Microbial pathogen removal from porcine semen with acoustophoresis

Microbial pathogens in semen used for artificial insemination (AI) do not only transmit diseases, but also reduce semen quality. A continuous pathogen removal separation technique overcomes the limits of current processing and separation techniques. We have applied acoustophoresis to separate bacteria and viruses from porcine semen while maintaining spermatozoa viability and achieved high spermatozoa recovery

Effect of microfluidic processing on the viability of boar and bull spermatozoa

The use of microfluidics in artificial reproductive technologies for manipulation or assessment of spermatozoa is unique in the sense that it is not always an end point measurement and the sample may be used afterward. During microfluidic processing, spermatozoa are exposed to shear stress, which may harm viability and functioning of spermatozoa. The shear stresses during general microfluidic processing steps were calculated and compared to estimated shear stresses during ejaculation. The viability of boar and bull spermatozoa after microfluidic processing was studied and compared to the typical handling method (centrifugation) and to a control (the sample in a tube at the same temperature). The boar spermatozoa showed a small but significant decrease in viability of 6% after microfluidic handling. Bull spermatozoa proved to be less susceptible to shear stress and were not significantly affected by microfluidic processing. These data indicate that the impact of microfluidic processing on the viability of boar and bull spermatozoa is less than the literature values reported for flow cytometry and comparable to the impact of centrifugatio

Using short read sequencing to characterise balanced reciprocal translocations in pigs

Background: A balanced constitutional reciprocal translocation (RT) is a mutual exchange of terminal segments of two non-homologous chromosomes without any loss or gain of DNA in germline cells. Carriers of balanced RTs are viable individuals with no apparent phenotypical consequences. These animals produce, however, unbalanced gametes and show therefore reduced fertility and offspring with congenital abnormalities. This cytogenetic abnormality is usually detected using chromosome staining techniques. The aim of this study was to test the possibilities of using paired end short read sequencing for detection of balanced RTs in boars and investigate their breakpoints and junctions. Results: Balanced RTs were recovered in a blinded analysis, using structural variant calling software DELLY, in 6 of the 7 carriers with 30 fold short read paired end sequencing. In 15 non-carriers we did not detect any RTs. Reducing the coverage to 20 fold, 15 fold and 10 fold showed that at least 20 fold coverage is required to obtain good results. One RT was not detected using the blind screening, however, a highly likely RT was discovered after unblinding. This RT was located in a repetitive region, showing the limitations of short read sequence data. The detailed analysis of the breakpoints and junctions suggested three junctions showing microhomology, three junctions with blunt-end ligation, and three micro-insertions at the breakpoint junctions. The RTs detected also showed to disrupt genes. Conclusions: We conclude that paired end short read sequence data can be used to detect and characterize balanced reciprocal translocations, if sequencing depth is at least 20 fold coverage. However, translocations in repetitive areas may require large fragments or even long read sequence data.</p

Effect of Microfluidic Processing on the Viability of Boar and Bull Spermatozoa

In this study, we evaluate the impact of microfluidic processing on the viability of spermatozoa and compare shear stress in various chip configurations and connection tubing. In contrast to other microfluidic devices for cell analysis, the spermatozoa are still used after processing in our application. During this processing, the semen is exposed to shear stress which might harm the viability and functioning of the spermatozoa. Our results indicate that the impact of microfluidic processing on the viability of boar and bull spermatozoa is less than literature reported for flow cytometry and that it is comparable to the impact of conventional centrifugation

Label-Free Microfluidic Impedance Cytometry for Acrosome Integrity Assessment of Boar Spermatozoa

Microfluidics and lab-on-chip technologies have been used in a wide range of biomedical applications. They are known as versatile, rapid, and low-cost alternatives for expensive equipment and time-intensive processing. The veterinary industry and human fertility clinics could greatly benefit from label-free and standardized methods for semen analysis. We developed a tool to determine the acrosome integrity of spermatozoa using microfluidic impedance cytometry. Spermatozoa from boars were treated with the calcium ionophore A23187 to induce acrosome reaction. The magnitude, phase and opacity of individual treated and non-treated (control) spermatozoa were analyzed and compared to conventional staining for acrosome integrity. The results show that the opacity at 19 MHz over 0.5 MHz is associated with acrosome integrity with a cut-off threshold at 0.86 (sensitivity 98%, specificity 97%). In short, we have demonstrated that acrosome integrity can be determined using opacity, illustrating that microfluidic impedance cytometers have the potential to become a versatile and efficient alternative in semen analysis and for fertility treatments in the veterinary industry and human fertility clinics

Virus removal from semen with a pinched-flow fractionation microfluidic chip

In the veterinary industry, due to the use of artificial insemination, a single infected boar can transfer a disease to a multitude of sows which can lead to a disease outbreak [1]. An example is the epidemic of classical swine fever (CSF) in the Netherlands in 1997-1998, where the semen of infected boars led to CSF outbreaks in farms all over the country and beyond [2]. To reduce the chance of such an event happening in the future, we present a pinched-flow fractionation device to remove viruses from semen while retaining a high percentage of spermatozoa

Relationship between the estimated breeding values for litter traits at birth and ovarian and embryonic traits and their additive genetic variance in gilts at 35 days of pregnancy

We investigated (1) the relationship between the estimated breeding values (EBVs) for litter traits at birth and ovulation rate (OR), average corpora luteal weight, uterine length and embryonic survival and development traits in gilts at 35 days of pregnancy by linear regression, (2) the genetic variance of OR, average corpora lutea (CL) weight, uterine length and embryonic survival and development traits at 35 days of pregnancy, and (3) the genetic correlations between these traits. Landrace (n = 86) and Yorkshire × Landrace (n = 304) gilts were inseminated and slaughtered at 35 days of pregnancy. OR was assessed by dissection of the CL on both ovaries. Individual CL was weighed and the average CL weight calculated. The number of embryos (total and vital) were counted and the vital embryos were individually weighed for calculation of within litter average and standard deviation (SD) of the embryo weight. Length of the uterine implantation site of the vital embryos was measured and the average per gilt calculated. Results suggests that increasing the EBV for total number of piglets born would proportionally increase OR and number of embryos, while decreasing the average CL weight. On the contrary, increasing the EBV for average piglet birth weight and for within litter birth weight standard deviation would increase the average CL weight. There was no relationship between the EBVs for BW and for BWSD and vital embryonic weight at 35 days of pregnancy. OR, average CL weight, number of embryos, average weight and implantation length of the vital embryos had all moderate to high heritabilities, ranging from 0.36 (±0.18) to 0.70 (±0.17). Thus, results indicate that there is ample genetic variation in OR, average CL weight and embryonic development traits. This knowledge could be used to optimize the balance between selection for litter size, average piglets birth weight and within litter birth weight uniformity

Virus removal from semen with a pinched flow fractionation microfluidic chip

Nowadays pigs are bred with artificial insemination to reduce costs and transportation. To prevent the spread of diseases, it is important to test semen samples for viruses. Screening techniques applied are enzyme-linked immunosorbent assays and/or polymerase chain reaction, which are labor-intensive and expensive methods. In contrast to the current used screening techniques, it is possible to remove viruses physically from semen. However, existing methods for virus removal techniques have a low yield of spermatozoa. Therefore, we have developed a microfluidic chip that performs size-based separation of viruses and spermatozoa in boar semen samples, thereby having the potential to reduce the risk of disease spreading in the context of artificial insemination in the veterinary industry. As the head of a spermatozoon is at least twenty times larger than a virus particle, the particle size can be used to achieve separation, resulting in a semen sample with lower viral load and of higher quality. To achieve the size separation, our microfluidic device is based on pinched-flow fractionation. A model virus, cowpea chlorotic mottle virus, was used and spiked to porcine semen samples. With the proposed microfluidic chip and the optimized flow parameters, at least 84 ± 4% of the model viruses were removed from the semen. The remaining virus contamination is caused by the model virus adhering to spermatozoa instead of the separation technique. The spermatozoa recovery was 86 ± 6%, which is an enormous improvement in yield compared to existing virus removal techniques

Weighted single-step GWAS and gene network analysis reveal new candidate genes for semen traits in pigs

Background: In recent years, there has been increased interest in the study of the molecular processes that affect semen traits. In this study, our aim was to identify quantitative trait loci (QTL) regions associated with four semen traits (motility, progressive motility, number of sperm cells per ejaculate and total morphological defects) in two commercial pig lines (L1: Large White type and L2: Landrace type). Since the number of animals with both phenotypes and genotypes was relatively small in our dataset, we conducted a weighted single-step genome-wide association study, which also allows unequal variances for single nucleotide polymorphisms. In addition, our aim was also to identify candidate genes within QTL regions that explained the highest proportions of genetic variance. Subsequently, we performed gene network analyses to investigate the biological processes shared by genes that were identified for the same semen traits across lines. Results: We identified QTL regions that explained up to 10.8% of the genetic variance of the semen traits on 12 chromosomes in L1 and 11 chromosomes in L2. Sixteen QTL regions in L1 and six QTL regions in L2 were associated with two or more traits within the population. Candidate genes SCN8A, PTGS2, PLA2G4A, DNAI2, IQCG and LOC102167830 were identified in L1 and NME5, AZIN2, SPATA7, METTL3 and HPGDS in L2. No regions overlapped between these two lines. However, the gene network analysis for progressive motility revealed two genes in L1 (PLA2G4A and PTGS2) and one gene in L2 (HPGDS) that were involved in two biological processes i.e. eicosanoid biosynthesis and arachidonic acid metabolism. PTGS2 and HPGDS were also involved in the cyclooxygenase pathway. Conclusions: We identified several QTL regions associated with semen traits in two pig lines, which confirms the assumption of a complex genetic determinism for these traits. A large part of the genetic variance of the semen traits under study was explained by different genes in the two evaluated lines. Nevertheless, the gene network analysis revealed candidate genes that are involved in shared biological pathways that occur in mammalian testes, in both lines.</p