Is estradiol valerate an alternative to estradiol benzoate in promoting the synchronization of ovulation and timed artificial insemination in suckled Bos indicus beef cows?

The objective of the present study was to evaluate the effect of estradiol valerate administered at the beginning of the ovulation synchronization protocol on the pregnancy rate of Bos indicus cows. In the experiments, the following products from MSD, Sao Paulo, Brazil were used: estradiol valerate (EV), estradiol benzoate (EB), intravaginal progesterone device (P4), estradiol cypionate (EC), equine chorionic gonadotropin (eCG) and cloprostenol (PGF). In Experiment 1, Bos indicus cows (n=899) with a body condition score (BCS) of 2.76 ± 0.01 were included in a 3 (device) × 2 (protocol: 5 mg of EV or 2 mg of EB) factorial design. There were three types of P4 devices: a new device (New), a device previously used for 9 days (1×), and a device previously used for 18 days (2×). Nine days later (D9), the P4 device was removed, and cows received 300 IU of eCG. In addition, cows in the EB group received 1 mg of EC and 265 µg of PGF. Timed artificial insemination (TAI) was performed 48 h after P4 device removal in the EB group (TAI48) and 54 h after P4 device removal in the EV group (TAI54). In Experiment 2, Bos indicus cows (n=434) with a BCS of 2.62 ± 0.01 received a new P4 device or one previously used for 9 days and 5 mg of EV. On D9, all cows received 300 IU of eCG, and the P4 devices were removed and distributed in TAI48 and TAI54 cows. In Experiment 3, Bos indicus cows (n=429) with a BCS of 2.80 ± 0.01 were divided into the control and EV/EC groups. All cows received a P4 device. In addition, cows in the control group received 2 mg of EB, and cows in the EV/EC group received 5 mg of EV on D0. On D9, all cows received 1 mg of EC and 300 IU of eCG, and the P4 devices were removed. Cows in the control group also received 265 µg of PGF. All cows were inseminated 48 h after the removal of the P4 device. In Experiment 1, there was no effect of the interaction between protocol and P4 device on the occurrence of estrus (P=0.45) or on the pregnancy per artificial insemination ratio (P/AI; P=0.30). In addition, the occurrence of estrus and P/AI were not different between in the two estradiol groups (P=0.12 and P=0.82) and across the types of intravaginal P4 device (P=0.91 and P=0.47). In Experiment 2, the pregnancy rate was lower (tendency) in TAI48 cows (P=0.07). In Experiment 3, the estrus rate (P=0.12) and P/AI (P=0.56) were similar between the experimental groups. In summary, protocols using estradiol valerate without exogenous ovulation induction require adjustments in the timing of AI from 48 to 54 h after P4 device removal. However, a combination of estradiol valerate at the beginning of the protocol and estradiol cypionate nine days later successfully induced ovulation in Bos indicus cows inseminated 48 h after P4 device removal.

Efficacy of prepartum vaccination against neonatal calf diarrhea in Nelore dams as a prevention measure.

BACKGROUND: Neonatal calf diarrhea (NCD) is the leading cause of calf morbidity and mortality in beef cattle. Cow's vaccination in last stage of pregnancy is one of the most important measures to mitigate the risk of NCD outbreaks. The aim of this study was to evaluate the efficacy of prepartum single dose vaccination against NCD, especially Bovine Rotavirus type A (BoRVA) and Bovine Coronavirus (BCoV), in Nelore dams and offspring. A total of 117 pregnant cows (n = 81) and heifers (n = 36) were distributed in two groups, vaccinated (VAC: cows = 40; heifers = 19) and non-vaccinated (NVAC: cows = 41; heifers = 17). Vaccination occurred between 60 to 50 days before the expected calving date with a single dose of a water-in-oil (W/O) vaccine, and NVAC group received a dose of saline solution 0.9%. Blood samples were collected before vaccination and 30 days after to evaluate the antibody (Ab) response. Specific IgG1 Abs against BoRVA and BCoV were measured by using an Enzyme Linked Immuno Sorbent Assay (ELISA). Calves' births were monitored, and the transference of passive immunity was evaluated. Diarrhea was monitored in the first 30 days of age, and fecal samples were collected for identification of the etiological agent. RESULTS: Higher titers of IgG1 Ab against BoRVA and BCoV was observed in the VAC group than NVAC group in the cow (P < 0.0001) and total dams categories (P < 0.0001). The titer of specific IgG1 Abs in the calves' serum reflected the dams response, observing higher IgG1 Ab titers for BoRVA (P < 0.0016) and BCoV (P < 0.0095) in the offspring born to VAC cows and higher IgG1 Ab titers for BoRVA(P < 0.0171) and BCoV (P < 0.0200) in the offspring born to VAC total dams. The general incidence of diarrhea observed was 18.6% (11/59) and 29.3% (17/58) in the calves born to the VAC and NVAC group, respectively. CONCLUSIONS: Prepartum vaccination with a single dose of the vaccine tested increased the titers of IgG1 Ab against BCoV and BoRVA, and it could be used as a preventive strategy to decrease the NCD occurrence in Nelore calves.

Identification, molecular characterization and factors associated with occurrences of Cryptosporidium spp. in calves on dairy farms in Brazil.

Cattle are an important source of zoonotic species of Cryptosporidium for humans. The aim of this study was to investigate the presence of Cryptosporidium, identify the species and determine the risk factors relating to environment, animals and management among dairy calves in eight Brazilian states. A total of 408 fecal samples from calves aged 1-60 days were analyzed. An epidemiological questionnaire was completed. Sample screening was performed using Ziehl-Neelsen technique and the positive samples were subjected to nested PCR. Cryptosporidium species were identified by means of the PCR-RFLP technique, using SSPI, ASEI and MBOII enzymes. The Ziehl-Neelsen technique showed that 89.7% (35/39) of the farms and 52.9% (216/408) of the samples were positive. Through nested PCR, these protozoa were detected in 54.6% of the samples. The 56 samples subjected to PCR-RFLP presented Cryptosporidium parvum. There was higher prevalence of the parasite in animals aged 7 to 28 days (62.6%). Diarrhea, ages between seven and 28 days and a spring water source were factors associated with the risk of infection. The calf hutch-type management system was associated with reduced infection. These findings demonstrate the high level of Cryptosporidium spp. circulation in cattle herds and the predominance of the species C. parvum.

Identification, molecular characterization and factors associated with occurrences of Cryptosporidium spp. in calves on dairy farms in Brazil / Identificação, caracterização molecular e fatores associados à ocorrência de Cryptosporidium spp. em bezerros de propriedades leiteiras no Brasil

Abstract Cattle are an important source of zoonotic species of Cryptosporidium for humans. The aim of this study was to investigate the presence of Cryptosporidium, identify the species and determine the risk factors relating to environment, animals and management among dairy calves in eight Brazilian states. A total of 408 fecal samples from calves aged 1-60 days were analyzed. An epidemiological questionnaire was completed. Sample screening was performed using Ziehl-Neelsen technique and the positive samples were subjected to nested PCR. Cryptosporidium species were identified by means of the PCR-RFLP technique, using SSPI, ASEI and MBOII enzymes. The Ziehl-Neelsen technique showed that 89.7% (35/39) of the farms and 52.9% (216/408) of the samples were positive. Through nested PCR, these protozoa were detected in 54.6% of the samples. The 56 samples subjected to PCR-RFLP presented Cryptosporidium parvum. There was higher prevalence of the parasite in animals aged 7 to 28 days (62.6%). Diarrhea, ages between seven and 28 days and a spring water source were factors associated with the risk of infection. The calf hutch-type management system was associated with reduced infection. These findings demonstrate the high level of Cryptosporidium spp. circulation in cattle herds and the predominance of the species C. parvum.

Resumo O gado é uma fonte importante de espécies zoonóticas de Cryptosporidium para o homem. O objetivo deste estudo foi investigar a presença de Cryptosporidium, identificar a espécie e determinar os fatores de risco relacionados ao meio ambiente, aos animais e ao manejo em bezerros leiteiros em oito estados brasileiros. Um total de 408 amostras fecais de bezerros, com idade entre 1 e 60 dias, foram analisadas. Um questionário epidemiológico foi preenchido. A triagem das amostras foi realizada pela técnica de Ziehl-Neelsen, e as amostras positivas foram submetidas à "nested" PCR. As espécies de Cryptosporidium foram identificadas pela técnica de PCR-RFLP, utilizando-se as enzimas SSPI, ASEI e MBOII. A técnica de Ziehl-Neelsen mostrou que 89,7% (35/39) das fazendas e 52,9% (216/408) das amostras foram positivas. Por meio de nested PCR, esses protozoários foram detectados em 54,6% das amostras. As 56 amostras submetidas à PCR-RFLP apresentaram Cryptosporidium parvum. Houve maior prevalência do parasita em animais de 7 a 28 dias (62,6%). Diarreia, idade entre sete e 28 dias, e fonte de água mineral foram fatores associados ao risco de infecção. O sistema de manejo do tipo "casinha" para bezerros foi associado à redução da infecção. Esses achados demonstram o alto nível de Cryptosporidium spp. em circulação nos rebanhos bovinos e o predomínio da espécie C. parvum.

Prevalence and risk factors for agents causing diarrhea (Coronavirus, Rotavirus, Cryptosporidium spp., Eimeria spp., and nematodes helminthes) according to age in dairy calves from Brazil.

The present study attempted to verify the prevalence of and risk factors for diarrhea-causing agents in dairy calves from Brazil. Additionally, ages with a higher risk of occurrence for each agent were verified by means of the receiver operating characteristic (ROC) curve. The collections were performed on 39 farms, belonging to 29 municipalities located in eight states of Brazil. It was possible to conclude that the prevalence of Coronavirus, Rotavirus, Cryptosporidium spp., Eimeria spp., and nematodes was 7.20% (95% CI 4.54-9.78), 6.37% (95% CI 3.85-8.89), 51.52% (95% CI 45.26-55.57), 3.46% (95% CI 2.24-4.67), and 3.46% (95% CI 2.24-4.67), respectively. Ages with higher probabilities of occurrence of these diseases in calves were < 10, > 8, > 6, > 37, and > 36 days, respectively. Diarrhea occurred more significantly (P < 0.0001) in animals less than 21 days old and mainly on those receiving milk through automatic feeders (P < 0.001). Cryptosporidium spp. were a risk factor for the occurrence of Rotavirus, and vice versa (P = 0.0039) and presented a positive correlation with Coronavirus (P = 0.0089). Calves that drink water from rivers, streams, and ponds had a higher chance of being infected by Eimeria spp. (P < 0.0001), as well as developing infection by nematodes (P < 0.0001). The results found in this study highlight the importance of studying the agents of diarrhea together, once they act as coinfection where the losses triggered for the owners will involve some of these agents simultaneously.

Longitudinal surveillance of rotavirus A genotypes circulating in a high milk yield dairy cattle herd after the introduction of a rotavirus vaccine.

Worldwide, neonatal diarrhea is one of the most important health issues affecting dairy calves, and rotavirus A (RVA) is one of its primary causes. Among the measures to mitigate the risk of diarrhea outbreaks, cow vaccination stands out as one of the most important. However, the immune pressure resulting from routine vaccination may be able to select specific G and P genotypes in RVA field strains. This study aimed to determine the frequency and intensity of neonatal diarrhea and the incidence of RVA and attempted to monitor the G and P genotypes present in the RVA strains circulating in a high milk yield cattle herd vaccinated with RVA G6P[5] strain. Fecal samples (n = 1220) from 122 Holstein heifer calves between 0-30 days old that were born from RVA-vaccinated cows were collected at 10 different time points, regardless of the presence or absence of diarrhea. The presence of RVA in fecal samples was determined by the polyacrylamide gel electrophoresis (PAGE) technique and confirmed by reverse transcription polymerase chain reaction (RT-PCR). G and P amplicons from 10 RVA-positive fecal samples from calves of different ages and collections were subjected to nucleotide sequencing. The proportion of the calves and fecal samples that were positive for RVA were 62.3% (76/122) and 8.1% (99/1220), respectively. Using sequence analysis, all 10 RVA field strains presented genotype G10P[11]. The protection of G6P[5] vaccination is clear, as this genotype was not detected in this study, and it is known that vaccination against RVA reduces the incidence of diarrhea independent of genotype involved. This result demonstrates the importance of epidemiological monitoring of RVA genotypes circulating in vaccinated dairy cattle herds to the early detection of new potential pathogenic RVA strains.

Cross-sectional study of the G and P genotypes of rotavirus A field strains circulating in regularly vaccinated dairy cattle herds.

Neonatal diarrhea is the main cause of morbidity and mortality in calves up to 30 days old, and rotavirus A (RVA) is the main viral etiology. RVA vaccines are one of the main tools for diarrhea control in neonates. The aim of this cross-sectional study was to monitor by RT-PCR the G and P genotypes of RVA strains identified in dairy cattle herds regularly vaccinated with the RVA UK strain (G6P[5]). Of the 14 randomly selected herds, two were excluded because no calf was diagnosed with diarrhea on the day of fecal collection. Another six herds were also excluded from the study because all 20 diarrheic fecal samples evaluated were RT-PCR-negative. In the remaining six herds, 17 (25.4%) of the 67 diarrheic samples were RVA-positive. One G and P amplicon from each herd were selected for nucleotide sequencing. In the phylogenetic analysis, five RVA strains presented the G6P[11] genotype, and one presented the G10P[11] genotype. The G6 genotype present in all RVA field strains clustered into a distinct phylogenetic arrangement (lineage III) of the UK vaccine strain (lineage IV), characterizing the emergence of a phylogenetically distant G6 strain. In addition, we observed the emergence of strains with G10 and P[11] genotypes characterizing failure in heterologous immune protection. These results show the epidemiological importance of constant monitoring of RVA strains in vaccinated cattle herds and the low frequencies of diarrhea and diagnosis of RVA suggest that a regular vaccination program reduces the frequency and severity of RVA diarrhea in suckling calves.

Reference gene selection for gene expression analysis of oocytes collected from dairy cattle and buffaloes during winter and summer.

Oocytes from dairy cattle and buffaloes have severely compromised developmental competence during summer. While analysis of gene expression is a powerful technique for understanding the factors affecting developmental hindrance in oocytes, analysis by real-time reverse transcription PCR (RT-PCR) relies on the correct normalization by reference genes showing stable expression. Furthermore, several studies have found that genes commonly used as reference standards do not behave as expected depending on cell type and experimental design. Hence, it is recommended to evaluate expression stability of candidate reference genes for a specific experimental condition before employing them as internal controls. In acknowledgment of the importance of seasonal effects on oocyte gene expression, the aim of this study was to evaluate the stability of expression levels of ten well-known reference genes (ACTB, GAPDH, GUSB, HIST1H2AG, HPRT1, PPIA, RPL15, SDHA, TBP and YWHAZ) using oocytes collected from different categories of dairy cattle and buffaloes during winter and summer. A normalization factor was provided for cattle (RPL15, PPIA and GUSB) and buffaloes (YWHAZ, GUSB and GAPDH) based on the expression of the three most stable reference genes in each species. Normalization of non-reference target genes by these reference genes was shown to be considerably different from normalization by less stable reference genes, further highlighting the need for careful selection of internal controls. Therefore, due to the high variability of reference genes among experimental groups, we conclude that data normalized by internal controls can be misleading and should be compared to not normalized data or to data normalized by an external control in order to better interpret the biological relevance of gene expression analysis.