Department of Large Animal Clinical Sciences

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https://hdl.handle.net/10919/24261

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Browsing Department of Large Animal Clinical Sciences by Department "Dairy Science"

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    Frequency of off-targeting in genome edited pigs produced via direct injection of the CRISPR/Cas9 system into developing embryos
    Carey, Kayla; Ryu, Junghyun; Uh, Kyungjun; Lengi, Andrea J.; Clark-Deener, Sherrie; Corl, Benjamin A.; Lee, Kiho (2019-05-06)
    Background The CRISPR/Cas9 system can effectively introduce site-specific modifications to the genome. The efficiency is high enough to induce targeted genome modifications during embryogenesis, thus increasing the efficiency of producing genetically modified animal models and having potential clinical applications as an assisted reproductive technology. Because most of the CRISPR/Cas9 systems introduce site-specific double-stranded breaks (DSBs) to induce site-specific modifications, a major concern is its potential off-targeting activity, which may hinder the application of the technology in clinics. In this study, we investigated off-targeting events in genome edited pigs/fetuses that were generated through direct injection of the CRISPR/Cas9 system into developing embryos; off-targeting activity of four different sgRNAs targeting RAG2, IL2RG, SCD5, and Ig Heavy chain were examined. Results First, bioinformatics analysis was applied to identify 27 potential off-targeting genes from the sgRNAs. Then, PCR amplification followed by sequencing analysis was used to verify the presence of off-targeting events. Off-targeting events were only identified from the sgRNA used to disrupt Ig Heavy chain in pigs; frequency of off-targeting was 80 and 70% on AR and RBFOX1 locus respectively. A potential PAM sequence was present in both of the off-targeting genes adjacent to probable sgRNA binding sites. Mismatches against sgRNA were present only on the 5′ side of AR, suggesting that off-targeting activities are systematic events. However, the mismatches on RBFOX1 were not limited to the 5′ side, indicating unpredictability of the events. Conclusions The prevalence of off-targeting is low via direct injection of CRISPR/Cas9 system into developing embryos, but the events cannot be accurately predicted. Off-targeting frequency of each CRISPR/Cas9 system should be deliberately assessed prior to its application in clinics.
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    Ruminal volatile fatty acid absorption is affected by elevated ambient temperature
    Bedford, Andrea; Beckett, Linda; Harthan, Laura; Wang, Chong; Jiang, Ning; Schramm, Hollie H.; Guan, Leluo; Daniels, Kristy M.; Hanigan, Mark D.; White, Robin R. (2020-08-04)
    The objective of this study was to investigate the effect of short-term elevated ambient temperature on ruminal volatile fatty acid (VFA) dynamics and rumen epithelium gene expression associated with the transport and metabolism of VFA. Eight ruminally cannulated Holstein heifers (200 kg) were used in a factorial, repeated measures experiment with two treatments and two periods. During the first period, animals were provided with feed ad libitum and housed at 20 degrees C. During the second period, one group (HS) was housed at 30 degrees C and fed ad libitum. The other group (PF) was housed at 20 degrees C and pair-fed to match the intake of the HS group. During each period, animals were kept on treatment for 10 day, with sample collection on the final day. In the second period, indicators of heat stress were significantly different between PF and HS animals (P<0.05). There was a thermal environment effect on butyrate production (P<0.01) that was not associated with feed intake (P=0.43). Butyrate absorption decreased in HS animals (P<0.05) but increased in PF animals (P<0.05) from period 1 to period 2. There was a feed intake effect on BHD1 expression (P=0.04) and a tendency for a thermal environment effect (P=0.08), with expression increasing in both cases. Expression of MCT4 was affected by feed intake (P=0.003) as were all NHE genes (NHE1, NHE2, and NHE3; P<0.05). These results indicate that with low feed intake and heat stress, there are shifts in rumen VFA dynamics and in the capacity of the rumen epithelium to absorb and transport VFA.
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    Short Communication: Association between neonatal calf diarrhea and lying behaviors
    Swartz, Turner H.; Schramm, Hollie H.; Petersson-Wolfe, Christina S. (Elsevier, 2020-06-01)
    The objective of this study was to determine the association of neonatal calf diarrhea (NCD) with step activity and lying behaviors in pre-weaned dairy calves. Calves were housed in individual hutches for the first 6 days of life, and then moved into a group pen. On the day of birth, calves (n = 30) were fitted with an accelerometer, and step activity and lying behaviors were recorded. Calves were assigned a fecal score (FS) twice daily using a 0 to 3 scale, and were diagnosed with NCD (n = 10) when the score was a 3. To ensure the only association noted was due to NCD, calves that had any other health complications were excluded from analyses (n = 1). Calves with NCD were pair matched by age, breed, and birthdate to a healthy calf. Day 0 was designated as the date of NCD diagnosis. Calves with NCD spent less time lying (P < 0.05) and displayed more lying bouts (P < 0.05) of a shorter duration (P < 0.01) than healthy calves. Specifically, calves with NCD displayed more lying bouts on days -7 (P < 0.05), -6 (P < 0.01), -5 (P < 0.01), -4 (P < 0.01), and -3 (P < 0.05). Similarly, lying bout duration was shorter for calves with NCD on days -6 (P < 0.05), -5 (P < 0.05), -4 (P < 0.01), and -3 (P < 0.01). Additional research is needed to examine if these tools can be used to identify diseased calves prospectively.