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In mice, microinjection is the most common gene transfer method used. Unfortunately, this strategy does not translate as well to livestock. Another potential method is sperm-mediated gene transfer, which takes advantage of sperm’s natural ability to bind to naked DNA. Gene transfer using sperm-mediated gene transfer has been shown in pigs (Gandolfi et al. 1989 J. Reprod. Fert. Abstr. Ser. 4) and cattle (Perez et al. 1991 Biotecnol. Apl. 8, 90–94). Based on these observations, we examined the efficiency of exogenous DNA binding to sperm using liposomes. In this experiment, we analysed methods to select thawed bovine sperm for DNA binding and evaluated the binding of exogenous DNA to those sperm. To determine the optimal sperm-selection method, the sperm were analysed using a computer-assisted sperm analyzer (CASA), the parameters selected were: total motility, rapid motility, and progressive motility. To measure the binding of DNA we used an indirect analysis using NanoDrop technology (Thermo Scientific, Wilmington, DE, USA) to compare the different DNA concentrations among groups. Liposome preparation was done using a cationic lipid, 3-(trimethyl ammonium iodide) 1,2 dimystryl-propanediate and a neutral lipid, l-a Dioleoyl phosphatidyl-ethanolamine prepared according to the protocol of Russell (1997). Percoll or swim-up methods were used to select sperm after thawing (Rubessa et al. 2016), followed by incubation (3 h) with the liposome-DNA complexes according to liposome preparation protocol (Russell, 1997). We used enhanced green fluorescent protein in combination with the liposomes as a marker for exogenous DNA binding. Five treatments per selection method were analysed: 1) immediately after processing (Control), 2) After 3 h of incubation with no liposomes, 3) incubation with liposomes and no DNA, 4) incubation with 1 ng of DNA, and 5) incubation with 10 ng of DNA. This was repeated five times. The CASA results for total motility and rapid motility showed a greater amount of significant differences (P

Reproductive biotechnology frequently requires improvements in our ability to isolate DNA from fewer and fewer cells. It is now possible to find several commercially available kits to meet this demand, but they frequently come at a relatively high cost. The aim of this project was to evaluate the efficiency of a simple handmade protocol to extract DNA from a small number of cells. To evaluate this method we chose 3 different bovine cell lines, Mac T-cells, sperm, and fibroblasts, for the analysis. Five different quantities (30, 100, 150, 300, and 500 cells) were evaluated for each cell line. The endpoints examined were the purity of the extracted DNA by NanoDrop analysis and visualisation of bands on agarose gels after PCR. The samples were initially diluted in 20 µL of PBS. The cell lysis buffer contained 15 mM Tris-HCl (pH 8.9), 50 mM KCl, 2.5 mM MgCl2, 0.1% Triton X-100, and 150 µg mL−1 of proteinase K. Twenty microlitres of lysis buffer were added to each sample. The tubes were then incubated at 55°C for 1 h. Last, proteinase K was inactivated by incubation at 90°C for 10 min. After extraction, 3 samples from each cell quantity for each replicate were evaluated by NanoDrop. After NanoDrop analysis, a standard PCR procedure was carried out using the homologue gene, tRep-137, as the amplification target. As a control, the Gene Wizard DNA extraction kit was used for all cell treatments. The DNA fragments resulting from PCR were separated for visualisation on agarose gels. Five replicates of the experiment were performed with 3 individual samples for each cell quantity evaluated. There were 80 total samples evaluated, including 5 control samples (1 per replicate). The PCR results were analysed using chi-square analysis with Fisher correction. An α-level of 0.05 was selected a priori for significant differences. The agarose gel analysis showed that each cell line gave different results. The data presented in Table 1 shows that this protocol is less effective for Mac T-cells, but satisfactory for both sperm and fibroblast cells. When the quality of the DNA extracted was evaluated with the NanoDrop method, DNA was found in all samples, but the purity of the samples was very low. None of the samples had a 260/280 ratio over 1, the range being 0.6 to 0.8. This high impurity could explain why not all samples showed PCR product bands on the gel. The next step will be to purify the DNA by removing all of the protein from the samples after isolation from the cells. In conclusion, these data show that it is possible to extract DNA at a low cost from a small number of cells. Table 1. The mean (SD in parentheses) percentage of samples with PCR bands for each cell line and each cell quantity

In cattle, a mutation in the NHL-repeat containing 2 genes causes a heritable abnormality referred to as developmental duplications. Calves homozygous for this mutation are affected with a broad range of phenotypes resulting from abnormal neural crest cell migration, most commonly manifested as polymelia, the presence of additional limbs. This mutation has become highly prevalent in Angus beef cattle, as lines of cattle with high genetic merit have been shown to have an increased allele frequency of the mutation. The mutation has been identified as a single nucleotide polymorphism resulting in a valine to alanine substitution in a highly conserved protein-coding region of the gene. CRISPR/Cas9 genome editing technology has been shown to induce changes in the genome by using a guide RNA to target a specific site paired with a Cas-9 protein to create a break in the DNA. These breaks are repaired by either nonhomologous end joining or homology-directed repair. The aim of this preliminary study was to determine the editing efficiency of CRISPR/Cas-9 proteins paired with site-specific guide RNA using cell lines derived from animals homozygous and heterozygous for the NHLRC2 mutation. Bovine fetal fibroblasts of both genotypes were grown in DMEM/F10 media supplemented with 10% fetal bovine serum, 0.01 µg mL−1 of basic fibroblast growth factor, 1 mL L−1 penicillin streptomycin, and 1 mL L−1 of amphotericin B. Cells were plated in a 6-well plate at 80,000 cells/well 48 h before transfection. Two 20-nucleotide guide RNA targeting the genome near the developmental duplications mutation were designed and ligated into pSpCas9(BB)-2A-GFP CRISPR plasmids. Six microliters of Fugene 6 was added to 150 µL of Opti-MEM followed by 2 µg of plasmid DNA and complexed at 37°C for 15 min before being added to each well. At 24 h after transfection, cells were detached with trypsin and sorted by fluorescence-activated cell sorting. When wells were confluent, DNA was extracted using 65 µL of QuickExtract DNA extraction solution. The 500-bp fragment surrounding the mutation was amplified and subjected to a restriction enzyme digest. Fragments with the exact sequence of the mutation were cleaved, whereas normal genotypes or edited genotypes without the mutation sequence remain uncleaved. Fragments were size separated on a 2% agarose gel. Band intensity under ultraviolet illumination was calculated with GelReader and the ratios of cleaved versus uncleaved fragments were compared with the ratio of control (unedited cell lines) for both guide RNA and both heterozygous and homozygous cell lines. Based on this preliminary data, CRISPRs with guide RNA 1 edited the genome at the target site at 13.3 and 12.2% for heterozygous and homozygous cell lines, respectively, and guide RNA 2 affected the target site at 2.5 and 4.1% for heterozygous and homozygous, respectively. These data show that the designed guide RNA paired with CRISPRs are able to elicit changes at the desired locus. In order to understand what repair mechanisms were employed at these loci, the next step is to subclone and sequence PCR products.

Sperm selection is a fundamental step for IVF, influencing treatment and the health of offspring. Over the years, several techniques have been tested to recover highly motile sperm, including centrifugation with discontinuous gradients and numerous filtration procedures. In our previous paper, we showed that it is possible, in ~20 min, to select a high quality sperm population using an electric charge in a microchannel device. This method produced the same blastocyst rates as a discontinuous gradient (DG), but in less time [25 min less; Rubessa et al. 2016 Andrology (Los Angeles) 5]. This technique takes advantage of one of the physiological characteristics of high quality sperm; that is, the head has a negative electric charge. The aim of the present experiment was to analyse the characteristics of the sperm selected with our device. We compared 2 standard sperm procedures and our device, with semen assessed using a computer-assisted sperm analyzer to evaluate the principal sperm parameters. Standard procedures were used to select sperm by DG and swim-up (SU). Thawed semen was pipetted into the microchannel of our device and then exposed to the electric charge (10 V) for 10 min [Rubessa et al. 2016 Andrology (Los Angeles) 5]. For each replicate, 3 straws of frozen bovine semen were thawed at 37°C for 40 s and pooled. The pooled sample was divided into 3 aliquots: one for each technique, 5 replicates of the experiment were performed. Data were analysed by ANOVA using the general linear model (GLM) procedure (SPSS Inc., Chicago, IL, USA). Bonferroni’s post hoc test was used to perform statistical multiple comparison. The α-level was set at 0.05. The results show an effect of the method all motility parameters are not significantly different, although some of the motility parameters were statistically different (P

In 2050, the expected size of the human population is 9 billion, the demand for food will increase, and the demand for milk will increase along with it. Genetically modifying animals is a tool that can be used to meet this growing demand. In the United States, Holstein is the leading breed for milk production and Holsteins produce on average 24 291 pounds of milk per year, whereas Jerseys, the other major dairy breed, produce on average 16 997 pounds. Their ability to produce large quantities of milk is linked to 2 mutations. These mutations are on the α-lactalbumin (α-lac) gene; the α-lac exon (+1) corresponds to the transcription start point of α-lac, (+15) and (–1689) are the positions corresponding to the single nucleotide polymorphism associated with increased milk production. Holstein cows have an adenine at both of these positions in contrast to the other cattle breeds with lower milk production, which have either a cytosine or guanine at either position. Inserting an adenine at position (+15) and (–1689) in cows without this mutation could lead to increased milk production and a better response to market demands. The purpose of this experiment was to test the cutting efficiency of candidate clustered regularly interspaced short palindromic repeats (CRISPR) that will later be used in knock-in experiments. CRISPRs were used because the CRISPR-Cas9 system is inexpensive, easily programmed, and efficient. In this preliminary study, we worked with Holstein MAC-T cells, which already contain the mutation at both positions. CRISPRs were used on this cell line to cut the DNA at a site near the mutation. Based on the genomic DNA sequence of these MAC-T cells, 3 guide RNAs were designed. Cells were then transfected with the designed CRISPRs by a variety of transfection methods, including Fugene™ (Promega, Madison, WI, USA), electroporation, and Lipofectamine (ThermoFisher Scientific, Waltham, MA, USA). Green fluorescent protein was used to determine the efficiency of transfection; 30% efficiency was seen for Fugene™, whereas electroporation and Lipofectamine™ had 70% efficiency. To select for successfully transfected cells, puromycin selection was applied. The DNA was later extracted and sent in for sequencing. Next, the website TIDE was used to compare the transfected MAC-T cells to normal MAC-T cells. The TIDE software measures the editing efficiency and looks for major insertions or deletions in pools of DNA by comparing 2 sequences to quantify the editing efficacy of CRISPR-Cas9. Our results showed that CRISPRs successfully cut the DNA near the α-lac mutation region with a total efficiency of 13.8%. The desired next step will be to insert a single-strand oligonucleotide (ssODN) donor to make a single basepair mutation. The ultimate aim of this research would be to insert these mutations into other cattle species in order to increase their milk production.

Bovine leukosis virus (BLV) is a pathogen that affects the bovine immune system and leads to lymphosarcoma, leukemia, decreased milk production, and increased culling rates in cattle. BLV-infected cattle herds can be found worldwide; in the United States, specifically, 38% of beef herds, 84% of all dairy herds, and 100% of large-scale dairy operation herds are infected (Buehring et al. 2014 Emerg. Infect. Dis. 5, 772–782). The main transmission between cattle in herds is affected leukocytes in blood. Several farm practices, such as dehorning, rectal palpation, and vaccinating can lead to the pathogen transmission. Due to international trade laws and biosecurity concerns, semen from a BLV-positive bull is illegal to sell within certain countries. Prior studies have looked at use of seropositive bulls in AI with little risk in affecting the dam (Burger et al. 2000 AVJR 60, 819). Other studies used semen that was artificially infected with the virus then used for IVF (Bielanski et al. 2000 Vet. Rec. 146, 255–256). The aim of this research was to evaluate naturally infected BLV donor semen using abattoir-derived oocytes and the possible contamination of in vitro-produced (IVP) embryos. Semen was collected and frozen by a private company. Three seropositive bulls and 1 negative control bull were selected. All positive bulls were selected based on availability of seropositive BLV status. Prior to the experiment, all bulls used were evaluated for motility, concentration, and morphology. The negative control was used in prior IVF experiments that produced acceptable results for use in this experiment. Frozen sperm were thawed at 37°C for 40 s and pelleted by centrifugation (25 min at 300 × g) on a Percoll discontinuous gradient (45–80% in Tyrode’s modified medium without glucose and BSA). The matured oocytes were purchased from DeSoto Biosciences (Seymour, TN, USA) and were IVF according to standard procedures (Rubessa et al. 2011 Theriogenology 76, 1347–1355). Using 200 oocytes per replicate, the 3 positive bulls and 1 control bull were allocated 50 oocytes per bull in each replicate. After 20 to 22 h of gametes co-incubation, zygotes were denuded and cultured for 7 days in SOF, followed by the evaluation of embryos (from tight morula until hatching blastocyst). Positive bull #1 produced and tested 48 embryos. Positive bull #2 produced and tested 41 embryos. Positive bull #3 produced and tested 46 embryos. The negative control produced and tested 55 embryos. Embryonic DNA extraction was performed using standard procedures (Sattar et al. 2011 Reprod. Domest. Anim. 46, 1090–1097). Nested PCR followed the Fechner evaluations methods (Fechner et al. 1996 J. Vet. Med. B 43, 621–630). To detect BLV presence, electrophoresis was used with a 2% agarose gel containing 0.1% ethidium bromide. A total of 190 embryos were evaluated that were produced in 3 replicates. All samples analysed showed no evidence of BLV. In conclusion, use of BLV seropositive donor semen showed no transmission of the virus upon IVF of the oocytes.

Lactoferrin (LF) is an antimicrobial protein that is produced at high levels in human secretions, including milk, but is present at very low levels in the milk of ruminants. Lactoferrin is an iron-binding protein, and because iron is required by some bacteria to grow, lactoferrin can reduce the growth of certain bacteria. Lactoferrin can inhibit the growth of gram-negative bacteria, gram-positive bacteria, yeast, and some antibiotic-resistant pathogens. Lactoferrin also has bacteriocidal activity, killing the organism by inducing membrane perturbations often in conjunction with other antimicrobial agents such as lysozyme. Due to these properties, lactoferrin has the potential to increase the health of lactating animals as well as extend the shelf life of the milk. A herd of cows were genetically engineered to produce milk containing recombinant human lactoferrin (rhLF-milk) at approximately the same concentration found in human milk. In this study, growth of bacteria in raw milk samples from 2 control cows was compared with that from 2 cows that produce rhLF-milk. The experiment examined the shelf life of milk at room temperature over a 72-h period and the shelf life of milk left at 4°C over 3 weeks by plating aliquots on agar plates and counting the number of bacterial colonies that grew on plates after 24 and 48 h. The milk left at room temperature was plated at 0, 24, 48, and 72 h. The milk left in at 4°C was measured at 0, 4, 7, 11, 14, 18, and 21 days. Statistical evaluations were conducted using SAS statistical software, version 9.3 (SAS Institute Inc., Cary, NC, USA). Bacterial data were compared using the repeated-measures function in SAS and the Greenhouse-Geisser epsilon test to determine differences. For all tests, P-values