Acquisition of fertilization competence in guinea pig spermatozoa under different capacitation protocols.

Guinea pig in vitro fertilization (IVF) are poorly developed due to the limited accessibility to oocytes and the lack of an efficient method of sperm capacitation. Thus, we aimed to evaluate different capacitation protocols that we validated through sperm analysis and using heterologous (He) IVF with zona-intact bovine oocytes. Spermatozoa of guinea pigs were collected and processed separately by 4 different protocols: A) Spermatozoa were obtained by flushing the lumen of one cauda epididymis and incubated in a minimal culture medium (MCM); B) One epididymis was placed in a prewarmed of M2 medium and gently minced with fine scissors. Spermatozoa were incubated in a modified human tubal fluid medium (HTF). In both protocols, the spermatozoa were capacitated at 37 °C under an atmosphere of 5% CO2 for 2 h. In the protocols C and D, the spermatozoa were collected by flushing the lumen of the cauda epididymis and selected by commercial density gradient Bovipure® (Nidacon Laboratories AB, Göthenborg, Sweden), according to the manufacturer's instructions. Then for Protocol C) spermatozoa were incubated in MCM medium supplemented with 10 mg/mL heparin (MCM-Hep); while for Protocol D) spermatozoa were incubated in FERT medium supplemented 10 mg/mL heparin (FERT-Hep). Incubation of C and D protocols were performed at 38.5 °C under an atmosphere of 5% CO2 for 2 h. Capacitation protocols C and D showed a higher percentage of viability, total and hyperactive-like motility, and acrosome reaction compared to protocols A and B. For this reason, protocols C and D were used for further He-IVF analysis. Guinea pig sperm and matured zona-intact bovine oocytes were co-incubated at 5% CO2 and 38.5 °C. Sperm-oocyte interaction was assessed at 2.5 h post-insemination (hpi) and pronuclear formation (PrF) were evaluated at 18, 20, 22, 24 and 26 hpi, while the cleavage rate was evaluated at 48 hpi. In protocol D, PrF was significantly higher than in protocol C (P ≤ 0.05) at every time point evaluated. Also, the cleavage rate at 48 hpi was higher (P ≤ 0.05) in He-IVF protocol D (69.8 ± 1.7%) compared to He-IVF protocol C (49.1 ± 1.1%). In conclusion, we determined the most adequate sperm capacitation conditions for guinea pig that allow zona-intact bovine oocyte penetration and lead to hybrid embryo formation, suggesting that these conditions could be optimal to develop IVF in guinea pigs.

Seroprevalence and risk factors associated with bovine leukemia virus (BLV) seropositivity in cattle herds from Ecuador / Seroprevalencia y factores de riesgo asociados a la seropositividad al virus de la leucemia bovina (BLV) en explotaciones de producción bovina en Ecuador / Seroprevalência e fatores de risco associados à soropositividade do vírus da leucemia bovina (BLV) em rebanhos bovinos do Equador

Abstract Background: Bovine leukemia virus (BLV) is the causative agent of enzootic bovine leukosis (EBL). This disease mainly affects cattle, causing severe economic losses to producers. Objective: To establish individual and herd seroprevalence and determine the risk factors associated with BLV seropositivity for dairy and dual-purpose cattle herds in Ecuador. Methods: A total of 2,668 serum samples from 386 herds were collected. A questionnaire, including variables related to cattle health, management and the environment was completed by each herd. A commercial blocking enzyme-linked immunosorbent assay (ELISA) test was used to determine seropositivity. A generalized estimating equation model (GEE) was developed to determine the factors associated with BLV seropositivity. Results: Individual seroprevalence of BLV infection in Ecuador was 17.3% (CI95% = 15.86-18.74%). Herd prevalence was 37.8% (CI95% = 33.0-42.6%), and intra-herd prevalence ranged between 12.5 and 100% (median: 37.5%). The risk factors associated with BLV seropositivity were artificial insemination (OR: 2,215; CI95% =1.402-3.501), concrete floors (OR: 2.178; CI95% = 1.217-3.889), presence of wild ruminants (OR: 2.998; CI95% = 1.788-5.027), and sampling season (wet; OR: 1.996; CI95% = 1.140-3.497). Conclusions: Results indicate that BLV is widespread in cattle herds in Ecuador. In addition, the study suggests that a control program to fight BLV infection should focus on controlling the risk factors identified.

Resumen Antecedentes: El virus de la leucosis bovina (BLV) es el principal agente etiológico causante de la leucosis enzoótica bovina (EBL). Esta enfermedad afecta a los bovinos causando grandes pérdidas económicas a los productores. Objetivo: Establecer la seroprevalencia y dispersión del BLV, así como los factores de riesgo asociados a la seropositividad en explotaciones lecheras y de doble propósito en Ecuador. Métodos: Se recolectó un total de 2.668 muestras de suero de 386 explotaciones. Se aplicó un cuestionario que incluyó variables relacionadas con la salud del hato, medidas de manejo, y características ambientales de cada explotación. Para los análisis serológicos se utilizó un test inmunológico ligado a enzimas (ELISA). Para definir los factores de riesgo asociados a la seropositividad a BLV se desarrolló un modelo utilizando ecuaciones de estimación generalizadas (GEE). Resultados: La seroprevalencia de BLV en Ecuador fue de 17,3% (IC95% = 15,86-18,74%). La dispersión fue de 37,8% (IC95%= 33,0-42,6%), y la prevalencia intra-hato alcanzó rangos entre 12,5-100% (media: 37,5%). Los factores de riesgo asociados a la seropositividad a BLV fueron: inseminación artificial (OR: 2,215; IC95% = 1,402-3,501), piso de concreto (OR: 2,178; IC95% = 1,217-3,889), presencia de rumiantes salvajes (OR: 2,998; IC95% = 1,788-5,027), y temporada de muestreo (húmeda; OR: 1,996; IC95% = 1,140-3,497). Conclusiones: Los resultados indican que el BLV se encuentra disperso en las explotaciones de Ecuador. Adicionalmente, se sugiere la implementación de un programa de control para la lucha contra el BLV, debiéndose considerar medidas que se enfoquen al control de los factores de riesgo identificados en esta investigación.

Resumo Antecedentes: O vírus da leucemia bovina (BLV) é o principal agente causador da leucose enzoótica bovina (EBL). Esta doença afeta o gado causando graves prejuízos econômicos aos produtores. Objetivo: Estabelecer a soroprevalência e dispersão do BLV, assim como os fatores de risco associados à soropositividade nas produções leiteiras e de duplo propósito no Equador. Métodos: Um total de 2.668 amostras de soro de 386 explorações foram coletadas. Foi aplicado um questionário que incluía variáveis relacionadas à saúde do rebanho, medidas de manejo e ambiente para cada exploração. Para a análise sorológica foi utilizado um teste imunológico sobre enzimas (ELISA) para determinação da soropositividade. Para definir os fatores de risco associados à soropositividade a BLV, foi utilizado um modelo de equações estimativas generalizadas (GEE). Resultados: A soroprevalência de BLVno Equador é de 17,3% (IC95% = 15,86-18,74%). La dispersão de 37,8% (IC95% = 33,0-42,6%), e a prevalência intra-rebanho alcançou entre 12,5-100% (media: 37,5%). Os fatores de risco associados à soropositividade a BLV foram inseminação artificial (OR: 2,215; IC95% = 1,402-3,501), chão de concreto (OR: 2,178; IC95% = 1,217-3,889), presença de ruminantes selvagens (OR: 2,998; IC95% = 1,788-5,027) e época da amostragem (úmida; OR: 1,996; IC95% = 1,140-3,497). Conclusões: Os resultados indicam que o BLV se encontra disseminado nas explorações no Equador. Adicionalmente, o estudo pode contribuir para a implementação de um programa de controle para a luta contra o BLV, devendo-se considerar ações de controle dos fatores de risco identificados nesta investigação.