School of Animal Sciences

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The School of Animal Sciences merged Dairy Science and Animal and Poultry Science in 2022.

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Biallelic modification of IL2RG leads to severe combined immunodeficiency in pigs
Clark-Deener, Sherrie; Kang, Jung-Taek; Cho, Bumrae; Ryu, Junghyun; Ray, Caitlin; Lee, Eun-Jin; Ahn, SunMi; Lee, JinSeok; Ji, Dal-Young; Jue, Nathaniel; Lee, Kiho; Park, Kwang-Wook (2016-11-03)
Background: Pigs with SCID can be a useful model in regenerative medicine, xenotransplantation, and cancer cell transplantation studies. Utilizing genome editing technologies such as CRISPR/Cas9 system allows us to generate genetically engineered pigs at a higher efficiency. In this study, we report generation and phenotypic characterization of IL2RG knockout female pigs produced through combination of CRISPR/Cas9 system and SCNT. As expected, pigs lacking IL2RG presented SCID phenotype. Methods: First, specific CRISPR/Cas9 systems targeting IL2RG were introduced into developing pig embryos then the embryos were transferred into surrogates. A total of six fetuses were obtained from the embryo transfer and fetal fibroblast cell lines were established. Then IL2RG knockout female cells carrying biallelic genetic modification were used as donor cells for SCNT, followed by embryo transfer. Results: Three live cloned female piglets carrying biallelic mutations in IL2RG were produced. All cloned piglets completely lacked thymus and they had a significantly reduced level of mature T, B and NK cells in their blood and spleen. Conclusions: Here, we generated IL2RG knockout female pigs showing phenotypic characterization of SCID. This IL2RG knockout female pigs will be a promising model for biomedical and translational research.
Comparison of reproductive performance of AI- and natural service-sired beef females under commercial management
Marrella, Mackenzie A.; White, Robin R.; Dias, Nicholas W.; Timlin, Claire; Pancini, Stefania; Currin, John F.; Clark, Sherrie G.; Stewart, Jamie L.; Mercadante, Vitor R. G.; Bradford, Heather L. (Oxford University Press, 2021-07)
The objective of this study was to assess differences in reproductive performance of natural service and artificial insemination (AI) sired beef females based on pregnancy outcomes, age at first calving, and calving interval. Data were sourced from 8,938 cows sired by AI bulls and 3,320 cows sired by natural service bulls between 2010 and 2017. All cows were in a commercial Angus herd with 17 management units located throughout Virginia and represented spring and fall calving seasons. All calves were born to dams managed with estrus synchronization. Pregnancy was analyzed with generalized linear mixed models and other reproductive measures with linear mixed models in R. Six models were evaluated with the dependent variables of pregnancy status at the first diagnosis, pregnancy status at the second diagnosis, pregnancy type (AI or natural service) at the first diagnosis, pregnancy type at the second diagnosis, calving interval, and age at first calving. Independent variables differed by model but included sire type of the female (AI or natural service), prebreeding measures of age, weight, and body condition score, postpartum interval, sex of the calf nursing the cow, and management group. No differences were observed between AI- and natural service-sired females based on pregnancy status at first and second pregnancy diagnosis (P > 0.05). Sire type was only found to be significant for age at first calving (P < 0.05) with AI-sired females being 26.6 ± 1.6 d older at their first calving, which was expected because AI-sired females were born early in the calving season making them older at breeding. Surprisingly, age and body condition score were not significant predictors of pregnancy (P > 0.05). Body weight at breeding was not significant for pregnancy (P > 0.05) but was significant for age at first calving (P < 0.05). These data suggested that lighter heifers calved earlier which contradicts our original hypothesis. Overall, commercial Angus females sired by AI or natural service bulls had similar reproductive performance. Factors that were commonly associated with reproductive success were not significant in this commercial Angus herd managed with estrus synchronization. Given the size of these data, the importance of body condition, age, and weight should be reassessed in modern genetics and management practices.
Effects of mid-gestational l-citrulline supplementation to twin-bearing ewes on umbilical blood flow, placental development, and lamb production traits
Kott, Michelle L.; Pancini, Stefania; Speckhart, Savannah L.; Kimble, Lauren N.; White, Robin R.; Stewart, Jamie L.; Johnson, Sally E.; Ealy, Alan D. (Oxford University Press, 2021-07)
The objective of the study was to examine how l-citrulline supplementation to ewes during mid-gestation influences placental activity, placental blood flow, lamb body weight, and carcass characteristics. Two studies were completed. A pharmacokinetic study to compare circulating plasma amino acid concentrations after a single intravenous injection of 155 µmol/kg BW l-citrulline or after an isonitrogenous amount of l-alanine (control; 465 µmol/kg BW). Increases (P < 0.05) in circulating citrulline concentrations were detected for 8 h after l-citrulline injection versus the control. Similarly, increases (P < 0.05) in circulating arginine concentrations were detected for 24 h after l-citrulline treatment. The second study used 12 ewes with twin pregnancies. Daily intravenous injections of either l-citrulline or l-alanine were administered for 39 d from d 42-45 to 81-84 of gestation. Ewes were limit-fed at 85% daily energy requirements during the injection period. A decrease (P < 0.0001) in body weight was observed in both treatment groups during this period. No treatment differences were observed in circulating pregnancy-specific protein B concentrations or placental blood flow during the treatment and post-treatment gestational period. No treatment differences were observed in lamb survival nor in lamb birth, weaning and slaughter weights. Treatment did not influence lamb carcass composition or organ weights. However, there was a tendency (P = 0.10) for an increase in antral follicle numbers in ovaries from ewe lambs derived from ewes treated with l-citrulline. In summary, a daily l-citrulline injection increased both circulating citrulline and arginine concentrations in ewes, but daily l-citrulline injections during mid-gestation did not produce any detectable changes in placental activity and blood flow, neonatal and postnatal lamb development, and lamb carcass composition at slaughter. In conclusion, no benefits in placental function and lamb development were observed after providing l-citrulline during mid-gestation in ewes exposed to a mild energy restriction, but there was an indication that follicle numbers in ewe lambs were positively influenced by l-citrulline treatment during fetal development.
Establishing an immunocompromised porcine model of human cancer for novel therapy development with pancreatic adenocarcinoma and irreversible electroporation
Hendricks-Wenger, Alissa; Aycock, Kenneth N.; Nagai-Singer, Margaret A.; Coutermarsh-Ott, Sheryl; Lorenzo, Melvin F.; Gannon, Jessica; Uh, Kyungjun; Farrell, Kayla; Beitel-White, Natalie; Brock, Rebecca M.; Simon, Alexander; Morrison, Holly A.; Tuohy, Joanne L.; Clark-Deener, Sherrie; Vlaisavljevich, Eli; Davalos, Rafael V.; Lee, Kiho; Allen, Irving C. (Nature Research, 2021-04-07)
New therapies to treat pancreatic cancer are direly needed. However, efficacious interventions lack a strong preclinical model that can recapitulate patients’ anatomy and physiology. Likewise, the availability of human primary malignant tissue for ex vivo studies is limited. These are significant limitations in the biomedical device field. We have developed RAG2/IL2RG deficient pigs using CRISPR/Cas9 as a large animal model with the novel application of cancer xenograft studies of human pancreatic adenocarcinoma. In this proof-of-concept study, these pigs were successfully generated using on-demand genetic modifications in embryos, circumventing the need for breeding and husbandry. Human Panc01 cells injected subcutaneously into the ears of RAG2/IL2RG deficient pigs demonstrated 100% engraftment with growth rates similar to those typically observed in mouse models. Histopathology revealed no immune cell infiltration and tumor morphology was highly consistent with the mouse models. The electrical properties and response to irreversible electroporation of the tumor tissue were found to be similar to excised human pancreatic cancer tumors. The ample tumor tissue produced enabled improved accuracy and modeling of the electrical properties of tumor tissue. Together, this suggests that this model will be useful and capable of bridging the gap of translating therapies from the bench to clinical application.
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.
Growth hormone stimulates hepatic expression of bovine growth hormone receptor messenger ribonucleic acid through signal transducer and activator of transcription 5 activation of a major growth hormone receptor gene promoter
Jiang, Honglin; Wang, Ying; Wu, Miaozong; Gu, Zhiliang; Frank, Stuart J.; Torres-Diaz, Roberto (Endocrine Society, 2007-07)
The objective of this study was to determine whether and how GH regulates hepatic expression of GH receptor (GHR) mRNA in cattle. Ribonuclease protection assays revealed that injection of GH in a slow-release formula increased both hepatic GHR and IGF-I mRNAs 1 wk after the injection. The increases in GHR and IGF-I mRNAs were highly correlated. Western blot analysis showed that the injection also increased liver GHR protein level. In cattle and other mammals, hepatic GHR mRNA is expressed as variants that differ in the 5'-untranslated region due to the use of different promoters in transcription and/or alternative splicing. We found that GH increased the expression of the liver-specific GHR mRNA variant GHR1A without affecting the other two major GHR mRNA variants in the bovine liver, GHR1B and GHR1C. In transient transfection analyses, GH could robustly activate reporter gene expression from a 2.7-kb GHR1A promoter, suggesting that GH augmentation of GHR1A mRNA expression in the liver is at least partially mediated at the transcriptional level. Additional transfection analyses of serially 5'-truncated fragments of this promoter narrowed the GH-responsive sequence element down to a 210-bp region that contained a putative signal transducer and activator of transcription 5 (STAT5) binding site. EMSAs demonstrated that this putative STAT5 binding site was able to bind to STAT5b protein. In cotransfection assays, deletion of this putative STAT5 binding site abolished most of the GH response of the GHR1A promoter. Like 1-wk GH action, 6-h (i.e. short-term) GH action also increased liver expression of GHR1A and total GHR mRNAs in cattle. These observations together suggest that GH directly stimulates the expression of one GHR mRNA variant, GHR1A, through binding STAT5 to its promoter, thereby increasing GHR mRNA and protein expression in the bovine liver.
Habituation Protocols Improve Behavioral and Physiological Responses of Beef Cattle Exposed to Students in an Animal Handling Class
Ujita, Aska; Seekford, Zachary; Kott, Michelle L.; Goncherenko, Guillermo; Dias, Nicholas W.; Feuerbacher, Erica N.; Bergamasco, Luciana; Jacobs, Leonie; Eversole, Dan Edward; Negrão, João A.; Mercadante, Vitor R. G. (MDPI, 2021-07-21)
Our objective was to determine the impact of different habituation protocols on beef cattle behavior, physiology, and temperament in response to human handling. Beef heifers were exposed to three habituation strategies: (1) tactile stimulation (brushing) in the working chute for seven consecutive days (STI; n = 18); (2) passage through the working chute for seven consecutive days (CHU; n = 19) and; (3) no habituation (CON; n = 19). Individual heifer respiratory rate (RR; n/min), internal vaginal temperature (VAGT; °C), and blood cortisol were measured. Further, behavior parameters were observed to generate a behavior score, and heifer interaction with students and their behavioral responses were recorded. Habituation with STI and CHU resulted in improved numerical behavioral scores compared to CON, and greater (p ≤ 0.05) handling latencies. Vaginal temperature was decreased in STI compared to CHU and CONT (p ≤ 0.05). Cortisol concentration did not differ among treatments, but decreased (p ≤ 0.05) from the start of the experiment to 14 days after treatment initiation. Both habituation protocols showed benefits, but heifers that received the positive tactile stimulation in the chute had the greatest behavior improvements. Furthermore, these heifers responded more calmly during student-animal interactions in class, which is beneficial for the students’ and animals’ safety.
Immunological castration temporarily reduces testis size and function without long-term effects on libido and sperm quality in boars.
Clark-Deener, Sherrie; Lugar, Drew William; Rhoads, Michelle; Callahan, Stuart Russell; Prusa, Ken J.; Estienne, Mark J. (2016-10-27)
The objective was to determine the effects of immunization against gonadotropin releasing hormone on reproductive characteristics in boars. Seventy-two boars were used in a randomized design with three treatments: single immunization (SI) or double immunization (DI) with Improvest® (Zoetis Animal Health, Florham Park, NJ, USA) and intact controls (no Improvest®; CNT) (n = 24/group). At 10, 15, 20, 25, and 40 wk of age, blood was collected and serum harvested to evaluate testosterone concentrations. Testosterone concentrations were less for DI boars compared to CNT boars and SI boars at 20 and 25 wk (P < 0.001), but not 40 wk of age. At wk 25, 18 pigs (n = 6/ group) were sacrificed and testes were removed, weighed, and measured and seminiferous tubules were examined and scored using histological slides of testes parenchyma. A sample of backfat was assessed for boar taint aroma. All testicular measurements and weights and seminiferous tubule scores were less for DI boars compared to SI and CNT boars (P < 0.001). More (P < 0.05) SI and CNT boars had detectable boar taint aroma than DI boars. Libido was assessed at 32, 36, 47, 60, and 63 wk of age and semen collected at 60 wk of age was analyzed for indicators of quality. There was no treatment effect (P = 0.41) on libido. Semen volume, gel weight and total number of sperm cells were not different among treatments. Sperm concentration was greater for DI than SI (P = 0.01), and tended to be greater for DI compared to CNT (P = 0.10). Sperm motility tended to be greater for DI boars compared to CNT boars (P = 0.066). The results show that there are no permanent effects of immunocastration on reproductive characteristics in boars.
Increased and prolonged human norovirus infection in RAG2/IL2RG deficient gnotobiotic pigs with severe combined immunodeficiency
Lei, Shaohua; Ryu, Junghyun; Wen, Ke; Twitchell, Erica; Bui, Tammy; Ramesh, Ashwin; Weiss, Mariah; Li, Guohua; Samuel, Helen; Clark-Deener, Sherrie; Jiang, Xi; Lee, Kiho; Yuan, Lijuan (Nature Publishing Group, 2016-04-27)
Application of genetically engineered (GE) large animals carrying multi-allelic modifications has been hampered by low efficiency in production and extended gestation period compared to rodents. Here, we rapidly generated RAG2/IL2RG double knockout pigs using direct injection of CRISPR/Cas9 system into developing embryos. RAG2/IL2RG deficient pigs were immunodeficient, characterized by depletion of lymphocytes and either absence of or structurally abnormal immune organs. Pigs were maintained in gnotobiotic facility and evaluated for human norovirus (HuNoV) infection. HuNoV shedding lasted for 16 days in wild type pigs, compared to 27 days (until the end of trials) in RAG2/IL2RG deficient pigs. Additionally, higher HuNoV titers were detected in intestinal tissues and contents and in blood, indicating increased and prolonged HuNoV infection in RAG2/IL2RG deficient pigs and the importance of lymphocytes in HuNoV clearance. These results suggest that GE immunodeficient gnotobiotic pigs serve as a novel model for biomedical research and will facilitate HuNoV studies.
Quantification of zearalenone and α-zearalenol in swine liver and reproductive tissues using GC-MS
Pack, Erica; Stewart, Jacob; Rhoads, Michelle; Knight, James W.; De Vita, Raffaella; Clark-Deener, Sherrie; Schmale, David G. III (Elsevier, 2020-12-01)
The mycotoxin zearalenone (ZEN) is a common contaminant of swine feed which has been related to a wide range of reproductive anomalies in swine, such as pelvic organ prolapse, anestrous, and pseudopregnancy. New information is needed to understand how ZEN and related metabolites accumulate in swine reproductive tissues. We conducted a feeding study to track ZEN and the metabolite α-zearalenol (α-ZEL) in swine liver and reproductive tissues. Thirty pubertal gilts were randomly assigned one of three treatments, with ten pigs in each treatment group: (1) base feed with solvent for 21 days, (2) ZEN-spiked feed for seven days followed by base feed with solvent for 14 days, and (3) ZEN-spiked feed for 21 days. At the end of the trial, liver, anterior vagina, posterior vagina, cervix, uterus, ovaries, and broad ligament were collected from pigs. ZEN was found in the anterior vagina, posterior vagina, cervix, and ovaries, with significantly higher concentrations in the cervix relative to other reproductive tissues. ZEN and α-ZEL were found in liver tissue from pigs in each treatment group. Our results show that ZEN accumulates more in the cervix than other reproductive tissues. The presence of ZEN in reproductive tissues may be indicative of ZEN-related reproductive symptoms. Future work could examine how ZEN concentrations vary in reproductive tissues as a factor of the pigs age, weight, sex, or parity, to establish parameters that make pig more sensitive to ZEN.
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.
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.
Variation in Eimeria oocyst count and species composition in weanling beef heifers
Lucas, A. S.; Swecker, William S.; Scaglia, G.; Lindsay, David S.; Zajac, Anne M. (American Society of Parasitology, 2006-10)
Rectal fecal samples were collected daily on 10 consecutive days in November 2004 from I I weaned beef heifers to assess daily variation in fecal oocyst count and species composition. Subsequent samples were collected from the same animals on 15 April 2005 and 9 June 2005. Oocyst numbers were determined by the modified McMaster's test, and species were identified by examination of oocysts recovered with the Wisconsin sugar flotation technique. Soil samples were collected from the heifer pasture on 8 June 2005, and oocysts were quantified and identified to species. Mean fecal oocyst counts varied little at all sampling dates ranging from 134-377 oocysts/g. Ten Eimeria spp. were identified in fecal samples collected in November and April and 11 in June. Eimeria bovis was the most common species identified at all samplings. Mean species composition showed little variation during the 10-day sampling period in November, remained similar in April, and varied slightly in June. Twelve Eimeria spp. were identified in soil samples in proportions similar to those seen in fecal samples. The results indicate that clinically normal weanling beef heifers are likely to be infected with a diverse, but relatively stable, community of Eimeria spp.

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