Association between Neospora caninum seropositivity and reproductive disorders in girolando cows / [Associação entre a soropositividade para Neospora caninum e distúrbios reprodutivos em vacas girolando]

The aim of this study was to investigate the presence of anti-Neospora caninum antibodies in Girolando cows, in order to evaluate the association between seropositivity and reproductive disorders. Blood samples were collected from 40 dairy cows in their reproductive phase from the cranial superficial epigastric vein. The blood samples were tested using the Indirect Fluorescence Antibody Test (IFAT) to detect anti-N.caninum antibodies. The serological results were used to verify whether there was any association with the manifestation of reproductive disorders based on data from the records of reproductive history from 2017 to 2018 as well as the clinical observations of the herd throughout this study. The Fisher exact test was used to verify the existence of an association between the serology and reproductive disorders, adopting a 95% confidence level. The serological results showed a 27.5% seroprevalence in the herd for N. caninum, however, after statistical analysis, no association between seropositivity and reproductive disorders was found in the evaluated herd. Although the studied population is infected with Neospora caninum, we can infer that anti-Neospora caninum antibodies present in Girolando dairy cows at the UFRRJ Dairy Cattle Facility are not associated with the occurrence of reproductive disorders.(AU)

O objetivo deste estudo foi investigar a presença de anticorpos anti-Neospora caninum em vacas Girolando, avaliando-se a associação entre a soropositividade e os distúrbios reprodutivos. Foram coletadas amostras de sangue da veia epigástrica superficial cranial de 40 vacas leiteiras em fase reprodutiva, sendo as amostras testadas pela reação de imunofluorescência indireta (RIFI) para detecção de anticorpos anti-N. caninum. A partir dos resultados sorológicos, foi realizada a verificação de associação, ou não, com a manifestação de distúrbios reprodutivos, coletados nos registros de histórico reprodutivo entre 2017 e 2018 e observações do rebanho no transcorrer do estudo. O teste exato de Fisher foi utilizado para verificar a existência de associação entre a sorologia e os distúrbios reprodutivos, adotando-se nível de confiança de 95%. O resultado do estudo demonstrou uma soroprevalência no rebanho de 27,5% para N. caninum, contudo, após análise estatística, não foi confirmada a associação entre soropositividade e distúrbios reprodutivos no rebanho avaliado. Apesar de a população estudada estar infectada com o Neospora caninum, pode-se inferir que anticorpos anti-Neospora caninum presentes em vacas leiteiras Girolando do Setor de Bovinocultura de Leite da UFRRJ não estão associados à ocorrência de distúrbios reprodutivos.(AU)

Early pregnancy diagnosis at 21 days post artificial insemination using corpus luteum vascular perfusion compared to corpus luteum diameter and/or echogenicity in Nelore heifers.

The objective was to compare the use of corpus luteum (CL) vascular perfusion to CL diameter and/or echogenicity to diagnose pregnancy at 21 d after timed-AI. Ovaries of Nelore heifers were assessed using ultrasonography in B-mode and color Doppler simultaneously 21 d after timed-AI (n = 113). Objective evaluations were performed using an image processing software to extract the number of colored pixels (ColorPix), diameter (mm) and echogenicity/mm² (EchoPix) of the CL. Subjective evaluations of the CL were performed by five evaluators using scores of estimated vascular perfusion area of color Doppler scan videos and estimated CL size and qualitative echogenicity of B-mode scan videos. The reference pregnancy diagnosis was performed 33 d after timed-AI using an ultrasonic device. Corpus luteum ColorPix, diameter and EchoPix were highly correlated (P < 0.001) with pregnancy. Pregnancy diagnosis accuracy, sensitivity, and negative predictive value were not different for CL ColorPix and diameter and was less with use of EchoPix compared to the other parameters. Size and vascular perfusion scores were correlated to the greatest extent (0.88-0.94) with the respective objective values within evaluator. The results from the ROC curve analysis indicated a smaller area under the curve for qualitative echogenicity compared to CL size and vascular perfusion. Corpus luteum vascular perfusion was the only subjective evaluation that when assessed there were no false negative pregnancy diagnoses. In conclusion, the use of the objective CL diameter resulted in the same efficacy as CL vascular perfusion evaluations for early pregnancy diagnosis in Nelore heifers.

Use of different pressures for transvaginal follicular aspiration in mares / Uso de diferentes pressões para aspiração folicular transvaginal em éguas

The success of transvaginal follicular aspiration in mares can be influenced by several factors, such as vacuum pump pressure levels. The present study aimed to investigate the effect of different negative pressures (150, 280 and 400mmHg) of the vacuum pump on the oocyte recovery in the mares. The mares (n=10) were undergoing follicular aspiration using three different negative pressures for three consecutive estrous cycles as follows: G150 = 150mmHg (n = 10); G280 = 280mmHg (n = 10); G400 = 400mmHg (n = 10). Every estrous cycle, the group that the mare would participate was drawn, and each animal participated once in each group. Only preovulatory follicle was used, about 30 to 36 hours after application of hCG. To compare the results, the chi-square test was used (5% significance) and Fisher exact test, when recommended. Thirty preovulatory follicles (diameter 36.1±1.80mm) were aspirated and ten oocytes were recovered (33.3%). There was no statistical difference between the experimental groups (p=0.59). Thus, accord to the results observed in this study, we could conclude that the negative pressure of the vacuum pump used was not efficient to increase oocyte recovery.(AU)

O sucesso da técnica de aspiração folicular transvaginal em éguas pode ser influenciado de maneira determinante por diversos fatores, tais como níveis de pressão da bomba de vácuo. Diante disso, o presente experimento visou investigar o efeito de diferentes pressões negativas (150, 280 e 400mmHg) da bomba de vácuo sobre a taxa de recuperação de oócitos em éguas. As éguas (n=10) foram submetidas à aspiração folicular utilizando-se três diferentes pressões negativas por três ciclos estrais consecutivos, da seguinte maneira: G150= 150mmHg (n=10); G280= 280mmHg (n=10); G400= 400mmHg (n=10). A cada ciclo estral, sorteava-se o grupo do qual a égua participaria, sendo que cada animal integrou um grupo somente uma vez. Foi puncionado somente folículo pré-ovulatório, em torno de 30 a 36 horas após a aplicação do hCG. Os resultados foram comparados utilizando-se o teste qui-quadrado (a 5% de significância) e o Fisher Exato, quando recomendados. Foram aspirados 30 folículos pré-ovulatórios (diâmetro 36,1±1,80mm) e recuperados 10 oócitos (33,3%). Não houve diferença estatística entre os grupos experimentais (P=0,59). Dessa forma, mediante os resultados obtidos no presente estudo, foi possível concluir que a pressão negativa da bomba de vácuo utilizada não se mostrou determinante para elevar a recuperação oocitária.(AU)

Production of a cloned calf from a fetal fibroblast cell line

The present study examined the in vitro and in vivo development of bovine nuclear-transferred embryos. A bovine fetal fibroblast culture was established and used as nucleus donor. Slaughterhouse oocytes were matured in vitro for 18 h before enucleation. Enucleated oocytes were fused with fetal fibroblasts with an electric stimulus and treated with cytochalasin D and cycloheximide for 1 h followed by cycloheximide alone for 4 h. Reconstructed embryos were cultured for 7-9 days and those which developed to blastocysts were transferred to recipient cows. Of 191 enucleated oocytes, 83 (43.5 percent) were successfully fused and 24 (28.9 percent) developed to blastocysts. Eighteen freshly cloned blastocysts were transferred to 14 recipients, 5 (27.8 percent) of which were pregnant on day 35 and 3 (16.7 percent) on day 90. Of the three cows that reached the third trimester, one recipient died of hydrallantois 2 months before term, one aborted fetus was recovered at 8 months of gestation, and one delivered by cesarian section a healthy cloned calf. Today, the cloned calf is 15 months old and presents normal body development (378 kg) and sexual behavior (libido and semen characteristics).

Production of a cloned calf from a fetal fibroblast cell line.

The present study examined the in vitro and in vivo development of bovine nuclear-transferred embryos. A bovine fetal fibroblast culture was established and used as nucleus donor. Slaughterhouse oocytes were matured in vitro for 18 h before enucleation. Enucleated oocytes were fused with fetal fibroblasts with an electric stimulus and treated with cytochalasin D and cycloheximide for 1 h followed by cycloheximide alone for 4 h. Reconstructed embryos were cultured for 7-9 days and those which developed to blastocysts were transferred to recipient cows. Of 191 enucleated oocytes, 83 (43.5%) were successfully fused and 24 (28.9%) developed to blastocysts. Eighteen freshly cloned blastocysts were transferred to 14 recipients, 5 (27.8%) of which were pregnant on day 35 and 3 (16.7%) on day 90. Of the three cows that reached the third trimester, one recipient died of hydrallantois 2 months before term, one aborted fetus was recovered at 8 months of gestation, and one delivered by cesarian section a healthy cloned calf. Today, the cloned calf is 15 months old and presents normal body development (378 kg) and sexual behavior (libido and semen characteristics).

Establishment of new murine embryonic stem cell lines for the generation of mouse models of human genetic diseases.

Embryonic stem cells are totipotent cells derived from the inner cell mass of blastocysts. Recently, the development of appropriate culture conditions for the differentiation of these cells into specific cell types has permitted their use as potential therapeutic agents for several diseases. In addition, manipulation of their genome in vitro allows the creation of animal models of human genetic diseases and for the study of gene function in vivo. We report the establishment of new lines of murine embryonic stem cells from preimplantation stage embryos of 129/Sv mice. Most of these cells had a normal karyotype and an XY sex chromosome composition. The pluripotent properties of the cell lines obtained were analyzed on the basis of their alkaline phosphatase activity and their capacity to form complex embryoid bodies with rhythmically contracting cardiomyocytes. Two lines, USP-1 and USP-3, with the best in vitro characteristics of pluripotency were used in chimera-generating experiments. The capacity to contribute to the germ line was demonstrated by the USP-1 cell line. This cell line is currently being used to generate mouse models of human diseases.

Establishment of new murine embryonic stem cell lines for the generation of mouse models of human genetic diseases

Embryonic stem cells are totipotent cells derived from the inner cell mass of blastocysts. Recently, the development of appropriate culture conditions for the differentiation of these cells into specific cell types has permitted their use as potential therapeutic agents for several diseases. In addition, manipulation of their genome in vitro allows the creation of animal models of human genetic diseases and for the study of gene function in vivo. We report the establishment of new lines of murine embryonic stem cells from preimplantation stage embryos of 129/Sv mice. Most of these cells had a normal karyotype and an XY sex chromosome composition. The pluripotent properties of the cell lines obtained were analyzed on the basis of their alkaline phosphatase activity and their capacity to form complex embryoid bodies with rhythmically contracting cardiomyocytes. Two lines, USP-1 and USP-3, with the best in vitro characteristics of pluripotency were used in chimera-generating experiments. The capacity to contribute to the germ line was demonstrated by the USP-1 cell line. This cell line is currently being used to generate mouse models of human diseases

Cryopreservation of Bos taurus vs Bos indicus embryos: are they really different?

Cryopreservation with storage at very low temperatures is essential to make full use of this technology for both biological and commercial reasons. However, most mammalian cells will die if exposed to these temperatures unless they are exposed to cryoprotectant solutions and cooled and warmed at specific rates. Lowering temperature below 0 degree C introduces the risk of intracellular ice formation, which likely increases rapidly as the temperature falls. Evidence indicates that ice formation during cooling can cause significantly more damage than ice formation during warming. Comparisons of the toxicity of various cryoprotectants indicated that ethylene glycol (EG) is a nontoxic compound for murine and bovine embryos. The 3.6 M EG solution resulted in similar high survival rates when compared with nonfrozen embryos; deleterious effects of high concentrations of EG became apparent at 7.2 M. The use of EG provides a nontoxic method for the rapid and simplified controlled freezing of in vivo bovine compact morulae-early blastocyst, avoiding the risk of injury caused by high concentrations of cryoprotectants usually required for vitrification. However, in vivo embryos used for freezing and thawing require further studies to understand the ultrastructural changes during the freezing procedure with EG as the single cryoprotectant, especially between Holstein and Nelore cows. This paper describes the ultrastructure of bovine compact morulae-early blastocysts derived by in vivo methods from Holstein and Nelore cows to investigate the fresh morphology as well as that after exposure to cryoprotectant and cryopreservation by conventional slow freezing, quick freezing (nitrogen vapor), and vitrification.