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.     

Effect of the Type and Number of Prostaglandin Treatments on Corpus Luteum, the Largest Follicle and Progesterone Concentration in Dairy Cows

Lactating dairy cows (n = 72) with a mature corpus luteum (CL) (diameter of > or = 17 mm) determined by ultrasonography and having a follicle with a diameter of > or = 10 mm were randomly assigned to four groups. Cows were treated with cloprostenol i.m. once or twice, or with dinoprost i.m. once or twice with an 8-h interval. The ovaries of each cow were scanned daily by transrectal ultrasonography to measure the changes in the areas of CL and the largest follicle and to determine the occurrence of ovulation. Oestrus was verified twice daily. In addition, blood sample was withdrawn from each cow daily for measuring progesterone (P4) concentrations. Significant decreases in the percentage changes relative to areas of CL and P4 concentrations or increases in the percentage changes in the area of the largest follicle on day 0 were detected in each group during the experiment. However, the type of the drug and the number of the treatments had no significant effect on those parameters. Cows ovulated with or without showing oestrus (group A) and cows exhibiting no oestrus and ovulation (group B) were also evaluated. In contrast to the mean area of the CL and the mean concentration of P4 on day 0, the mean area of the largest follicles between the two groups on day 0 differed significantly. Significant decreases in the percentage changes relative to the area of the CL and P4 concentration or increases in the percentage changes relative to the area of the largest follicle during the experiment were detected in both groups; however, there were no group differences. Treatment of dairy cows with two injections of prostaglandins (cloprostenol or dinoprost) at an 8-h interval resulted in more cows being observed in oestrus within 5 days after treatment and having significantly higher pregnancy rate than those treated with a single prostaglandin injection.     

Coagulase gene typing of Staphylococcus aureus isolated from cows with mastitis in southeastern Brazil

A typing procedure based on polymorphism of the coagulase gene (coa) was used to discriminate Staphylococcus aureus isolated from Minas Gerais dairy cows with mastitis. Amplification of the gene from the 64 S. aureus isolates produced 27 different polymerase chain reaction (PCR) products; 60 isolates showed only 1 amplicon, and 4 showed 2 amplicons. The isolates were grouped into 49 types by analyzing the restriction fragment length polymorphism (RFLP) of the coa gene; the 10 most common types accounted for 39% of the isolates. The results demonstrate that many variants of the coa gene are present in the studied region, although only a few predominate.     

Bacterial and fungal pathogens isolated from corneal ulcerations in domesticated elephants (Elephas maximus maximus) in Sri Lanka

Infective corneal diseases are common among the domesticated elephants in Sri Lanka, causing morbidity including blindness leading to loss of revenue to their owners. From 140 elephants of different ages and both sexes, 36 animals (25.7%) had evidence of keratitis, corneal ulcers, corneal opacities, and some had foreign bodies in their eyes. Nine elephants (6.4%) had lesions in both eyes (6.41%). Cultures for both bacteria and fungi were obtained from 26 corneal ulcers, including the nine elephants with bilateral lesions. The other 10 animals could not be restrained for sample collection. Swabs from the normal corneas of an additional 20 elephants without signs of any ophthalmic diseases were also collected. Twenty-three of the 35 (65.71%) samples from affected corneas yielded bacterial pathogens, and 14 (40%) also had fungal isolates. None of them yielded a fungal isolate alone. The predominant bacteria isolated were Staphylococcus aureus , beta hemolytic streptococci and coliforms. Fusarium , Cladosporium , Curvularia and Aspergillus species were the primary fungal isolates. No bacteria or filamentous fungi were isolated from the eyes with the normal corneas. Microbial identification including fungal isolations is suggested in the management of infective corneal diseases in elephants.     

Some aspects of crop variation in ‘Golden Delicious’ apple trees

Fruit-set of ‘Golden Delicious’ apple trees was found to be largely independent of seasonal differences and much more closely related to the initial number of blossom clusters, when compared with many other apple cultivars under English conditions. In one young orchard, yield became increasingly a function of trunk girth during the first 3 cropping-years, but the trees then abruptly developed a biennial rhythm. Thereafter, blossom production was negatively correlated with the number of clusters in the previous year rather than with the number of fruits.Applications of daminozide or GA₃ appeared to increase or decrease, respectively, the amount of return bloom without altering the negative correlation with the number of clusters in the previous year. Chemical fruit-thinning with a mixture of carbaryl and NAA also appeared to be effective in increasing return bloom.     

Use of artificial neural networks to estimate production variables of broilers breeders in the production phase

1. Although the poultry industry uses state-of-the-art equipment and up-to-date services, in Brazil it generally makes decisions involving all its production variables based on purely subjective criteria. This paper reports the use of artificial neural networks to estimate performance in production birds belonging to a South Brazilian poultry farm. 2. Recorded data from 22 broiler production breeder flocks were obtained, from April, 1998 to December, 1999, which corresponded to 689 data lines of weekly recordings. 3. These data were processed by artificial neural networks using the software NeuroShell 2 version 4.0 (Ward Systems Group). The artificial neural network models generated were compared and selected based on their largest determination coefficient (R2), lowest Mean Squared Error (MSE), as well as on a uniform scatter in the residual plots. The authors conclude that it is possible to explain the performance variables of production birds, with the use of artificial neural networks. 4. The method allows the decisions made by the technical staff to be based on objective, scientific criteria, allows simulations of the consequences related to these decisions, and reports the contribution of each variable to the variables under study.     

Histopathologic patterns of nephropathy in naturally acquired canine visceral leishmaniasis

Although the nephropathy of visceral leishmaniasis (VL) is known both in humans and dogs, histopathologic alterations have not been thoroughly studied. We examined renal alterations in 55 dogs with naturally acquired VL compared with five noninfected dogs from an endemic area in northeastern Brazil. Glomerulonephritis was found in 55 dogs, interstitial alterations in 53 dogs, and tubular changes in 43 dogs with VL. The glomerular alterations found were minor glomerular abnormalities (n = 8, 14.5%), focal segmental glomerulosclerosis (n = 10, 18.2%), mesangial proliferative glomerulonephritis (n = 17, 32.7%), membranoproliferative glomerulonephritis, (n = 18, 30.9%), crescentic glomerulonephritis (n = 1, 1.8%), and chronic glomerulonephritis (n = 1, 1.8%). Morphometric and ultrastructural studies complemented the analysis. The five control animals exhibited no glomerular alterations. The glomerular lesions were related to functional alterations. Considering that the alterations of canine and human nephropathy in VL are very similar, the data obtained in this study constitute an important contribution to the understanding of canine and human VL nephropathy.     

Antigens from Rhipicephalus sanguineus ticks elicit potent cell-mediated immune responses in resistant but not in susceptible animals

In the present study we compared the immunological reactions between Rhipicephalus sanguineus tick-infested susceptible (dogs and mice) and tick-resistant hosts (guinea pigs), elucidating some of the components of efficient protective responses against ticks. We found that T-cells from guinea pigs infested with adult ticks proliferate vigorously in the presence of concanavalin A (ConA), whereas ConA-induced cell proliferation of tick-infested mice and dogs was significantly decreased at 43.1 and 94.0%, respectively, compared to non-infested controls. Moreover, cells from mice and dogs submitted to one or three successive infestations did not exhibit a T-cell proliferative response to tick antigens, whilst cells from thrice tick-infested guinea pigs, when cultured with either a tick extract or tick saliva, displayed a significant increase in cell proliferation. Also, we evaluated the response of tick-infested mice to a cutaneous hypersensitivity test induced by a tick extract. Tick-infested mice developed a significant immediate reaction, whereby a 29.9% increase in the footpad thickness was observed. No delayed-type hypersensitivity (DTH) reaction was detected. Finally, the differential cell count at the tick attachment site in repeatedly infested mice exhibited a 6.6- and 4.1-fold increase in the percentage of eosinophils and neutrophils, respectively, compared to non-infested animals, while a decrease of 77.0-40.9 in the percentage of mononuclear cells was observed. The results of the cutaneous hypersensitivity test and the cellular counts at the tick feeding site for mice support the view that tick-infested mice develop an immune response to R. sanguineus ticks very similar to dogs, the natural host of this species of tick, but very different from guinea pigs (resistant host), which develop a DTH reaction in addition to a basophil and mononuclear cell infiltration at the tick-attachment site. In conclusion, saliva introduced during tick infestations reduces the ability of a susceptible animal host to respond to tick antigens that could stimulate a protective immune response. As a consequence, the animals present a lack of DTH response and disturbed cellular migration to tick feeding site, which can represent a deficient response against ticks.     

Disseminated pythiosis in three horses

Three cases of equine subcutaneous pythiosis with dissemination to the internal organs were investigated. The subcutaneous lesions were observed on the mammary gland, nostrils and limbs of the infected horses. Histopathological analysis of the infected tissues revealed a strong eosinophilic reaction, with macrophages, mast cells and giant cells. Sparsely septated hyphal filaments of 4-6 microm diameter were identified in the center of the eosinophilic areas. Specific fluorescent antibody against Pythium insidiosum confirmed the hyphae in the infected tissues in three examined horses. In one of the three cases, the DNA sequences amplified from the infected subcutaneous tissues and internal organs, revealed that P. insidiosum's 18S SSU rDNA amplicons shared 100% identity with those sequences deposit in GenBank. This is the first report confirming by immunochemical and genetic techniques that P. insidiosum can disseminated from superficial to deep structures.