Manipulation of follicle development to ensure optimal oocyte quality and conception rates in cattle

Over the last several decades, a number of therapies have been developed that manipulate ovarian follicle growth to improve oocyte quality and conception rates in cattle. Various strategies have been proposed to improve the responses to reproductive biotechnologies following timed artificial insemination (TAI), superovulation (SOV) or ovum pickup (OPU) programmes. During TAI protocols, final follicular growth and size of the ovulatory follicle are key factors that may significantly influence oocyte quality, ovulation, the uterine environment and consequently pregnancy outcomes. Progesterone concentrations during SOV protocols influence follicular growth, oocyte quality and embryo quality; therefore, several adjustments to SOV protocols have been proposed depending on the animal category and breed. In addition, the success of in vitro embryo production is directly related to the number and quality of cumulus oocyte complexes harvested by OPU. Control of follicle development has a significant impact on the OPU outcome. This article discusses a number of key points related to the manipulation of ovarian follicular growth to maximize oocyte quality and improve conception rates following TAI and embryo transfer of in vivo-and in vitro-derived embryos in cattle.

Fluorescence polarization assay, competitive enzyme-linked immunosorbent assay (ELISA-C) and indirect ELISA for the diagnosis of brucellosis in buffaloes (Bubalus bubalis)

The objective of the present study was to compare the performance of three serological tests for diagnosis of Brucella abortus infections in buffaloes (Bubalus bubalis). Serum samples collected from 696 adult females were submitted to the competitive enzyme-linked immunosorbent assay (ELISAC), (I-ELISA), fluorescence polarization test (FPA), 2-mercaptoethanol test (2-ME) and complement fixation test (CFT). The gold standard was the combination of CFT and 2-ME, considering as positive the reactors in both CFT and 2-ME, and as negative those non-reactors. ROC analyses were done for C-ELISA, I-ELISA and FPA and the Kappa agreement index were also calculated. The best combinations of relative sensitivity (SEr) and relative specificity (SPr) and Kappa were given by C-ELISA (96.9%, 99.1%, and 0.932, respectively) and FPA (92.2%, 97.6 and 0.836, respectively). The C-ELISA and FPA were the most promising confirmatory tests for the serological diagnosis of brucellosis in buffaloes, and for these tests, cut-off values for buffaloes may be the same as those used for bovines.

Use of Leptospira spp. strains isolated in Brazil in the microscopic agglutination test applied to diagnosis of leptospirosis in cattle herds in eight brazilian states

Sarmento A.M.C., Azevedo S.S., Morais Z.M., Souza G.O., Oliveira F.C.S., Goncales A.P., Miraglia F. & Vasconcellos S.A. 2012. [Use of Leptospira spp. strains isolated in Brazil in the microscopic agglutination test applied to diagnosis of leptospirosis in cattle herds in eight brazilian states.] Emprego de estirpes Leptospira spp. isoladas no Brasil na microtecnica de soroaglutinacao microscopica aplicada ao diagnostico da leptospirose em rebanhos bovinos de oito estados brasileiros. Pesquisa Veterinaria Brasileira 32(7);601-606. Universidade de Sao Paulo, Faculdade de Medicina Veterinaria e Zootecnia, Departamento de Medicina Veterinaria Preventiva e Saude Animal, Av. Prof. Dr. Orlando Marques de Paiva 87, Sao Paulo, SP 05508-270, Brazil. E-mail: savasco@usp.br The aim of this study was to investigate the adequacy of the use of autochthonous strains of leptospires isolated in Brazil, added to antigen collection of the microscopic agglutination test (MAT) applied to the diagnosis of bovine leptospirosis. By means of non-probability sampling, 109 farms and 9,820 cattle, females at reproductive age were chosen from 85 municipalities in the states of Goias, Mato Grosso, Mato Grosso do Sul, Minas Gerais, Parana, Rio Grande do Sul, Santa Catarina and Sao Paulo. Among the 9,820 examined animals, 5,806 (59.12%) were reactants at MAT for at least one serovar using the 23 reference serovars. Employing the collection of reference serovars and the ten autochthonous strains, 6,400 (65.24%) reactants and significant difference (p=0.001) was found. The most probable serovars identified by the collection of reference antigens were Hardjo (43.03%), Shermani (20%), Wolfi (9.96%), Grippothyphosa (5.42%) and Pomona (4.28%). With the collection amplified with the ten strains isolated in Brazil, the most probable serovars were Hardjo (31%), Guaricura-M4/84 (22.50%), Shermani (15.43%), Wolffi (4.76%), Grippothyphosa (3.71%) and Autumnalis (3.24%). The serovar Guaricura, strain M4/84, isolated from bovines and buffaloes in the State of Sao Paulo, was ranked as one of the three most probable serovars in the states of Goias, Mato Grosso, Mato Grosso do Sul, Minas Gerais and Sao Paulo. The addition of autochthonous strains to the MAT antigen collection provided the confirmation of the diagnosis of leptospirosis in 594 cattle (6%) which have been classified as non-reactants by the reference collection (p=0.001).

Molecular confirmation of ovine herpesvirus 2-induced malignant catarrhal fever lesions in cattle from Rio Grande do Norte, Brazil

Molecular findings that confirmed the participation of ovine herpesvirus 2 (OVH-2) in the lesions that were consistent with those observed in malignant catarrhal fever of cattle are described. Three mixed-breed cattle from Rio Grande do Norte state demonstrated clinical manifestations that included mucopurulent nasal discharge, corneal opacity and motor incoordination. Routine necropsy examination demonstrated ulcerations and hemorrhage of the oral cavity, corneal opacity, and lymph node enlargement. Significant histopathological findings included widespread necrotizing vasculitis, non-suppurative meningoencephalitis, lymphocytic interstitial nephritis and hepatitis, and thrombosis. PCR assay performed on DNA extracted from kidney and mesenteric lymph node of one animal amplified a product of 423 base pairs corresponding to a target sequence within the ovine herpesvirus 2 (OVH-2) tegument protein gene. Direct sequencing of the PCR products, from extracted DNA of the kidney and mesenteric lymph node of one cow, amplified the partial nucleotide sequences (423 base pairs) of OVH-2 tegument protein gene. Blast analysis confirmed that these sequences have 98-100% identity with similar OVH-2 sequences deposited in GenBank. Phylogenetic analyses, based on the deduced amino acid sequences, demonstrated that the strain of OVH-2 circulating in ruminants from the Brazilian states of Rio Grande do Norte and Minas Gerais are similar to that identified in other geographical locations. These findings confirmed the active participation of OVH-2 in the classical manifestations of sheep associated malignant catarrhal fever.

Genome wide scan for quantitative trait loci affecting tick resistance in cattle (Bos taurus × Bos indicus)

Abstract Background In tropical countries, losses caused by bovine tick Rhipicephalus (Boophilus) microplus infestation have a tremendous economic impact on cattle production systems. Genetic variation between Bos taurus and Bos indicus to tick resistance and molecular biology tools might allow for the identification of molecular markers linked to resistance traits that could be used as an auxiliary tool in selection programs. The objective of this work was to identify QTL associated with tick resistance/susceptibility in a bovine F2 population derived from the Gyr (Bos indicus) × Holstein (Bos taurus) cross. Results Through a whole genome scan with microsatellite markers, we were able to map six genomic regions associated with bovine tick resistance. For most QTL, we have found that depending on the tick evaluation season (dry and rainy) different sets of genes could be involved in the resistance mechanism. We identified dry season specific QTL on BTA 2 and 10, rainy season specific QTL on BTA 5, 11 and 27. We also found a highly significant genome wide QTL for both dry and rainy seasons in the central region of BTA 23. Conclusions The experimental F2 population derived from Gyr × Holstein cross successfully allowed the identification of six highly significant QTL associated with tick resistance in cattle. QTL located on BTA 23 might be related with the bovine histocompatibility complex. Further investigation of these QTL will help to isolate candidate genes involved with tick resistance in cattle.

Luteal function and follicular growth following follicular aspiration during the peri-luteolysis period in bos indicus and crossbred cattle

Follicular estradiol triggers luteolysis in cattle. Therefore, the control of follicle growth and steroidogenesis is expected to modulate luteal function and might be used as an anti-luteolytic strategy to improve embryo survival. Objectives were to evaluate follicular dynamics, plasma concentrations of estradiol and luteal lifespan in Bos indicus and crossbred cows subjected to sequential follicular aspirations. From D13 to D25 of a synchronized cycle (ovulation = D1), Nelore or crossbred, non-pregnant and non-lactating cows were submitted to daily ultrasound-guided aspiration of follicles >6 mm (n = 10) or to sham aspirations (n = 8). Diameter of the largest follicle on the day of luteolysis (7.4 +/- 1.0 vs 9.7 +/- 1.0 mm; mean +/- SEM), number of days in which follicles >6 mm were present (2.3 +/- 0.4 vs 4.6 +/- 0.5 days) and daily mean diameter of the largest follicle between D15 and D19 (6.4 +/- 0.2 vs 8.5 +/- 0.3 mm) were smaller (p <0.01) in the aspirated group compared with the control group, respectively. Aspiration tended to reduce (p< 0.10) plasma estradiol concentrations between D18 and D20 (2.95 +/- 0.54 vs 4.30 +/- 0.55 pg/ml). The luteal lifespan was similar (p > 0.10) between the groups (19.6 +/- 0.4 days), whereas the oestrous cycle was longer (p <0.01) in the aspirated group (31.4 +/- 1.2 vs 21.2 +/- 1.3 days). Hyperechogenic structures were present at the sites of aspiration and were associated with increase in concentration of progesterone between luteolysis and oestrus. It is concluded that follicular aspiration extended the oestrous cycle and decreased the average follicular diameter on the peri-luteolysis period but failed to delay luteolysis.

Developmental and Epigenetic Anomalies in Cloned Cattle

Many of the developmental anomalies observed in cloned animals are related to foetal and placental overgrowth, a phenomenon known as the 'large offspring syndrome' (LOS) in ruminants. It has been hypothesized that the epigenetic control of imprinted genes, that is, genes that are expressed in a parental-specific manner, is at the root of LOS. Our recent research has focused on understanding epigenetic alterations to imprinted genes that are associated with assisted reproductive technologies (ART), such as early embryo in vitro culture (IVC) and somatic cell nuclear transfer (SCNT) in cattle. We have sought and identified single nucleotide polymorphisms in Bos indicus DNA useful for the analysis of parental-specific alleles and their respective transcripts in tissues from hybrid embryos derived by crossing Bos indicus and Bos taurus cattle. By analysing differentially methylated regions (DMRs) of imprinted genes SNRPN, H19 and the IGF2R in cattle, we demonstrated that there is a generalized hypomethylation of the imprinted allele and the biallelic expression of embryos produced by SCNT when compared to the methylation patterns observed in vivo (artificially inseminated). Together, these results indicate that imprinting marks are erased during the reprogramming of the somatic cell nucleus during early development, indicating that such epigenetic anomalies may play a key role in mortality and morbidity of cloned animals.

Differential gene expression and developmental competence in in vitro produced bovine embryos

The embryonic developmental block occurs at the 8-cell stage in cattle and is characterized by a lengthening of the cell cycle and an increased number of embryos that stop development. The maternal-embryonic transition arises at the same stage resulting in the transcription of many genes. Gene expression studies during this stage may contribute to the understanding of the physiological mechanisms involved in the maternal-embryonic transition. Herein we identified genes differentially expressed between embryos with high or low developmental competence to reach the blastocyst stage using differential display PCR. Embryos were analysed according to developmental kinetics: fast cleavage embryos showing 8 cells at 48 h post insemination (hpi) with high potential of development (F8), and embryos with slow cleavage presenting 4 cells at 48 hpi (54) and 8 cells at 90 hpi (S8), both with reduced rates of development to blastocyst. The fluorescence DDPCR method was applied and allowed the recovery of 176 differentially expressed bands with similar proportion between high and low development potential groups (52% to F8 and 48% in S4 and S8 groups). A total of 27 isolated fragments were cloned and sequenced, confirming the expected primer sequences and allowing the identification of 27 gene transcripts. PI3KCA and ITM2B were chosen for relative quantification of mRNA using real-time PCR and showed a kinetic and a time-related pattern of expression respectively. The observed results suggest the existence of two different embryonic genome activation mechanisms: fast-developing embryos activate genes related to embryonic development, and slow-developing embryos activate genes related to cellular survival and/or death.

Real-time PCR assay targeting the actin gene for the detection of Cryptosporidium parvum in calf fecal samples

Cryptosporidium parvum infection is very important with respect to public health, owing to foodborne and waterborne outbreaks and gastrointestinal illness in immunocompetent and immunocompromised persons. In cattle, infection with this species manifests either as a subclinical disease or with diarrheal illness, which occurs more often in the presence of other infectious agents than when alone. The aim of this study was to develop a real-time polymerase chain reaction (PCR) assay for the detection of C. parvum in calf fecal samples and to compare the results of this assay with those of the method routinely used for the diagnosis of Cryptosporidium spp., nested PCR targeting the 18S rRNA gene. Two hundred and nine fecal samples from calves ranging in age from 1 day to 6 months were examined using real-time PCR specific for the actin gene of C. parvum and by a nested PCR targeting the 18S rRNA gene of Cryptosporidium spp. Using real-time PCR detection, 73.2% (153 out of 209) of the samples were positive for C. parvum, while 56.5% (118 out of 209) of the samples were positive for Cryptosporidium spp. when the nested PCR amplification method was used for the detection. The analytical sensitivity of the real-time PCR was approximately one C. parvum oocyst. There was no significant nonspecific DNA amplification of any of the following species and genotype: Cryptosporidium andersoni, Cryptosporidium baileyi, Cryptosporidium bovis, Cryptosporidium canis, Cryptosporidium galli, Cryptosporidium ryanae, Cryptosporidium serpentis, or avian genotype II. Thus, we conclude that real-time PCR targeting the actin gene is a sensitive and specific method for the detection of C. parvum in calf fecal samples.

Prevalência e fatores de risco de leptospirose bovina no Estado do Maranhão

Silva F.J., Conceicao W. L. F., Fagliari J.J., Girio R.J.S., Dias R. A., Borba M. R. & Mathias L. A. 2012. [Prevalence and risk factors of bovine leptospirosis in the State of Maranhao, Brazil.] Prevalencia e fatores de risco de leptospirose bovina no Estado do Maranhao. Pesquisa Veterineria Brasileira 32(4): 303-312. Departamento de Medicina Veterinaria Preventiva e Reproducao Animal, Faculdade de Ciencias Agrarias e Veterinarias, Universidade Estadual Paulista, Via de Acesso Professor Paulo Donato Castellane s/n, Zona Rural, Jaboticabal, SP 14884-900, Brazil. E-mail: fjsepi@gmail.com Prevalence and risk factors of bovine leptospirosis in the State of Maranhao were investigated. Based on production parameters that vary across different production systems, management practices, the purpose of exploitation, the average size of herds and market systems, the state was divided in four sampling circuits. The study aimed to investigate the epidemiological features of bovine leptospirosis in the State of Maranhao, in order to determine the prevalence of the infection in cattle and herds, to determine the occurrence of serovars of Leptospira spp., to identify risk factors associated with leptospirosis in cattle and to differentiate the livestock circuits itself regarding the prevalence of leptospirosis. The survey was conducted in 136 herds in the circuit I, in which 841 >= 24 months old females were analyzed; 238 in the circuit II and 2,582 females were analyzed; 122 in the circuit III and 869 females were analyzed; 77 in the circuit IV and 540 females were analyzed; a total of 573 herds and 4,832 females were analyzed. The presence of antibodies against Leptospira spp. was verified by microscopic agglutination test (MAT). Of the 4,832 cows examined, 1,904 (35.94%, CI 95% = 33.01% -38.98%) were positive. Of the 573 herds, 380 (64.81%, CI 95% = 61.10% -68.35%) were positive. Serovars Hardjo and Wolffi were the most frequent in the state. The circuit III showed the lowest prevalence of leptospirosis in all comparisons. The variables presence of horses (p = 0.000), presence of capybaras (p = 0.034) and herds with up to 32 adult females (p = 0.002) were identified as risk factors for leptospirosis.

Essays on the history of Brazilian dipterology. III. Three remarkable notices from the 18th century, mainly related to myiasis-producing flies (Cochliomyia and Dermatobia)

Essays on the history of Brazilian dipterology. III. Three remarkable notices from the 18th century, mainly related to myiasis-producing flies (Cochliomyia and Dermatobia). This paper registers reports about dipterans made by three Portuguese who lived in Brazil during the 18th century. Luiz Gomes Ferreira, in his book "Erario mineral" ["Mineral revenue"], wrote curious passages related with myiasis-causing flies of the genus Cochliomyia. Jose Rodrigues de Mello registered, in Latin verses, the folklore for curing myiases caused by Cochliomyia hominivorax in cattle. Luiz dos Santos Vilhena, in the last of his twenty letters dealing with several aspects of life in Brazil, made reference to horseflies, human bot flies and mosquitos.

Morphology and Involution of the Yolk Sac during Early Gestation Bovine (Bos indicus)

Background: The bovine yolk sac derives from visceral endoderm and its development occurs between days 18-23 of gestation. The study of this membrane is important for comparative data and has already been performed in rodents, sheep and in cattle, especially Bos taunts. In species Bos indicus the yolk sac has not quite been studied and is believed that there are morphological differences between these species. The yolk sac undergoes a process of involution and degeneration during embryonic development and none vestige of it is found in late gestation. The period in which occurs the involution of the yolk sac coincides with the period of increased pregnancy loss in cattle, and changes in the morphology of this membrane may indicate the reasons for such high loss rates. Thus, considering that the yolk sac is important for embryonic circulation and metabolic transmission, besides participating actively in the process of cattle placentation, this study aimed characterize morphologically the involution of the bovine yolk sac. Materials, Methods & Results: The early gestational period was determined between days 20 and 70 post-insemination (p.i), according to the exterior characteristics of embryo/fetus. For macroscopic analyzes the uterus was dissected to expose the fetal membranes and subsequently the embryo/fetus was photographed. The samples were fixed for light microscopy and transmission electron microscopy. The yolk sac that emerges from the ventral part of the embryo was prominent and composed by a central part with two thin peripheral projections of different lengths. The bovine yolk sac with about 9 cm on day 25 p. i. of pregnancy permanently decreased its total length during this study. Histologically, the yolk sac is composed of three cell layers: the mesothelium, the mesenchyme and the endoderm. In mesenchyme are found blood islets. In the endoderm are formed cells invaginations toward the mesenchyme originating small canaliculi. The ultrastructure of yolk cells presented many mitochondria, rough endoplasmic reticulum, vesicles, euchromatin and the presence of two nucleoli, Discussion: The real first blood circulation in the bovine is attached with the development of yolk sac, differently from other membranes, such as the corium, that does not present evidence of vascularization by the age of 20-30 days. The erythroblasts found in the yolk sac are related to vasculogenesis and the process of differentiation of blood cells during the erythropoiesis. It could be observed on the histology of the yolk sac, in embryos of 30-50 days old, the presence of canaliculi and small folds of the epithelium. The canaliculi collapse is associated with the degeneration of the endoderm wall of the yolk sac. The organelles present in the endoderm cells of the yolk sac are associated with the function of protein metabolism and in the exchange of substances between the mesenchyme and the mesothelium, For these findings, could be observed that the yolk sac epithelium is found active until the 50th day of gestation, and thereafter regresses. However, remnants of this membrane may be present until the 70th day, These features may represent a presence of an active chorionvitelline placenta in this period responsible for the maintenance of pregnancy whereas the chorioallantoic placenta is not definitively established.

Isolation and characterization of sodium 2-fluoroacetate from Mascagnia rigida using chromatography and infrared spectroscopy

Sodium monofluoroacetate was first identified in Dichapetalum cymosum, a South African plant that can cause livestock poisoning and death. After, several other plants also showed to contain this toxin, which leads to the "sudden death". Mascagnia rigida, a well identified poisonous plant, commonly found in northeast of Brazil also cause sudden death in cattle, which shows clinical signs similar to those produced by the ingestion of plants that contain monofluoroacetate. Our aim was to identify the toxic compound present in the aqueous extract of M. rigida. For this purpose, the dried and milled plant was extracted; the extract was lyophilized and submitted to successive chromatographic process, until the desired purity of the active compound was achieved. The study of this material by planar chromatography and by infrared spectrometry indicated that the toxin can be a mixture of mono, di and trifluoroacetate. (C) 2012 Elsevier Ltd. All rights reserved.

Essays on the history of brazilian dipterology: III. Three remarkable notices from the 18th century, mainly related to myiasis-producing flies (Cochliomyia and Dermatobia)

This paper registers reports about dipterans made by three Portuguese who lived in Brazil during the 18th century. Luiz Gomes Ferreira, in his book "Erário mineral" ["Mineral revenue"], wrote curious passages related with myiasis-causing flies of the genus Cochliomyia. José Rodrigues de Mello registered, in Latin verses, the folklore for curing myiases caused by Cochliomyia hominivorax in cattle. Luiz dos Santos Vilhena, in the last of his twenty letters dealing with several aspects of life in Brazil, made reference to horseflies, human bot flies and mosquitos.