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.

Heat stress and embryo production in high-producing dairy cows

Background: Heat stress (HS) can compromise the female reproductive system, resulting in a massive reduction of reproductive performance. During the period of follicular growth, HS may compromise the oocyte, either because of a direct effect of elevated temperature on the gamete or because of changes in follicular function that would damage oocyte quality. Oocytes harvested from Holstein cows during summer have reduced ability to develop to the blastocyst stage than those harvested during winter. Nevertheless, although there are reports showing a clear effect of HS on oocyte competence to development after IVF, the exact mechanisms by which the oocyte is compromised remain unknown. Review: Resistance to HS is dependent on cow's genotype. Bos indicus breeds (i.e. Gir) are more resistant to elevated temperature and humidity than breeds (i.e. Holstein) that evolved in a temperate climate. This resistant is, among other factors, due to the superior ability of certain breeds to regulate their body temperature. Thus, breed selection is present as a possible tool to minimize the effects of temperature and humidity on milk production and reproduction. On the other hand, Holstein cows are the most common breed used, even in tropical countries (i.e. Brazil), because of their yield in milk production. As a result, there is a considerable effect upon milk production and reproductive performance through the year in tropical and subtropical countries. A possible way to minimize this effect is the employment of reproductive biotechnologies such as fixed-time artificial insemination (FTAI) and embryo transfer (ET). FTAI has been shown to eliminate problems of estrus detection caused by HS. Nevertheless, it is not enough to complete restore herd pregnancy rates because oocytes and early embryos have already been damaged by HS. In contrast, embryos three days after conception orolder are less sensitive to HS. Thus, ET has been used to improve cow's reproductive performance during the hot months of the year (i.e. spring and summer). This is carried out by producing embryos during the fresh months but using them for ET during the HS period. Regarding fertility problems, it is recognized that repeat-breeder (RB) cows cause huge economic problems to the farmer. These cows are characterized by poor fertilization rates and/or early embryonic loss. It is still not certainly known why these cows become RB, when it is the critical period of the year for them, and what should be done to avoid this problem. We have observed that the conception rates in RB Holstein cows were greater after ET (41.7%) than after AI (17.9%). This indicates that ET may be an effective alternative to achieve satisfactory conception rates throughout the year in RB cows, especially during periods of HS. Moreover, when three categories of Holstein cows (heifers, high-producing cows in peak lactation and RB) were subjected to ovum pick up during the summer and the winter, blastocyst rates and quality were significantly affected by both, category and period, being RB cows mostly affected during the summer. Moreover, lower amounts of mitochondrial DNA were found in RB oocytes, suggesting that the low reproductive performance observed in these cows is related to some injure in oocyte quality. Conclusion: HS negatively affects the physiology and fertility of dairy cows and heifers. Concerning about the reproductive field, the effects of HS were detected in vitro and in vivo on oocytes and embryos and also on conception rates. Moreover, RB Holstein cows seem to be even more sensitive to HS and this can probably be related to their oocyte potential into develop in blastocysts. Therefore, efforts should be taken in order to improve the environmental conditions and heat resistance of dairy cows in tropical and subtropical areas. Studies are been conducted to understand the molecular level by which HS disrupt reproduction aiming to develop methods to attenuate or reverse HS consequences upon fertility of dairy cows.

O uso de FSH exógeno estimula o crescimento folicular final e a função luteínica de vacas Holandesas em lactação sincronizadas para Inseminação Artificial em Tempo Fixo? / Does exogenous FSH increase the final follicular growth and lutea function for TAI in lactating Holstein cows?

Vacas leiteira de alta produção têm apresentado declínio da eficiência reprodutiva. Essa redução é devido a causas multifatoriais, entre elas a baixa concentração de estradiol (E2) no proestro e a baixa concentração de progesterona (P4) no ciclo estral subsequentente. O objetivo deste trabalho foi comparar o uso de gonadotrofina exógena na dinâmica folicular e na taxa de prenhez de vacas submetidas ao protocolo Ovsynch (Experimento 1) ou a protocolos utilizando P4 e E2 (Experimento 2). No Experimento 1, animais de primeiro serviço foram pré-sincronizados com dois protocolos (Presynch ou Double-Ovsynch). Já os animais de segundo ou mais serviços foram resincronizados com o protocolo Resynch. Os animais receberam GnRH (1º GnRH), seguido 7 dias depois pela adiministração de prostaglandina F2α (PGF2α). Nesse momento os animais foram divididos homogeneamente por paridade e número de inseminação em um de dois tratamentos: sem FSH (Ovsynch, n = 561) ou com FSH (Ovsynch + FSH, n = 571). O segundo GnRH (2º GnRH) foi administrado 56 horas após a PGF2α e a inseminação em tempo fixo foi realizada 16 horas após. Amostras de sangue foram colhidas no 1º e no 2º GnRH, na PGF2α e 6 e 13 dias após o 2º GnRH para dosagem de P4. Ainda, no 2º GnRH dosou-se também E2 No Experimento 2, os animais foram sincronizados no dia 0 com um dispositivo de P4 associado a 2 mg de benzoato de estradiol. Oito dias após o dispositivo foi removido e os animais receberam uma dose de PGF2α. Neste mesmo momento, as vacas foram divididas homogeneamente por paridade, número de serviços prévios, escore de condição corporal e presença de CL no inicio do protocolo em três tratamentos: Controle (sem tratamento adicional; n = 232); eCG (400 UI de eCG; n = 232) e FSH (20 mg de FSH; n = 230). Todos os animais receberam GnRH e foram insemados 56h após a retirada do dispositivo. Foram colhidas amostras de sangue a cada 48h do dia 11 ao dia 22. No Experimento 1, não houve efeito do FSH na concetração sérica de E2 no 2º GnRH (P = 0,88), no tamanho do maior folículo no 2 º GnRH (P = 0,63), na taxa de ovulação ao 2º GnRH (P = 0,69) ou na concentração sérica de P4 no 6º (P = 0,15) e 13º (P = 0,36) dia após o 2º GnRH. A taxa de prenhez foi semelhante (P> 0,05) entre os animais tratados com Ovsynch (36,2%) e Ovsynch + FSH (39,1%). No Experimento 2, os tratamentos não alteraram o diâmetro do folículo ovulatório (P = 0,15), o intervalo entre a remoção do dispositivo de P4 e a ovulação (P = 0,30) e a taxa de ovulação (P = 0,44). Não houve efeito de tratamento na concentração sérica de P4 (P = 0,15). A taxa de prenhez foi diferente entre os tratamentos aos 30 dias após a IATF (Controle = 28,0a vs FSH = 18,7b vs eCG = 29,7a %; P = 0,01), mas não aos 60 dias (Controle = 21,6 vs FSH = 16,1 vs eCG = 24,1%; P = 0,08) e na perda de gestacional (Controle = 18,8 vs FSH = 14,0 vs eCG = 18,4%; P = 0,39). Assim, o tratamento com FSH não estimulou o crescimento folicular final e a função luteínica de vacas leiteiras de alta produção sincronizadas com os protocolos Ovsynch e P4/E2

Fertility in high-producing dairy cows has decreased over the years, which has been associated with reduced estradiol (E2) concentrations during proestrus and suboptimal progesterone (P4) concentrations during early stages of gestation. The objectives of the present study were to evaluate the effects of exogenous gonadotropins on follicular dynamics and risk of pregnancy per artificial insemination (P/AI) in cows subjected to the Ovsynch protocol (Experiment 1) or to a P4/E2-based timed AI protocol (experiment 2). In experiment 1, cows were enrolled in the Ovsynch protocol (GnRH, 7 d PGF2α, 56 h GnRH, 16 h IA) either after presynchronization (Presynch or Double-Ovsynch; first AI postpartum) or 32 d after previous AI. At the PGF2α injection, cows were blocked by parity and number of AI and, within each block, randomly allocated to eitherreceive 20 mg of FSH at the moment of the PGF2α(Ovsynch + FSH, n = 571) or to remain as untreated control (Ovsynch, n = 561). Blood was sampled at the 1st and 2nd GnRH and PGF2α injections, as well as on d 6 and 13 after the 2nd GnRH to access P4 and E2 (at the 2nd GnRH only) concentrations. In experiment 2, cows were received a P4 device and 2 mg of estradiol benzoate. The device was removed 8 d later concurrently with an injection of PGF2α, followed by an injection of GnRH and AI at 56 h.At the PGF2α injection, cows were blocked by parity, number of AI, body condition score, and presence of a CL at device insertion.Within each block, cows were randomly allocated to receive either 20 mg of FSH (FSH, n = 230), 400 IU of eCG (eCG, n = 232), or no additional treatment at the moment of the PGF2α (Control, n = 232). Blood was sampled at every 48 h from 1 to 12 d after AI. In experiment 1, there was no effect of FSH on serum E2 at the 2nd GnRH (P = 0.88), follicle diameter at the 2nd GnRH (P = 0.63), ovulatory response to the 2nd GnRH (P = 0.69), or serum P4 on d 6 (P = 0.15) and 13 (P = 0.36) after the 2nd GnRH. Also, P/AI was similar (P > 0.05) between Ovsynch (36.2%) and Ovsynch + FSH (39.1%). In experiment 2, treatment did not affect ovulatory diameter (P = 0.15), interval from P4 device removal and ovulation (P = 0.30), ovulatory response to the 2nd GnRH (P = 0.44), and serum P4 (P = 0.15). Interestingly, treatment with FSH reduced (P = 0.01) P/AI on d 30 (Control = 28.0a vs. FSH = 18.7b vs. eCG = 29.7a %), but not on d 60after timed AI (Control = 21.6 vs. FSH = 16.1 vs. eCG = 24.1%; P = 0.08).The risk of pregnancy loss was not affected by treatment (Control = 18.8 vs. FSH = 14.0 vs. eCG = 18.4%; P = 0.39). In conclusion, treatment with FSH failed to enhance the final growth of the ovulatory follicle and did not improve luteal function after AI in high-producing dairy cows synchronized with either the Ovsynch or P4/E2-based timed AI protocols

Validação do escore de condição corporal e seu impacto na eficiência reprodutiva de vacas Nelore (Bos indicus) inseminadas em tempo fixo / Validation of body condition score and its impact on reproductive efficiency of Nelore (Bos indicus) cows fixed-time inseminated

A presente dissertação foi dividida em 2 Capítulos. O objetivo do Capítulo 1 foi determinar a relação entre as variáveis: peso vivo (PV), avaliação do escore de condição corporal (ECC) e espessura da gordura subcutânea na garupa (EGPU) mensurada por ultra-sonografia. 266 vacas Nelores foram avaliadas quanto ao ECC, a EGPU e PV, em cinco momentos distintos: M1) no momento da desmame; M2) ao parto, M3) no dia da sincronização; M4) aos 82 dias e M5) aos 112 dias pós-parto. Foi atribuído um ECC para cada vaca na escala 1 a 5 (MACIEL, 2006). Para a obtenção das imagens ultra-sonográficas para mensuração EGPU, foi utilizado um transdutor linear com freqüência de 3,5 MHz. As imagens ultra-sonográficas obtidas foram congeladas e imediatamente analisadas. As relações entre ECC e as mensurações de EGPU foram investigadas utilizando modelos de regressão. Para a análise de semelhança entre os cinco modelos foi utilizado o proc MIXED do SAS. As correlações entre variáveis foram analisadas com o proc CORR do SAS. O ECC foi capaz de predizer a EGPU de vacas Nelores em diferentes momentos do ciclo de criação. Ainda, foi demonstrado que o ECC apresentou alta correlação (r = 0,82 a 0,93) e relação (R2 = 0,73 a 0,92) com a EGPU. Porém, tanto o ECC, quanto a EGPU apresentam baixa correlação e baixíssima relação com o peso. No Capítulo2, o objetivo foi determinar o impacto do ECC, da EGPU, do PV e de suas variações nos 5 momentos descritos acima na eficiência reprodutiva (ER) de vacas Nelore submetidas à IATF e repasse com o touro e determinar o(s) momento(s) de avaliação com maior impacto na ER. Foram utilizados os mesmas animais do Capítulo 1. No dia 0, todos os animais receberam implante auricular de Norgestomet, reutilizado e a aplicação de 2,0 mg de benzoato de estradiol (D42). Oito dias após (D50), os implantes foram retirados e foi administrado 400 UI de eCG e PGF. No dia seguinte (D51), foi administrado 1,0 mg de Benzoato de estradiol. Os animais foram inseminados 56 horas após a retirada do implante (D52). Após 10 dias da inseminação, houve a introdução de touros. O diagnóstico de gestação foi realizado aos 30 e 60 dias após à IATF. Os animais foram avaliados quanto ao PV, ECC e EGPU com a mesma metodologia do Capítulo 1. A ER foi avaliada pela taxa de concepção à IATF aos 82 e aos 112 dias pós-parto e a taxa de prenhez acumulada até 82 dias pós-parto. Os dados binomiais foram analizados com o proc GLIMMIX do SAS. Os modelos de regressão foram gerados através do Guide Data Analysis do SAS. As correlações entre variáveis foram analisadas com o proc CORR do SAS. Os resultados apontam que vacas com maior ECC, EGPU e ganho destas medidas entre desmame-parto apresentaram melhor ER e a avaliação do peso apresentou baixa correlação com a ER. Porém, a menor perda de ECC e gordura subcutânea entre parto-sincronização não melhorou a ER. Avaliação do ECC e a EGPU no momento do parto demonstraram ter o maior impacto na ER de vacas Nelore submetidas à IATF e repasse com touro. Porém, mais estudos são necessários para confirmar os dados deste estudo

The present study was divided in two Chapters. The objective of the first Chapter was to determine the relationship among the variables: body weight (BW), body condition score (BCS) and back fat thickness (BFT), measured by ultrasonography. Two hundred and six Nelore cows had their BW, BCS and BFT evaluated at five different moments: M1) weaning; M2) parturition, M3) synchronization; M4) 82 days post-partum and M5) 112 days post-partum. A BCS value was attributed for each cow following a 1 to 5 points scale (MACIEL, 2006). Ultrasound images for BFT measurement were obtained using a 3.5 MHz linear transducer. Images were immediately analyzed as soon as they were formed and frozen. The relationship between BCS and BFT values was investigated by regression models. The analysis of similarity among the five obtained models was performed using the proc MIXED from SAS and the correlations among variables were analyzed with proc CORR from SAS. The BCS was able to predict BFT in Nelore cows in all different moments evaluated. Also, it was shown that BCS presented high correlation (r = 0.82 to 0.93) and relationship (R2 = 0.73 to 0.92) with BFT. However, both BCS and BFT showed poor correlation and extremely pour relationship with BW. On Chapter 2, the objective of the study was to determine the impact of BCS, BFT and BW, and their variations in that five previously cited moments, on reproductive efficiency (RE) of Nelore cows submitted to FTAI followed by exposition to clean-up bulls. Also, this work aimed to determine the moment(s) of evaluation that presented greater impact on RE. The same animals used on Chapter 1 were used here. All animals received a norgestomet ear implant (previously used for 9 days) associated to 2.0 mg of estradiol benzoate (D42). Eight days after (D50), the implants were removed and 400 IU of eCG plus PGF were administered. On the following day (D51), cows received 1.0 mg of estradiol benzoate and all animals were inseminated 56 hours after implant withdrawal (D52). Ten days after insemination females were exposed to clean-up bulls. Pregnancy diagnosis was performed 30 and 60 days post-FTAI. All animals had their BW, BCS and BFT measured using the same methods described on Chapter 1. Reproductive efficiency was evaluated considering pregnancy per AI at 82 and 112 days post-partum and cumulative pregnancy until 82 days post-partum. Binomial data were analyzed using the proc GLIMMIX from SAS, regression models were generated via Guide Data Analysis from SAS, and correlations among variables were analyzed with proc CORR from SAS. The obtained results evidenced that cows with higher BCS and BFT and greater gain of these measures during the interval weaning-partum presented better RE. However, the decreased loss of BCS and BFT between parturition and synchronization did not increase the RE. Also, BW evaluation showed poor correlation with RE. The evaluation of BCS and BFT at partum demonstrated that these parameters had great impact on RE of Nelore cows submitted to FTAI followed exposition to bulls. However, more studies are demanded in order to reaffirm the results obtained in this particular study