Investigation of the vaginal microbiota of dairy cows through genetic sequencing of short (Illumina) and long (PacBio) reads and associations with gestational status.

The vaginal microbiota has been shown to be important in local immune regulation and may play a role in reproduction and fertility. Next-generation sequencing (NGS) technologies have been used to characterize the bovine vaginal microbiota, mainly using short-read sequencing (Illumina). However, the main limitation of this technique is its inability to classify bacteria at the species level. The objective of this study was to characterize the bovine vaginal microbiota at the species level using long-read sequencing (PacBio) and to compare it with the results of short-read sequencing. In addition, the vaginal microbiota of cows that became pregnant after artificial insemination (AI) was compared with that of infertile animals. Thirteen Holstein cows had vaginal swabs collected prior to AI. DNA was extracted and subjected to Illumina and PacBio sequencing to characterize the V4 region and the entire 16S rRNA gene, respectively. PacBio sequencing yielded 366,509 reads that were assigned to 476 species from 27 phyla. However, none of the most abundant reads (>1%) could be classified at the species level. Illumina sequencing yielded more reads and consequently was able to detect a more observed species, but PacBio sequencing was able to detect more unique and rare species. The composition of the vaginal microbiota varies according to the sequencing method used, which might complicate the interpretation of results obtained in the majority of the current studies. The present study expands on the current knowledge of bovine microbiota, highlighting the need for further efforts to improve the current databanks.

Synchronization of stage of follicle development before OPU improves embryo production in cows with large antral follicle counts.

In the present study, there was an evaluation of in vitro embryo production (IVEP) in Bos indicus donor cows with small or large antral follicle counts (AFCs) when there was synchronization of follicular dynamics among cows before ovum pick-up (OPU). Donor cows classified as having small or large AFC were submitted to OPU/IVEP program (Experiment-I) or had follicular-stage synchronization imposed before OPU/IVEP (Experiment-II). In Experiment-I, the cows with a large AFC had a greater (P < 0.01) mean of embryos developing to the blastocyst stage compared to those with a small AFC. In Experiment-II, percentage of viable oocytes/OPU were not affected (P = 0.33) by synchronization of follicular dynamics, but the AFC had an effect (P < 0.0001). There was an interaction (P = 0.01) indicating the larger AFC, with or without imposing of a synchronization treatment regimen, resulted in the most desirable outcome. The number of embryos was affected (P < 0.001) by follicular-stage synchronization and AFC, with there being an interaction (P = 0.002) with the most desirable results for the large AFC-synchronized group. Number of pregnancies was greater (P ≤ 0.02) for recipient females with embryos from synchronized donors and with a large AFC. There was an interaction (P = 0.03) with there being a greater pregnancy percentage for cows with synchronized follicular stages and the large AFC. Bos indicus donor with a large AFC when associated with the synchronization of stage of follicular dynamics pre-OPU results in improvement of the efficacy of IVEP.

Follicular wave synchronization prior to ovum pick-up.

Among the reproductive biotechnologies, in vitro embryo production (IVEP) is an important tool for multiplying genetic material of superior merit. Recently, the number of embryos produced and transferred in vitro became significantly higher than that produced in vivo worldwide. In this context, the enhancement was attributable to ovum pick-up (OPU). With the advent of genomic technology, shortened breeding intervals, and increased selection accuracy, IVEP has attracted increasing attention for commercial use. The IVEP technique is well-established, but the embryo production rate has reached a plateau at 30-40%. Despite constant advances, the OPU/IVEP programs face some challenges that hinder the efficient application of the technique. Previous studies have shown that the quantity and quality of aspirated oocytes are essential factors for successful IVEP. This paper presents a brief overview of alternatives that can be employed to improve the process-seeking methods that assist in the recovery of better-quality oocytes and higher competence in OPU to improve embryo production. These strategies include using follicular wave synchronization prior to OPU, employing the influence of antral follicle populations, using the pre-OPU gonadotrophic stimulus and applying non-hormonal methods for selecting female donors.

Intensive use of IVF by large-scale dairy programs.

The number of embryos produced by in vitro fertilization (IVF) has grown exponentially in recent years. Recently, for the first time, the number of embryos produced and transferred in vitro was significantly higher than the number developed in vivo worldwide. In this context, a particular boost occurred with ovum pick-up (OPU) and in vitro embryos produced in North America, and this technology is becoming more prominent for commercial dairy farms. However, despite many advances in recent decades, laboratories and companies are looking for methods and alternatives that can be used in collaboration with the existing process to improve it. Among the strategies used to improve the dairy industry are the use of genomic analysis for the selection of animals with desired traits or as an evaluation tool of oocyte and embryo quality, the optimization of the collection and use of gametes from prepubertal females and males, the effective use of sexed semen, and improvements in culture media and methods of embryo cryopreservation. Thus, this review aims to discuss the highlights of the commercial use of IVF and some strategies to increase the application of this technique in large-scale dairy programs.

Ovarian antral follicle populations and embryo production in cattle.

Reproductive biotechniques such as embryo production are important tools to increase the reproductive performance in cattle in a short time. In this context, the antral follicle count (AFC), which reflects the population of antral follicles present in an ovary, has been indicated as an important phenotypic characteristic related to female fertility and closely correlated to the performance of in vivo and in vitro embryo production (IVEP). A positive correlation was evidenced between AFC and oocyte retrieval by ovum pick up (OPU) sessions and and with the number of embryos produced. Several studies have reported that females with a high AFC had greater embryo yields compared to those with medium and low AFC. However, controversial results were obtained by studies conducted in different bovine breeds. Many conflicting data may be due to the differences in the experimental design, particularly regarding the classification of animals in AFC groups, subspecies particularities, herd aptitude or even issues related to animal management. Therefore, aspects such as the choice of donor, type of aspirated follicles and the stage of follicular wave need to be clarified. Thus, this text aims to discuss the use of AFC as a reproductive tool and its applications in the in vivo and in vitro production of embryos, besides describing consistent results and new challenges regarding AFC and embryo production.

Cattle Tick Rhipicephalus microplus-Host Interface: A Review of Resistant and Susceptible Host Responses.

Ticks are able to transmit tick-borne infectious agents to vertebrate hosts which cause major constraints to public and livestock health. The costs associated with mortality, relapse, treatments, and decreased production yields are economically significant. Ticks adapted to a hematophagous existence after the vertebrate hemostatic system evolved into a multi-layered defense system against foreign invasion (pathogens and ectoparasites), blood loss, and immune responses. Subsequently, ticks evolved by developing an ability to suppress the vertebrate host immune system with a devastating impact particularly for exotic and crossbred cattle. Host genetics defines the immune responsiveness against ticks and tick-borne pathogens. To gain an insight into the naturally acquired resistant and susceptible cattle breed against ticks, studies have been conducted comparing the incidence of tick infestation on bovine hosts from divergent genetic backgrounds. It is well-documented that purebred and crossbred Bos taurus indicus cattle are more resistant to ticks and tick-borne pathogens compared to purebred European Bos taurus taurus cattle. Genetic studies identifying Quantitative Trait Loci markers using microsatellites and SNPs have been inconsistent with very low percentages relating phenotypic variation with tick infestation. Several skin gene expression and immunological studies have been undertaken using different breeds, different samples (peripheral blood, skin with tick feeding), infestation protocols and geographic environments. Susceptible breeds were commonly found to be associated with the increased expression of toll like receptors, MHC Class II, calcium binding proteins, and complement factors with an increased presence of neutrophils in the skin following tick feeding. Resistant breeds had higher levels of T cells present in the skin prior to tick infestation and thus seem to respond to ticks more efficiently. The skin of resistant breeds also contained higher numbers of eosinophils, mast cells and basophils with up-regulated proteases, cathepsins, keratins, collagens and extracellular matrix proteins in response to feeding ticks. Here we review immunological and molecular determinants that explore the cattle tick Rhipicephalus microplus-host resistance phenomenon as well as contemplating new insights and future directions to study tick resistance and susceptibility, in order to facilitate interventions for tick control.

Molecular detection and occurrence of 'Candidatus Mycoplasma haemobos' in dairy cattle of Southern Brazil.

Bovine hemoplasmas are bacteria found on the erythrocyte surface or free in the plasma of cattle. The aim of the present study was to evaluate the occurrence of 'Candidatus Mycoplasma haemobos' ('C. M. haemobos') in Holstein and Jersey cattle raised in Londrina and surroundings, northern region of the State of Parana, Southern Brazil. PCR testing directed to 16S rRNA gene fragment was performed to investigate the occurrence and characterize the molecular identity of 'C. M. haemobos'. A total of 264/433 (60.97%) blood samples were positive by PCR. Further alignment of 500-bp amplicons to available sequences at the GenBank database showed high identity (100%) to 'C. M. haemobos'. To the author's knowledge, this is the first molecular confirmation of the hemoplasma 'C. M. haemobos' in cattle from Brazil. Moreover, 'C. M. haemobos' was observed in high occurrence in dairy cattle, and may have significant impact in livestock production.

Molecular detection and occurrence of 'Candidatus Mycoplasma haemobos' in dairy cattle of Southern Brazil / Detecção molecular e ocorrência de 'Candidatus Mycoplasma haemobos' em bovinos de leite do Sul do Brasil

Bovine hemoplasmas are bacteria found on the erythrocyte surface or free in the plasma of cattle. The aim of the present study was to evaluate the occurrence of 'Candidatus Mycoplasma haemobos' ('C. M. haemobos') in Holstein and Jersey cattle raised in Londrina and surroundings, northern region of the State of Parana, Southern Brazil. PCR testing directed to 16S rRNA gene fragment was performed to investigate the occurrence and characterize the molecular identity of 'C. M. haemobos'. A total of 264/433 (60.97%) blood samples were positive by PCR. Further alignment of 500-bp amplicons to available sequences at the GenBank database showed high identity (100%) to 'C. M. haemobos'. To the author's knowledge, this is the first molecular confirmation of the hemoplasma 'C. M. haemobos' in cattle from Brazil. Moreover, 'C. M. haemobos' was observed in high occurrence in dairy cattle, and may have significant impact in livestock production.

Hemoplasmas de bovinos são bactérias encontradas na superfície de hemácias, ou livre no plasma de bovinos. O objetivo do presente estudo foi avaliar a ocorrência de Candidatus Mycoplasma haemobos' ('C. M. haemobos') em bovinos das raças Holandesa e Jersey da região de Londrina, norte do Paraná, sul do Brasil. Para investigar a ocorrência e caracterizar a identidade molecular do 'C. M haemobos' uma PCR baseada no fragmento do gene 16S rRNA foi realizada. A PCR identificou como positivas 264/433 (61%) amostras de sangue testadas. O alinhamento deste fragmento de 500 pb com seqüências disponíveis no GenBank mostrou 100% de identidade 'C. M. haemobos'. Pela bibliografia consultada, esta é a primeira confirmação molecular do hemoplasma 'C. M. haemobos' em bovinos no Brasil. Além disso, foi observada uma alta prevalência deste hemoplasma em bovinos de leite, que pode ter um impacto importante na pecuária bovina.