In Vitro Development and Cell Allocation After Aggregation of Syngeneic Wild Type and Fluorescence-Expressing Bovine Cloned Embryos

Background: The in vitro production (IVP) of embryos by in vitro fertilization or cloning procedures has been known to cause epigenetic changes in the conceptus that in turn are associated with abnormalities in pre- and postnatal development. Handmade cloning (HMC) procedures and the culture of zona-free embryos in individual microwells provide excellent tools for studies in developmental biology, since embryo development and cell allocation patterns can be evaluated under a wide range of embryo reconstruction arrangements and in in vitro embryo culture conditions. As disturbances in embryonic cell allocation after in vitro embryo manipulations and unusual in vivo conditions during the first third of pregnancy appear to be associated with large offspring, embryo aggregation procedures may allow a compensation for epigenetic defects between aggregated embryos or even may influence more favorable cell allocation in embryonic lineages, favoring subsequent development. Thus, the aim of this study was to evaluate in vitro embryo developmental potential and the pattern of cell allocation in blastocysts developed after the aggregation of handmade cloned embryos produced using syngeneic wild type and/or transgenic somatic cells. Materials, Methods & Results: In vitro-matured bovine cumulus-oocyte complexes (COC) were manually bisected after cumulus and zona pellucida removal; then, two enucleated hemi-oocytes were paired and fused with either a wild type (WT) or a GFP-expressing (GFP) fetal skin cell at the 11th and 19th passages, respectively. Following chemical activation, reconstructed cloned embryos and zona-free parthenote embryos were in vitro-cultured in microwells, for 7 days, either individually (1 x 100%) or after the aggregation of two structures (2 x 100%) per microwell, as follows: (G1) one WT cloned embryo; (G2) two aggregated WT embryos; (G3) one GFP cloned embryo; (G4) two aggregated GFP embryos; (G5) aggregation of a WT embryo and a GFP embryo; (G6) one parthenote embryo; or (G7) two aggregated parthenote embryos. Fusion (clones), cleavage (Day 2), and blastocyst (Day 7) rates, and embryonic cell allocation were compared by the. 2 or Fisher tests. Total cell number (TCN) in blastocysts was analyzed by the Student's test (P < 0.05). Fusion and cleavage rates, and cell allocation were similar between groups. On a per WOW basis, development to the blastocyst stage was similar between groups, except for lower rates of development seen in G3. However, when based on number of embryos per group (one or two), blastocyst development was higher in G1 than all other groups, which were similar between one another. Cloned GFP embryos had lower in vitro development to the blastocyst stage than WT embryos, which had more TCN than parthenote or aggregated chimeric WT/GFP embryos. Aggregated GFP embryos had fewer cells than the other embryo groups. Discussion: The in vitro development of GFP cloned embryos was lower than WT embryos, with no effects on cell allocation in resulting blastocysts. Differences in blastocyst rate between groups were likely due to lower GFP-expressing cell viability, as GFP donor cells were at high population cell doublings when used for cloning. On a per embryo basis, embryo aggregation on Day 1 resulted in blastocyst development similar to non-aggregated embryos on Day 7, with no differences in cell proportion between groups. The use of GFP-expressing cells was proven a promising strategy for the study of cell allocation during embryo development, which may assist in the elucidation of mechanisms of abnormalities after in vitro embryo manipulations, leading to the development of improved protocols for the in vitro production (IVP) of bovine embryos.Universal Grant [2008-2010/CNPq/Brazil]Universal GrantCAV/UDESC [1.01.717/06]CAV/UDESCCNPq (Brazil)CNPq/Brazi

Supplementary files - Investigation of Bovine Viral Diarrhea Virus (BVDV) (2023)

<p>BVDV is a productive and reproductive virus that causes high global economic impact in dairy production systems. The aim of this research was to investigate the prevalence of BVDV at herd and individual level in the most dairy productive region from Brazil. The frequency of BVDV at herd-level was investigated by using RT-qPCR from bulk milk tank monitored in 289 dairy farms between August 2020 to January 2022. Among these farms, 68 production systems were selected to investigate the prevalence of persistently infected (PI) animals by using 2 antigen-ELISA, apart 21 days, from ear-notches samples. A total of 2,902 RT-qPCR and 23,466 antigen ELISA were realized. At herd level, 23.87% (69/289) dairy-farms were considered infected, presenting at least one qPCR test positive. At individual-level, 41.17% (28/68) of a subgroup of selected farms had at least one animal positive in the antigen-ELISA test. Association between tests allows the classification of farms into four categories: level 0 - negative in both tests (41.17%; 28/68); level 1- RT-qPCR positive and Ag-ELISA negative (17.64%; 12/68); level 2- RT-qPCR negative and Ag-ELISA positive (13.23%; 9/68); level 3- positive for both tests (27.94%; 19/68). Multiple Correspondence Analysis (MCA) suggests a possible association between BVDV positivity with large-size farms, average daily milk production (herd), breed and different levels of somatic cells count. The confinement and intensification of animals from different categories, in addition to the use of artificial bedding, are associated with BVDV infection. Use of waste milk or bulk tank milk are also a risk factors for the BVDV positive results in the RT-qPCR and antigen-ELISA. Despite of the use of reproductive vaccines for the most of producers, the use of vaccines seems to be associated with farms positive for BVDV. This research gives epidemiological frequencies of BVDV at herd and individual level in the Campos Gerais paranaense region, tops the ranking milk-producing municipalities in Brazil. In addition, the association of BVDV tests and farms characteristics indicates the farm risk for BVDV and guide specific control programs</p&gt

Supplementary files - Investigation of Bovine Viral Diarrhea Virus (BVDV) (2023)

<p>BVDV is a productive and reproductive virus that causes high global economic impact in dairy production systems. The aim of this research was to investigate the prevalence of BVDV at herd and individual level in the most dairy productive region from Brazil. The frequency of BVDV at herd-level was investigated by using RT-qPCR from bulk milk tank monitored in 289 dairy farms between August 2020 to January 2022. Among these farms, 68 production systems were selected to investigate the prevalence of persistently infected (PI) animals by using 2 antigen-ELISA, apart 21 days, from ear-notches samples. A total of 2,902 RT-qPCR and 23,466 antigen ELISA were realized. At herd level, 23.87% (69/289) dairy-farms were considered infected, presenting at least one qPCR test positive. At individual-level, 41.17% (28/68) of a subgroup of selected farms had at least one animal positive in the antigen-ELISA test. Association between tests allows the classification of farms into four categories: level 0 - negative in both tests (41.17%; 28/68); level 1- RT-qPCR positive and Ag-ELISA negative (17.64%; 12/68); level 2- RT-qPCR negative and Ag-ELISA positive (13.23%; 9/68); level 3- positive for both tests (27.94%; 19/68). Multiple Correspondence Analysis (MCA) suggests a possible association between BVDV positivity with large-size farms, average daily milk production (herd), breed and different levels of somatic cells count. The confinement and intensification of animals from different categories, in addition to the use of artificial bedding, are associated with BVDV infection. Use of waste milk or bulk tank milk are also a risk factors for the BVDV positive results in the RT-qPCR and antigen-ELISA. Despite of the use of reproductive vaccines for the most of producers, the use of vaccines seems to be associated with farms positive for BVDV. This research gives epidemiological frequencies of BVDV at herd and individual level in the Campos Gerais paranaense region, tops the ranking milk-producing municipalities in Brazil. In addition, the association of BVDV tests and farms characteristics indicates the farm risk for BVDV and guide specific control programs</p&gt

Supplementary file - Ferreira et al. 2023

<p>A biosseguridade é o conjunto de práticas em um sistema de produção a fim de prevenir e/ou controlar a entrada, disseminação e saída de agentes biológicos nocivos. O objetivo geral deste estudo foi levantar práticas de biosseguridade e associá-las de acordo com o tamanho dos sistemas de produção. Para isso, aplicou-se um formulário de avaliação de risco biológico em 69 propriedades da região de Campos Gerais no Paraná. O questionário era dividido em duas seções: geral e específico sobre os vírus BVDV e BoHV-1. A seção geral contempla tópicos sobre controle de tráfego, quarentena e isolamento animal, práticas de higiene, descarte de carcaças e monitoramento/controle. A seção específica é composta por perguntas acerca fatores reprodutivos, respiratórios, uso de antimicrobianos e calendário vacinal. As 69 propriedades foram classificadas em pequenas (≤ 61), médias (de 62 a 201) e grandes (≥ 202 vacas em lactação). Neste estudo foram avaliados bovinos da raça Holandesa que apresentavam em média 183 vacas em lactação que produziam 31 litros/dia e apresentavam uma média da contagem de células somáticas de 218.000 células/ml e 197 dias em lactação. A produção diária total dos rebanhos foi 459.447 ± 1.147 litros de leite. Foi determinado uma pontuação (0-10) para cada questão e determinado o grau de biosseguridade, classificando em alto (≥290 pontos), médio (231 a 289 pontos) ou baixo (≤230 pontos). Nas propriedades pequenas a pontuação variou de 150-320 pontos, nas propriedades médias de 200-350 e nas grandes de 210-340 pontos. A média geral do escore de risco biológico dos rebanhos obtido em nosso estudo foi de 263 pontos, variando de 150-350 pontos. Os principais fatores de risco e variabilidade observados estavam relacionados ao controle de tráfego de pessoas, animais e veículos/equipamentos; quarentena/isolamento animal e práticas de higiene. A partir do MCA foi possível notar que propriedades de pequeno porte estavam comumente associadas a ausência de medidas de biosseguridade como aquelas relacionadas ao controle de tráfego, quarentena animal e higiene. Em propriedades de médias verificou-se contato de animais bovinos de diferentes idades e dificuldade no isolamento de animais como alguns dos principais fatores de risco. Já em propriedades grandes, o isolamento de animais doentes ocorria facilmente, mas a compra de animais bovinos também era um potencial fator de risco. Esses resultados auxiliam a compreensão da relação da biosseguridade com o tamanho das propriedades contribuindo para elaboração assertiva de planos de biosseguridade ao considerar tal característica.</p> <p>----</p> <p>Biosecurity is the set of practices in a production system in order to prevent and/or control the entry, spread and exit of harmful biological agents. The objective was to survey biosecurity practices and they associate with size of the production systems. For this purpose, teams applied a biological risk assessment form to 69 farms in the Campos Gerais region in Paraná. We divided the questionnaire into two sections: one general section and one specific to BVDV and BoHV-1 viruses. The general section covers topics on traffic control, animal quarantine and isolation, hygiene practices, carcass disposal and monitoring/control. The specific section is composed of questions about reproductive factors, respiratory factors, antimicrobial use and vaccination schedule. The 69 farms were classified as small (≤ 61), medium (62 to 201) and large (≥ 202 lactating cows). This study evaluated Holstein cattle with an average of 183 lactating cows that produced 31 liters/day and had an average somatic cell count of 218,000 cells/ml and 197 days in lactation. The total daily production of the herds was 459,447 ± 1,147 liters of milk. A score (0-10) was determined for each question and the degree of biosecurity was categorized as high (≥290 points), medium (231 to 289 points) or low (≤230 points). On small farms the score ranged from 150-320 points, on medium farms from 200-350 and on large farms from 210-340 points. The overall average biological risk score of the herds obtained in our study was 263 points, ranging from 150-350 points. Traffic control of people, animals and vehicles/equipment, animal quarantine/isolation and hygiene practices were main risk factors and variability observed. From the MCA it was possible to note that small farms were commonly associated with the absence of biosecurity measures such as those related to traffic control, animal quarantine and hygiene. In medium-sized properties, there was contact between bovine animals of different ages and difficulty in isolating animals as some of the main risk factors. In large farms, isolation of sick animals occurred easily, but the purchase of bovine animals was also a potential risk factor. These results help to understand the relationship between biosecurity and the size of the farms, contributing to the assertive elaboration of biosecurity plans when considering this characteristic.</p&gt