA). Firstly, our results show that cryopreservation substantially reduced (p < 0.001) sperm motility and plasma membrane and acrosome integrities. In addition, sperm heads were significantly smaller in cryopreserved spermatozoa than in the companion extended samples for area (32.05 microm2 vs 32.56 microm2; p < 0.05), length (8.46 microm vs 8.53 microm; p < 0.0001) and shape factor (1.833 vs 1.849; p < 0.0001) for all stags. These differences were found within 29 of 40 stags (75%) for at least three of the morphometric parameters. The individual variability (CV) of sperm head measurements from extended samples was negatively correlated (p < 0.005) with the per cent of change in sperm head measurements after cryopreservation for area (r = -0.465), width (r = -0.483) and perimeter (r = -0.375). Thus, the lower the sperm head variability in the extended samples, the greater the sperm change as a consequence of the cryopreservation. These results suggest that the variability (heterogeneity) in sperm head dimensions of individual stags may be a good indicator of sperm freezability.