VTechWorks Repository :: Browsing by Author "Ealy, Alan D."

Browsing by Author "Ealy, Alan D."

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Ablation of OCT4 function in cattle embryos by double electroporation of CRISPR-Cas for DNA and RNA targeting (CRISPR-DART)
Nix, Jada L.; Schettini, Gustavo P.; Speckhart, Savannah L.; Ealy, Alan D.; Biase, Fernando H. (Oxford University Press, 2023-11-01)
CRISPR-Cas ribonucleoproteins (RNPs) are important tools for gene editing in preimplantation embryos. However, the inefficient production of biallelic deletions in cattle zygotes has hindered mechanistic studies of gene function. In addition, the presence of maternal RNAs that support embryo development until embryonic genome activation may cause confounding phenotypes. Here, we aimed to improve the efficiency of biallelic deletions and deplete specific maternal RNAs in cattle zygotes using CRISPR-Cas editing technology. Two electroporation sessions with Cas9D10A RNPs targeting exon 1 and the promoter of OCT4 produced biallelic deletions in 91% of the embryos tested. In most cases, the deletions were longer than 1,000 nucleotides long. Electroporation of Cas13a RNPs prevents the production of the corresponding proteins. We electroporated Cas9D10A RNPs targeting exon 1, including the promoter region, of OCT4 in two sessions with inclusion of Cas13a RNPs targeting OCT4 mRNAs in the second session to ablate OCT4 function in cattle embryos. A lack of OCT4 resulted in embryos arresting development prior to blastocyst formation at a greater proportion (13%) than controls (31.6%, P < 0.001). The few embryos that developed past the morula stage did not form a normal inner cell mass. Transcriptome analysis of single blastocysts, confirmed to lack exon 1 and promoter region of OCT4, revealed a significant (False Discovery Rate, FDR < 0.1) reduction in transcript abundance of many genes functionally connected to stemness, including markers of pluripotency (CADHD1, DPPA4, GNL3, RRM2). The results confirm that OCT4 is a key regulator of genes that modulate pluripotency and is required to form a functional blastocyst in cattle.
Additive effects among uterine paracrine factors in promoting bovine trophoblast cell proliferation
Xie, Ming (Virginia Tech, 2014-06-10)
Several uterine-derived paracrine factors have been implicated as critical regulators of conceptus development in cattle, but it remains unclear how these factors work together to establish and maintain pregnancies. The primary objectives of this work were to establish if cooperative interactions between epidermal growth factor (EGF), fibroblast growth factor-2 (FGF2) and insulin-like growth factor-1 (IGF1) promote bovine trophoblast cell proliferation, and to decipher the intracellular signaling mechanisms employed by these growth factors to regulate cell proliferation. Pilot studies established effective concentrations for each growth factor on a bovine trophoblast cell line (CT1). The first set of studies examined how each factor worked individually or in conjunction with each other to impact CT1 proliferation. Mitotic index (percentage of EdU-positive nuclei after a 45 min challenge) was increased (P<0.05) by supplementation with 10 ng/ml EGF, 10 ng/ml FGF2, or 50 ng/ml IGF1 when compared with non-treated controls. In addition, a greater increase (P<0.05) was detected when all three factors were supplemented together. A follow-up study determined that supplementation of any two growth factors could not replicate the cooperative effect noted when all three factors were provided. A second set of studies was undertaken to examine how mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinase/AKT (PI3K/AKT) signaling systems mediate the independent and cooperative effects of these paracrine factors. Both EGF and IGF1 transiently activated mitogen-activated protein kinase3/1 (MAPK3/1) in CT1 cells as determined by Western Blot analysis. By contrast, FGF2 did not affect MAPK3/1 phosphorylation status, but increased AKT phosphorylation status. Neither EGF nor IGF1 impacted AKT activity. Supplementation with a pharmacological inhibitor of MAPK3/1 (PD98059) prevented EGF-, IGF1-, and FGF2-dependent increases in CT1 cell proliferation. This inhibitor also completely abolished the increases in cell proliferation observed when all three factors were supplemented together. Supplementation with a pharmacological inhibitor of AKT (Wortmannin) reduced FGF2-stimulated CT1 proliferation, but did not impact EGF- and IGF1 effects. The AKT inhibitor partially attenuated the cooperative effects of all three factors on CT1 cell proliferation. A final study examined how the combination of EGF, FGF2, and IGF1 affect bovine embryo development. In vitro produced bovine blastocysts were cultured either with the combination of growth factors or vehicle only from day 8 to day 12 post-in vitro fertilization (IVF). The combination of EGF, FGF2, and IGF1 increased (P<0.05) the percentage of hatched blastocysts and outgrowth formation versus controls. Increased (P<0.05) diameters were detected in blastocysts treated with the combination of three growth factors on day 12 post-IVF when compared to controls. Treatment with the combination of EGF, FGF2, and also IGF1 increased (P<0.05) the change of diameter from day 8 to 12 post-IVF. In summary, these observations provide evidence that cooperative interactions of uterine-derived factors promote trophoblast proliferation and conceptus development in ways that may promote the establishment and maintenance of pregnancy in cattle. The mechanisms utilized for these activities remain unresolved, but MAPK3/1 and PI3K/AKT signaling systems appear to play integral roles in some of these processes.
Associations of antral follicle count with fertility in cattle: A review
Alward, Kayla J.; Cockrum, Rebecca R.; Ealy, Alan D. (American Dairy Science Association, 2023-01-31)
Ovarian antral follicle count (AFC) is a marker of ovarian stimulatory response to superovulation protocols in cattle. This article reviews novel research from the past 10 years, focusing on the relationship between AFC and embryo production and cow fertility. Substantial evidence indicates a positive relationship between AFC with embryo production; however, conflicting findings exist regarding the relationship of AFC with conception and pregnancy rates. This lack of consistent association with pregnancy outcomes is perplexing given the differences detected in oocytes, embryos, and endometria from high- versus low-AFC animals. Those differences include markers of embryonic viability such as protein level, blastocyst development rates, cleavage rates, and blastocyst cell numbers that differ between high- and low-AFC groups, as well as differential gene expression at the cow and embryo level with genes associated with fertility. In addition, Bos indicus and Bos taurus cattle appear to have different fertility responses based on their AFC category. In summary, clearly more studies are needed to elucidate the true associations between AFC and cow fertility, but the data that have been accumulated thus far indicate that AFC has the potential to be a useful marker of lifetime cow fertility.
Bovine trophectoderm cells induced from bovine fibroblasts with induced pluripotent stem cell reprogramming factors
Talbot, Neil C.; Sparks, Wendy O.; Phillips, Caitlin E.; Ealy, Alan D.; Powell, Anne M.; Caperna, Thomas J.; Garrett, Wesley M.; Donovan, David M.; Blomberg, Le Ann (2017-06)
Thirteen independent induced bovine trophectroderm (iBT) cell lines were established by reprogramming bovine fetal liver-derived fibroblasts after viral-vector transduction with either six or eight factors, including POU5F1 (OCT4), KLF4, SOX2, MYC, NANOG, LIN28, SV40 large T antigen, and hTERT. Light-and electron-microscopy analysis showed that the iBT cells had epithelial cell morphology typical of bovine trophectoderm cells. Reverse-transcription-PCR assays indicated that all of the cell lines expressed interferon-tau (IFNT) at passages 1 or 2. At later passages (>= passage 8), however, immunoblot and antiviral activity assays revealed that more than half of the iBT cell lines had stopped expressing IFNT. Messenger RNAs specific to trophectoderm differentiation and function were found in the iBT cell lines, and 2-dimensional gel analysis for cellular proteins showed an expression pattern similar to that of trophectoderm cell lines derived from bovine blastocysts. Integration of some of the human reprogramming factors, including POU5F1, KLF4, SOX2, MYC, NANOG, and LIN28, were detected by PCR, but their transcription was mostly absent in the iBT cell lines. Gene expression assessment of endogenous bovine reprogramming factor orthologs revealed endogenous bLIN28 and bMYC transcripts in all; bSOX2 and bNANOG in none; and bKLF4 and bPOU5F1 in less than half of the iBT cell lines. These results demonstrate that bovine trophectoderm can be induced via reprogramming factor expression from bovine liver-derived fibroblasts, although other fibroblast populations-e.g., derived from fetal thigh tissue-may produce similar results, albeit at lower frequencies.
Consumption of Endophyte Infected Fescue During Gestation in Beef Cows
Oliver, Katherine Rene (Virginia Tech, 2016-07-11)
Tall fescue is a widely grown, cool season grass prevalent in the eastern United States that is known for its resistance to abiotic and biotic stresses. A main reason for tall fescue's resistance to these stresses is attributed to the presence of a fungal endophyte. Unfortunately, this endophyte also adversely affects cattle production. Cows consuming the ergot alkaloids produced by these endophytes can exhibit decreased feed intake, growth performance, organ vasoconstriction, and increased rectal temperature. This work is interested in examining how endophyte toxin exposure impacts pregnancy in cattle. Reduced blood flow to the fetus and inadequate maternal nutrition contributes to intra uterine growth restriction (IUGR), and this work proposed that fescue endophyte toxicity affects the gestating cow and fetus. Three studies were completed. In experiment 1, gestating cows grazed high or low endophyte fescue pastures during late gestation to determine if exposure to ergot alkaloids in utero results in IUGR and if calves from these pregnancies have altered growth performance. Creep feeding was evaluated as a mitigation strategy for impaired calf growth due to fescue toxicity, and feedlot performance was evaluated to determine if consuming fescue during gestation and creep feeding would affect feedlot performance. Calf BW was different (P < 0.01) by treatment x time. Birth weights of calves were similar , prior to creep feeding calves exposed to high endophyte fescue were lower, and post-supplementation creep fed calves had increased BW. Days on feed and dressing percentage were decreased in the supplemented group, and marbling score was decreased for both the supplemented and unsupplemented groups following the completion of the feedlot phase (P < 0.05). The second study was setup similar to study one, however cows were exposed to fescue pastures from d 170 of gestation until calving. Calf birth weights did not differ, but weights were increased in the supplemented group post creep feeding (P < 0.05). Average daily gains (ADG) of supplemented calves were greater during the supplementation period (P < 0.01). In the third study, indwelling vaginal temperature probes were used to evaluate differences in body temperature of cows fed fescue seed with high or low levels of ergot alkaloids during early gestation, and in varying environmental conditions. In the winter trial, body temperature was measured hourly from days 0-14 of gestation. In the summer trial, body temperature was measured hourly from days 0-32 of gestation. Body temperatures were different (P < 0.01) between treatments during both trials.
Cytokines That Serve as Embryokines in Cattle
Ealy, Alan D.; Speckhart, Savannah L.; Wooldridge, Lydia K. (MDPI, 2021-08-05)
The term “embryokine” has been used to denote molecules produced by the endometrium, oviduct, or by embryo itself that will influence embryo development. Several cytokines have been identified as embryokines in cattle and other mammals. This review will describe how these cytokines function as embryokines, with special emphasis being placed on their actions on in vitro produced (IVP) bovine embryos. Embryokines are being explored for their ability to overcome the poor development rates of IVP embryos and to limit post-transfer pregnancy retention efficiencies that exist in IVP embryos. This review will focus on describing two of the best-characterized cytokines, colony-stimulating factor 2 and interleukin 6, for their ability to modify bovine embryo quality and confirmation, promote normal fetal development, and generate healthy calves. Additional cytokines will also be discussed for their potential to serve as embryokines.
Dairy Pipeline, September 2019
Martel, Cynthia; Ealy, Alan D. (Virginia Cooperative Extension, 2019-08-29)
This issue has two articles. The first one deals with financial appraisals of farms, while the second one focuses on the importance of monitoring pregnant cattle early in gestation.
Development of an Interferon Bioassay and Primitive Endoderm Cell Lines to Study Lineage Specification During Early Bovine Embryogenesis
Mccoski, Sarah R. (Virginia Tech, 2015-01-09)
Embryonic wastage is rampant in cattle during early stages of pregnancy, particularly the first few weeks of gestation, a time recognized for significant remodeling of the embryo. Of particular interest to this laboratory are the first two lineage specification events, trophectoderm (TE) and primitive endoderm (PrE) specification, occurring between days 6 and 8 of gestation. The TE is responsible for uterine attachment and production of interferon-tau (IFNT), the factor of maternal recognition of pregnancy in ruminants. The PrE forms the yolk sac, which provides nutrients to the developing embryo. It is probable that developmental miscues during these differentiation events are responsible for the high rate of pregnancy loss, however, information on these early lineage processes is lacking in ruminants. The objective of the first study was to improve the current methods for detecting IFNT in biological samples. A novel interferon stimulatory response element (ISRE)-reporter assay was created, and provides adequate quantification to measure IFNT. Additionally, it has a shorter completion time than previous bioassays, and does not require the use of a live virus. The second study describes the development of a PrE cell line derived from bovine embryos. The PrE outgrowths can be produced at high rate, and can be maintained in a continuous culture system for about 6 weeks. As a true bovine PrE cell line does not currently exist, these lines hold great potential for the study of early development. Collectively, these studies improve knowledge of bovine embryogenesis, and provide insights that may be used to limit the pregnancy failures occurring in this species.
Developmental Hurdles That Can Compromise Pregnancy during the First Month of Gestation in Cattle
Speckhart, Savannah L.; Oliver, Mary A.; Ealy, Alan D. (MDPI, 2023-05-25)
Several key developmental events are associated with early embryonic pregnancy losses in beef and dairy cows. These developmental problems are observed at a greater frequency in pregnancies generated from in-vitro-produced bovine embryos. This review describes critical problems that arise during oocyte maturation, fertilization, early embryonic development, compaction and blastulation, embryonic cell lineage specification, elongation, gastrulation, and placentation. Additionally, discussed are potential remediation strategies, but unfortunately, corrective actions are not available for several of the problems being discussed. Further research is needed to produce bovine embryos that have a greater likelihood of surviving to term.
Dietary Supplementation of Omega-3 Fatty Acids Influences the Equine Maternal Uterine Environment and Embryonic Development
Jacobs, Robert David (Virginia Tech, 2015-08-03)
Adverse maternal events around the time of conception influence embryonic development. Thus, aberrations in the uterine environment during early pregnancy, such as those resulting from maternal metabolic or nutritional disruption, can alter gene expression in the developing embryo, leading to variations in its developmental trajectory. Dietary supplementation of long-chain omega-3 polyunsaturated fatty acids (LCPUFA), especially Docosahexaenoic acid (DHA) improves metabolic and reproductive health across species. The objective of this study was to evaluate the effects of peri-conceptual LCPUFA supplementation on endometrial gene expression, uterine health and embryonic gene expression in overweight horses. Thirteen non-lactating light horse mares (mean ± SEM age=13.56±0.11 yr; mean ± SEM BCS=7.07±0.21) were supplemented with concentrate (n=6) or an isocaloric diet containing 0.06 g/kg BW algae-derived omega-3 LCPUFA (n=7) beginning 60 d prior to sample collection. Four consecutive ovulatory cycles were monitored, and uterine endometrial samples were obtained 12 d post-ovulation in cycles 1, 3 and 4. Mares were bred and embryos were flushed 12 d post ovulation 2,3 and 4. Endometrial biopsies obtained from supplemented mares contained increased DHA and omega-3 fatty acids as a percent of total fat (P< 0.05). Endometrial biopsy scores were assigned to endometrial tissues and mares receiving the LCPUFA supplementation had improved scores during the first ovulatory period as compared to control animals (P=0.009). Candidate genes essential to inflammation, prostaglandin synthesis and embryonic development were evaluated by quantitative reverse transcriptase polymerase chain reaction. Data were log transformed and analyzed using the GLM procedure in SAS (v9.3). When examining the data independent of breeding and pregnancy status, endometrial obtained samples from LCPUFA supplemented mares contained reduced IL6 (P= 0.04) and TNFa (P=0.03) mRNA abundance and tended to have increased transcript abundance for Uterocalin (P= 0.09), SAA (P= 0.06) and IL10 (P= 0.06). Endometrial samples from mares fed LCPUFA pregnant in cycle 3 contained greater IL10 (P< 0.001), PTGFS (P=0.05), OXTR (P=0.05) and PLA2G3 mRNA (P= 0.009) and had a tendency for increased SAA (P= 0.08), PTGES (P=0.10) and SLCO2A1 (P=0.10) mRNA abundance. Supplemented mares bred but not pregnant at day 12 in cycle 3 had reduced expression of PTGER2 (P=0.001) and PTGS1 (P= <0.001) in endometrial samples. In embryos obtained post ovulatory cycle 3 and 4, relative transcript abundance of GATA4 and GATA6, markers of endoderm differentiation, along with GATA3 and ELF3, markers of trophectoderm differentiation were greater (P< 0.05) in embryos from LCPUFA supplemented mares (n=5), than controls (n=5). These results indicate that algae-derived LCPUFA supplementation during the peri-conceptual period alters the post-ovulatory uterine environment in the horse by modifying expression of genes related to inflammation and regulating prostaglandin synthesis. Additionally, embryos obtained from supplemented mares displayed differential gene expression related to embryonic lineage specification.
The direct injection of CRISPR/Cas9 system into porcine zygotes for genetically modified pig production
Ryu, Junghyun (Virginia Tech, 2019-07-16)
The pig has similar features to the human in aspects such as physiology, immunology, and organ size. Because of these similarities, genetically modified pigs have been generated for xenotransplantation. Also, when using the pig as a model for human diseases (e.g. cystic fibrosis transmembrane conductance regulator), the pig exhibited similar symptoms to those that human patients present. The main goal of this work was to examine the efficacy of direct injection of the CRISPR/Cas9 system (clustered regularly interspaced short palindromic repeats/ CRISPR associated protein 9) in pigs and to overcome shortcomings that resulted after direct injection into the cytoplasm of developing zygotes. By using direct injection of CRISPR/Cas9 into developing zygotes, we successfully generated fetuses and piglets containing 9 different mutations. The total number of aborted fetuses was 20 and of live piglets was 55. Moreover, one issue that was encountered during the production of mutated pigs was that insertion or deletion (indel) mutations did not always introduce a premature stop codon because it did not interfere with the codon read. As a result of these triplet indel(s) mutations, a hypomorphic phenotype was presented; consequently, the mutated gene was partially functional. To prevent this hypomorphic phenotype, we introduced two sgRNAs to generate an intended deletion that would remove a DNA fragment on the genome by causing two double-strand breaks (DSB) during non-homologous end joining (NHEJ). The injection of two sgRNAs successfully generated the intended deletion on the targeted genes in embryos and live piglets. Results after using intended deletions, in IL2RG mutation pigs, did not show hypomorphic phenotypes even when a premature stop codon was not present. After using the intended deletion approach, function of the targeted genes was completely disrupted regardless of the presence or absence of a premature stop codon. Our next aim was to introduce (i.e. knock-in) a portion of exogenous (donor) DNA sequence into a specific locus by utilizing the homology direct repair (HDR) pathway. Because of the cytotoxicity of the linear form of the donor DNA, the concentration of the injected donor DNA was adjusted. After concentration optimization, four different donor DNA fragments targeting four different genes were injected into zygotes. Efficiency of knock-in was an average of 35%. Another donor DNA was used in this study which is IL2RG-IA donor DNA carried 3kb of exogenous cassette. It showed 15.6% of knock-in efficiency. IL2RG-IA Donor DNA injected embryos were transferred into surrogates, and a total of 7 pigs were born from one surrogate, but none of the 7 were positive for the knock-in. Future experiments need to be developed to optimize this approach. Overall, the direct injection of CRISPR/Cas9 is advantageous in cost, time, and efficiency for large animal production and for biomedical research. However, there are still unsolved challenges (off-targeting effects, low efficiency of knock-in, and monoallelic target mutation) that need to be elucidated for future application in humans and other species.
The Effects of Dietary Fructose and Fat on the Reproductive Parameters of Prepubertal and Pregnant Gilts
McCracken, Victoria Lorraine (Virginia Tech, 2015-04-21)
Body adiposity is generally considered the most pertinent factor in puberty attainment; however, recent data suggests that pre-pubertal reproductive tract development may be altered by dietary sugar consumption. Two experiments were conducted to delineate the direct effects of fructose on the maturation of the pre-pubertal reproductive tract and fertility. At three weeks of age, forty gilts were placed on one of five dietary treatments (n=8) containing 15% fat (FAT), 35% fructose (FRU), both fat and fructose (HFHF), or two different controls: one standard industry (IND) diet meant to result in optimal lean growth and a second diet to account for the reduced lysine (LYS) intake in the treatment diets. Body weights did not differ amongst any of the five treatments on the day of sacrifice (P=0.32). As a percentage of BW, total reproductive tracts were heavier in fructose fed gilts (1.3±0.1 v. 0.8±0.1%; P=0.01) compared to non-fructose gilts. In the second experiment, starting at 130d of age, gilts were checked twice daily for puberty attainment. Gilts that attained puberty were artificially inseminated (AI) on their third estrous cycle. On gestational day 38±3, pregnant gilts were harvested for reproductive tract collection. Fewer fructose fed (FRU and HFHF) pigs became pregnant than non-fructose fed (IND, LYS, and FAT) gilts (25% v. 75% respectively; P=0.03). All HFHF gilts failed to become pregnant. Placental weights were greater in LYS fetuses than FAT fetuses (79.07 ± 6.55g v. 47.26 ± 6.45g, respectively, P= 0.04). Taken together, these results demonstrate that fructose consumption increases reproductive tract size, but that reproductive capabilities are reduced.
Effects of gestational heat stress on the lactational performance of gilts and growth performance and carcass characteristics of second-generation offspring
Wiegert, Jeffrey Glennon (Virginia Tech, 2016-01-19)
Pigs exposed to chronic intrauterine hyperthermia (gHS) experience greater fat deposition during life and yield carcasses with greater fat:lean content at slaughter compared to pigs gestated under thermoneutral conditions (gTN). The objectives of this study were to 1) determine whether gHS impacts the lactational performance of affected gilts (F1 generation), and 2) determine whether these effects of gHS are also evident in the next generation (F2 generation). Twenty-four gilts were bred and exposed to thermoneutral or heat stressed conditions for the entirety of gestation, and F1 female offspring were retained. At puberty, gHS and gTN gilts were bred to farrow in either spring (March / April) or summer (July / August). Colostrum and milk samples were collected at farrowing and on d 7, 14, and 21 of lactation. At weaning, four offspring (two male, two female) were retained and grown to market weight in mixed-pens under identical management conditions. Carcass characteristics were analyzed at slaughter. Milk nutrient analysis indicated that gHS gilts produced less lactose, and tended to produce greater protein, than did gTN gilts. There was no difference in the growth rate of F2 offspring, but pigs born of gHS dams did have a tendency for greater backfat thickness. The patterns of altered milk nutrient content observed in F1 gilts reflects a metabolic profile consistent with previous gHS research, and the greater backfat of F2 pigs at slaughter indicates the adipose-promoting effects of gHS may be diluted, but still evident, in the second generation.
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.
Effects of nutrient restriction on the metabolic profile of Bos indicus-influenced and B. taurus suckled beef cows
Fontes, Pedro L. P.; Oosthuizen, N.; Ciriaco, F. M.; Sanford, C. D.; Canal, L. B.; Cooke, Reinaldo F.; Pohler, K. G.; Henry, D. D.; Mercadante, Vitor R. G.; Ealy, Alan D.; Johnson, Sally E.; DiLorenzo, N.; Lamb, G. C. (2021-03)
Recent research from our group demonstrated that Bos indicus-influenced suckled beef cows had greater resilience to withstand nutrient restriction and establish pregnancy compared with B. taurus cows exposed to the same conditions. To further understand these findings, differences in metabolic profile between these same B. indicus-influenced and B. taurus females were explored. Suckled beef cows (n = 134) were enrolled in a completely randomized design with a 2 x 2 factorial arrangement of treatments. On day - 21, Angus (AN; Bos taurus) and Brangus (BN; B. indicus-influenced) cows were randomly assigned to 1) a diet that met daily energy maintenance requirements (MAINT), or 2) a diet that restricted intake to 70% of the daily energy maintenance requirements (RESTR). Cows were exposed to an estrus synchronization protocol and received an embryo 7 d after ovulation was pharmacologically induced on day 0. Blood samples were collected on days - 21 and 19 to determine circulating concentrations of non-esterified fatty acids (NEFA), beta-hydroxybutyrate (BHB), insulin, glucose, and IGF-1. Pregnancy status after embryo transfer was determined on day 28. As a consequence of the proposed diets, cows in the RESTR diet had less body condition score (BCS) on day 19 (P = 0.008) across breed types. Moreover, BCS change from day - 21 to 19 was included as independent covariate into subsequent analyses, allowing for the comparison of breed types under an equivalent level of body reserve mobilization. A breed x diet interaction was observed for plasma insulin (P = 0.03) and IGF-1 (P = 0.04) on day 19, where AN-RESTR cows had less plasma concentrations on day 19 compared with AN-MAINT cows. Diets did not impact (P > 0.10) plasma insulin and IGF-1 concentrations in BN cows. No diet or breed effects were observed in circulating concentrations of NEFA, BHB, and glucose (P > 0.10). Across breed types and nutritional treatment, there was positive linear effect (P = 0.04) of plasma concentrations of insulin and IGF-1 on the probability of pregnancy to fixed-time embryo transfer. In summary, the negative impacts of nutrient restriction on the somatotropic axis, independently of body tissue mobilization, were heightened in Bos taurus females compared with B. indicusinfluenced cohorts, which corroborate with the differences observed in fertility between these subspecies.
Effects of progesterone concentrations and follicular wave during growth of the ovulatory follicle on conceptus and endometrial transcriptome in dairy cows
Bisinotto, R. S.; Ribeiro, E. S.; Greco, L. F.; Taylor-Rodriguez, D.; Ealy, Alan D.; Ayres, H.; Lima, F. S.; Martinez, N.; Thatcher, W. W.; Santos, J. E. P. (Elsevier, 2022-01)
Objectives were to evaluate the effects of follicular wave and progesterone concentration on growth of the ovulatory follicle, conceptus elongation, uterine IFN-tau concentration, and transcriptome of conceptus and endometrium of pregnant cows on d 17 of gestation. Nonlactating nonpregnant Holstein cows were assigned randomly to one of 3 treatments: ovulation of a first-wave follicle (FW, n = 15); ovulation of a first-wave follicle and progesterone supplementation (FWP4, n = 12); and ovulation of a second-wave follicle (SW, n = 19). Ovulation of a first- or second-wave follicle was achieved by initiating the Ovsynch protocol (d -9 GnRH, d -2 and -1 PGF(2 alpha), d 0 GnRH and artificial insemination, d 0.7 artificial insemination) on d 0 or 6 of a presynchronized estrous cycle, respectively. Cows in FWP4 received 3 intravaginal inserts containing progesterone at 12, 24, and 48 h after the first GnRH injection that were removed on d -2. Cows were killed on d 17 for collection of the reproductive tract. Transcriptome was evaluated by microarray using the Affymetrix Bovine Array. Orthogonal contrasts were built to assess the effects of progesterone concentration during follicle growth (FW vs. FWP4 + SW) and follicular wave (FWP4 vs. SW). Progesterone concentrations (LSM +/- SEM) from d -9 to -2 were greater for SW, followed by FWP4 and FW (5.38 +/- 0.24, 4.26 +/- 0.28, and 1.17 +/- 0.27 ng/mL). Diameter of the ovulatory follicle (FW = 19.6 +/- 0.6; FWP4 = 15.6 +/- 0.6; SW = 15.2 +/- 0.5 mm) and concentrations of estradiol from d -2 to 1 (FW = 4.05 +/- 0.33; FWP4 = 2.73 +/- 0.35; SW = 2.48 +/- 0.30 pg/mL) were greater for FW compared with FWP4 and SW. Progesterone concentrations from d 3 to 16 were greater for FW compared with FWP4 and SW. A total of 28 singleton conceptuses were collected (FW, n = 8; FWP4, n = 8; SW, n = 12) and only intact conceptuses were included in the analyses of length (FW, n = 8; FWP4, n = 6; SW, n = 12). Although conceptuses were longer for FW compared with FWP4 and SW (FW = 16.6 +/- 2.3; FWP4 = 9.8 +/- 2.2; SW = 9.6 +/- 2.0 cm), treatment did not affect the amount of IFN-tau in uterine flushing. Transcriptome of conceptuses and endometrium of pregnant cows was not extensively affected by follicular wave (8 and 1 differentially expressed transcripts) or concentration of progesterone during follicle growth (0 and 3 differentially expressed transcripts), showing that these factors did not affect conceptuses and endometrium transcriptome in pregnancies that are maintained to d 17.
Equine Trophectoderm Cells and Their Role in Fetal-Maternal Recognition
Bonometti, Susana (Virginia Tech, 2019-01-18)
Establishment and maintenance of a successful pregnancy requires signaling from the embryo to the mare, a process known as maternal recognition. Six days after fertilization, the trophectoderm (TE), a placenta precursor is formed. Signals emanating from the TE to the uterine environment are critical to maternal recognition of pregnancy. The identity of factors necessary for this process remain unknown. A novel equine induced trophoblast cell line (iTr) that closely mimics the genotype and phenotype of native equine TE was created. Transcriptome analysis of iTr revealed increased expression of growth factor (GF) receptors for Epidermal GF (EGF), Hepatocyte GF (HGF), Fibroblast GF-2 (FGF-2) and Insulin GF (IGF-1), suggesting these GF may be important targets during TE development in the early embryo. We hypothesized that treatment of iTr cells with these GF would induce changes in cell proliferation and expression of genes likely involved in maternal recognition. The objectives of this experiment were to evaluate the effect of these GFs on iTr mitotic response and regulation of genes involved in steroidogenesis. Equine iTr cells (n = 3) were cultured with 10 ng/mL EGF, HGF, FGF-2 or IGF-1 for 24 hr, with 5-ethynyl-2'-deoxyuridine (EdU) supplementation during the final 2 hr. Subsequently, cells were fixed and EdU positive and total nuclei were enumerated. A parallel plate of iTr cells was treated in a similar manner and lysed for total RNA isolation. Quantitative PCR using gene-specific primers for CYP11A1, PTGS2, PTGES2, and PTGES3 was performed. Data were analyzed by ANOVA with Tukey's post hoc adjustment using the GLM procedure of SAS. Treatment with EGF, FGF-2, HGF, and IGF-1 increased (P < 0.05) iTr proliferation from control levels of 25.33 ± 1.03% to 38.58 ± 1.61%, 45.50 ± 2.94%, and 38.23 ± 2.01% respectively. The 2-ΔΔCT method was used to calculate the fold change (FC) using GAPDH as the reference gene for normalization. Expression of CYP11A2, PTGES2, and PTGES3 was not affected by GF, as measured by qPCR. By contrast, PTGS2 transcript abundance increased (P < 0.05) following FGF-2 (FC = 3.327 ± 0.8291) and HGF (FC = 11.88 ± 4.572) treatment. These results indicate that FGF-2 and HGF may simultaneously induce proliferation and prostaglandin production by TE cells. The combined results of these experiments will improve our understanding of TE morphogenesis and its response to uterine-derived growth factors.
Exposure to maternal obesity alters gene expression in the preimplantation ovine conceptus
McCoski, Sarah R.; Vailes, McCauley T.; Owens, Connor E.; Cockrum, Rebecca R.; Ealy, Alan D. (2018-10-11)
Background Embryonic and fetal exposure to maternal obesity causes several maladaptive morphological and epigenetic changes in exposed offspring. The timing of these events is unclear, but changes can be observed even after a short exposure to maternal obesity around the time of conception. The hypothesis of this work is that maternal obesity influences the ovine preimplantation conceptus early in pregnancy, and this exposure will affect gene expression in embryonic and extraembryonic tissues. Results Obese and lean ewe groups were established by overfeeding or normal feeding, respectively. Ewes were then bred to genetically similar rams. Conceptuses were collected at day 14 of gestation. Morphological assessments were made, conceptuses were sexed by genomic PCR analysis, and samples underwent RNA-sequencing analysis. While no obvious morphological differences existed between conceptuses, differentially expressed genes (≥ 2-fold; ≥ 0.2 RPKM; ≤ 0.05 FDR) were detected based on maternal obesity exposure (n = 21). Also, differential effects of maternal obesity were noted on each conceptus sex (n = 347). A large portion of differentially expressed genes were associated with embryogenesis and placental development. Conclusions Findings reveal that the preimplantation ovine conceptus genome responds to maternal obesity in a sex-dependent manner. The sexual dimorphism in response to the maternal environment coupled with changes in placental gene expression may explain aberrations in phenotype observed in offspring derived from obese females.
Factors affecting the quality and function of the bovine periovulatory follicle
Harl, Audra Whitney (Virginia Tech, 2018-11-15)
For many cattle operations, profitability depends on the success of reproductive management programs. Opportunities for improving fertility exist within the numerous challenges related to reproductive management. Non-conventional, creative tools for reproductive management could help producers overcome these challenges. In an effort to produce information that could be used to improve reproductive performance of cattle, the following studies were undertaken. The objectives of these studies were threefold: to determine whether GnRH administered as an epidural injection causes ovulation in healthy cows and heifers, to evaluate whether the follicular environment (specifically, follicle fluid) surrounding the oocyte during the maturation phase affects the ability of the cumulus-oocyte complex to progress through early embryonic development, and to investigate the relative effects of estradiol and progesterone on oocyte maturation and early embryo development. Ability of GnRH to elicit an ovulatory response when administered as an epidural was evaluated in crossbred angus cows and heifers. The preliminary study evaluated this route of administration in crossbred angus cows. Animals were assigned randomly to either intramuscular or epidural administration, and ovaries were visualized via transrectal ultrasound every 6 h until ovulation of the dominant follicle. Results indicated that epidural administration of GnRH was able to trigger an ovulatory response, but timing of ovulation was not measured. The main experiment evaluated incidence of ovulation, time to ovulation, and ovulatory follicle size in crossbred angus heifers administered GnRH either epidurally or intramuscularly. Heifers were randomly assigned to treatment and ovaries were visualized every 4 h via transrectal ultrasound until ovulation of the dominant follicle. Results indicated that epidural administration of GnRH was able to elicit an ovulatory response in heifers, and the timing of ovulation and ovulatory follicle size was not different between administration route. Further investigation is needed to determine if characteristics of the ovulatory response (such as the luteinizing hormone surge) and circulating concentrations of GnRH are altered by epidural administration, which may impact fertility. GnRH administration is standard practice in many estrous synchronization programs. For fixed-time artificial insemination programs, the detection of estrus prior to insemination has been shown to improve conception and decrease early embryonic loss. The impact of behavioral estrus expression on the oocyte and early embryo were evaluated. Oocytes were matured in vitro in follicle fluid collected from synchronized cows who were classified as having expressed behavioral estrus or not expressing estrus. Embryo cleavage was not affected by estrus expression, but there was a tendency for improved blastocyst development in embryos matured in follicle fluid from animals who had expressed estrus. Cell number was not affected by estrus expression, but future research is needed as to the effect on oocyte acquisition of competence and early embryonic development. Despite the progress that has been made in culture conditions for in vitro produced embryos, developmental capacity following fertilization is limited at best, with only around one-third of oocytes placed into maturation resulting in viable embryos. During in vivo maturation, the oocyte undergoes final maturation within the follicle, surrounded by a changing microenvironment of estradiol and progesterone. Although the effects of steroids on oocyte development in vitro have been studied on an individual basis, a direct comparison between the ratio of estrogen and progesterone relative to follicle size has not been investigated Effects of steroid hormones estradiol and progesterone on oocyte maturation and early embryonic development were evaluated. Oocytes were matured in vitro in media supplemented with either estradiol, progesterone, or a combination of estradiol and progesterone. Oocytes were fertilized after maturation and cultured for 7 d until development to blastocyst stage. Addition of estradiol alone did not support oocyte maturation or early embryonic development in vitro, and a combination of estradiol and progesterone exhibited an inhibitory effect on oocyte maturation and early embryonic development. Addition of progesterone alone resulted in improved development when compared with estradiol alone or a combination of estradiol and progesterone. These results indicate that efficiency of reproductive management programs is controlled by multi-faceted factors and opportunities for improvement of reproductive outcomes exist in all of these factors. Although ovulation can be elicited via epidural administration, the impact of this ovulatory trigger on fertility requires further investigation. Display of estrus after synchronization for fixed-time artificial insemination improves conception and decreases early embryonic loss and has a may improve blastocyst development. This effect on early embryo development could be the focus of future research, further improving fertility and possibly the efficacy of in vitro embryo production. Steroid hormones play crucial roles in oocyte competency and the addition of progesterone during in vitro maturation improves development compared with estradiol alone or a combination of estradiol and progesterone.
Fat and Fructose Consumption Affects Pre-pubertal Gilt Reproductive Tissues and Early Embryogenesis
Poole, Rebecca Kyle (Virginia Tech, 2016-07-19)
Infertility among women has become a growing issue in the world requiring a significant number of women to seek treatment by means of assisted reproductive technologies (ART). One suggested reason for the fertility issue is modern diet, leading to diseases such as obesity and type II diabetes. In this study, twenty gilts three weeks in age, were placed on one of five dietary treatments (n=4 gilts per treatment) containing 15% fat (FAT), 35% fructose (FRU), both fat and fructose (HFHF), or two different controls: one standard industry (IND) diet meant to result in optimal lean growth and a second diet to account for the reduced lysine (LYS) intake in the treatment diets. Two experiments were performed to assess the reproductive outcomes of pre-pubertal gilts consuming a high fat and/or high fructose diet and to demonstrate interactions between diet and infertility using pigs as a model. In the first experiment, follicular fluid was collected from these gilts and introduced into porcine in vitro maturation system to determine whether characteristics of the follicular fluid affect oocyte competence and embryo development. The follicular fluid of females consuming high fructose and fat diets did not alter nuclear maturation of oocytes (p>0.10). There were, however, detrimental effects on subsequent development of embryos, especially blastocyst formation, with the gilts having consumed the HFHF diet having reduced day 5 and 6 blastocysts formation when compared to the IVM follicular fluid free (FFF) group (p=0.03 and p=0.01, respectively). In regards to embryo quality, blastocysts from the FAT group had greater cell number when compared to all other groups. In the second experiment, the reproductive tissues; ovary, oviduct, and uterus were analyzed for genes of interest: estrogen receptor alpha (ESR1), estrogen receptor beta (ESR2), insulin like growth factor I (IGFI), insulin like growth factor I receptor (IGFIR), and growth differentiation factor 9 (GDF9). Resulting data was analyzed in three ways: 1) across all 5 treatments, 2) with gilts grouped by whether or not they consumed fat, or 3) with gilts grouped by whether or not they consumed fructose. There were no differences detected between individual treatments for ESR1 and ESR2. In the ovary samples, the fructose diets decreases ESR2 (p=0.05). Also, GDF9 ovarian expression tended to decrease with fructose consumption (p=0.07). Furthermore in the ovary, there was a positive correlation between ESR2 and GDF9 expression (r=0.92 and p<0.01). GDF9 expression was lower in the oviducts of gilts consuming fat diets when compared to non-fat diets (p=0.01). Neither IGFI nor IGFIR were altered in the reproductive tissues analyzed. Based on the results from both experiments, the consumption of fat and fructose alters both the developing embryo and gene expression in the reproductive tissues that support the growing embryo. Further investigation will provide more insight on the impact nutrition has on pre-pubertal reproductive development and subsequent fertility.

Browsing by Author "Ealy, Alan D."

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