Browsing by Author "Rhoads, Michelle"

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    Characterization of Phosphatidylserine Expression in Bovine Sperm
    Haines, Hannah (Virginia Tech, 2021-11-24)
    Many factors influence male fertility, and conventional fertility evaluations are not able to reliably identify sub-fertile animals. The overall goal of this work was to explore the expression of phosphatidylserine (PS) on bovine sperm and investigate what factors may impact it, as previous research demonstrated that phosphatidylserine (PS) plays a role in murine fertilization. Despite conventionally being an apoptotic marker, it is present on viable and fertilization-competent murine sperm, however, less is known of the possible role of PS in bovine fertilization. In experiment 1, viable bovine sperm cells expressing PS were identified and PS levels in fresh and frozen semen were compared. Phosphatidylserine levels in frozen samples were significantly less than in fresh samples. We conclude that the cryopreservation process has an impact on PS expression in sperm by altering the proportion of sperm cells which are capable of fertilization. Experiment 2 examined PS levels in bulls with varying fertility levels based on sire conception rate (SCR). There was no difference in PS levels between high and low fertility bulls. There was a significant difference in PS levels of uncapacitated samples and those capacitated for one hour. These results warrant further investigation into the role of phosphatidylserine in bovine fertilization.
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    Determining the Effects of Nerve Growth Factor Supplemented In-vitro Fertilization Media on Bovine Embryo Development
    Hellstern, Emily Anne (Virginia Tech, 2022-08-17)
    Scientists have developed techniques like ovum pick up (OPU) and follicular ablation as a large source of oocytes for creating IVP bovine embryos. These techniques have allowed for more efficient dissemination of valuable female genetics compared to traditional artificial insemination or embryo flushing. IVP embryos have lower embryo development rates and quality, leading to lower pregnancy rates. Nerve growth factor-beta (NGF), however, has been previously shown to improve 48-hour cleavage rates and the number of hatching/ hatched blastocysts out of total presumptive zygotes. We hypothesize that NGF will improve IVP embryo development by positively influencing cleavage and blastocyst rates. The first two experiments' objectives were to determine the effect of recombinant bovine (60 or 90% purity) and human NGF (97% purity) supplementation during in vitro fertilization on 24- and 48-hour cleavage and day 8 blastocyst development rates. The objective of the third experiment was to assess the effect of the supplementation of bovine NGF (90% purity) on heat shocked and non-heat shocked in vitro-matured cumulus-oocyte complexes, assessing cleavage rates at 48 and 72 hours post insemination and blastocyst development rates. The results of experiment 1 show there were no differences between any of the three treatment groups (bNGF60, hNGF95, and control) for 24 hour (P = 0.66) or 48 hour (P = 0.33) embryonic cleavage rates. Additionally, there were no differences between treatments in the total percentage of blastocysts per oocyte (P = 0.91) or the percentage of blastocysts per cleaved embryo (P = 0.32). The results of experiment 2 also showed no differences between any of the three treatment groups (bNGF90, hNGF95, and control) for 24 hour (P = 0.16) or 48 hour (P = 0.18) embryonic cleavage rates. Additionally, there were no differences between treatments in the total percentage of blastocysts per oocyte (P = 0.42) or the percentage of blastocysts per cleaved embryo (P = 0.57). In the 3rd experiment, there was not a significant effect of treatment (P ≤ 0.05) at all stages of embryonic development assessed. On the contrary, in the third experiment, non-heat stressed NGF treatment had an interestingly detrimental effect on early cleavage rates of embryos compared to the non-treated control embryos. These results showed that NGF could not improve in vitro embryonic development rates in standard conditions; however, this negative impact of NGF on early cleavage was not observed in heat-shocked embryos. Suggesting that there could be a protectant factor in NGF that warrants further investigation.
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    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.
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    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.
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    The Effect of Supplemental Grape Seed Extract on Pig Growth Performance and Body Composition During Heat Stress
    Smithson, Andrew Todd (Virginia Tech, 2016-07-08)
    Prolonged exposure to high ambient temperature without cooling causes heat stress (HS) resulting in altered growth, body composition and metabolic dysfunction in pigs. Grape seed extract (GSE) has been shown to reduce inflammation, and improve glucose transport and metabolism. Thus, GSE may be an effective supplement to combat the consequences of heat stress; however this possibility has not been evaluated in a large animal model. The objective of the current study was to examine the effect of GSE supplementation on pig performance and body composition during HS. Twenty-four female pigs (62.3± 8 kg BW) were randomly assigned to a 2X2 factorial experiment; thermal neutral (TN; 21-22°C) or heat stress conditions (HS; 33-34°C) for 7 days and fed either a control or a GSE supplemented diet (12mg/kg body weight). Body temperature (TB), respiration rate (RR) and feed intake (FI) were measured daily. Body composition was measured by dual-energy X-ray absorptiometry (DXA). Respiration rate and TB increased in the HS control group compared to the TN control group (p<0.05), however GSE did not alter these parameters compared to control for the duration of the 7 day period. HS decreased FI (P < 0.05). Fasting blood glucose concentrations were approximately 1.5-fold greater in the control diet compared to their GSE supplemented counterpart (p=0.067) on day 6 of the HS period, but did not differ between groups at the end of day 7 of HS. Body composition analysis indicated bone mineral density, bone mineral content, and percent change of fat remain unchanged between treatment groups. Percent change in weight was significantly reduced in HS. Lean tissue accretion was 45% greater in TN compared to HS groups (p<0.05). Endotoxin concentrations were approximately 2-fold lower in the HS-GSE group compared to the control (P=0.083). Grape seed extract supplementation does not appear to alter pig growth performance or body composition, but does appear to delay the onset of reduced feed intake by 1 day, reduce intestinal permeability, and improve insulin sensitivity during additional stress.
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    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.
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    Effects of feed additives on uterine morphology and selected reproductive attributes
    Soffa, Dallas Rae (Virginia Tech, 2022-07-01)
    Dairy cattle characteristically exhibit decreased milk yield and reproductive performance, as well as increased uterine infection rates during periods of high stress. Chromium supplementation has demonstrated efficacy as a feed additive capable of reducing the detrimental effects of stress. As such, its application in dairy production may help to alleviate economic losses associated with seasonal heat stress and the stress experienced during the early postpartum period. Therefore, the objective of the work described in this thesis was to evaluate the potential benefits of short-duration, high-dose chromium (Cr) supplementation in early postpartum dairy cows during the summer months. Multiparous, early postpartum cows (20.95 ± 0.21 DIM, 658.29 ± 13.61 kg) were assigned to one of two treatment groups: 1) normal TMR (Con; n=10) and 2) normal TMR + Cr propionate supplementation (CrPro; 12 mg/h/d Cr; n=12). Body weight (BW), milk yield, and feed intake were measured each day of the experiment. Ambient temperature and humidity were monitored, and the temperature-humidity index (THI) was calculated for the duration of the study as an indicator of the severity of the heat stress experienced by the cows. Transrectal ultrasonography was performed every three days to assess ovarian follicular and luteal dynamics. Respiration rates (RR), rectal temperatures (RT), and blood glucose were recorded concurrently with ovarian ultrasonography. Plasma was collected and used for analysis of progesterone concentrations. Every six days in conjunction with ultrasonography, endometrial cytology samples were collected via cytobrush from each cow to determine the incidences of subclinical endometritis (SCE), as determined by polymorphonuclear leukocyte (PMNL) %. There were no treatment-based differences in RR, RT, blood glucose, feed intake, milk yield, or BW. However, the supplementation strategy did improve reproductive parameters. Within the Con group, there was an increase in PMNL % between samples five and six. Furthermore, at cytology sample six, the Con group had a greater percentage of PMNL than the CrPro group (P=0.01). Chromium consumption did not affect the counts or sizes of most follicles, with the exception being the 6-9 mm category where the CrPro group had a greater average diameter and tended to have more follicles in this category. While CL numbers or size did not differ between treatments, the ratio of progesterone (P4) to corpus luteum (CL) volume was greater in the CrPro group compared to the Con group (P=0.03). The results from this study indicate that the proposed supplementation strategy does not influence DMI or milk yield in cows experiencing stress. Nonetheless, short-duration, high-dose Cr supplementation strategy could benefit reproductive performance and thereby limit economic losses experienced by dairy producers during periods of stress.
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    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.
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    Effects of heat-stress-induced physiological changes on the productivity of dairy cattle
    Baber, Taelor Xzavia (Virginia Tech, 2024-06-07)
    Dairy cattle exposed to summer heat stress (HS) typically exhibit altered metabolic characteristics, including hyperinsulinemia concurrent with hypoglycemia. The reasons for this change in glycemic status and its consequences are currently unknown. This project aimed to examine the relationship between summer HS, blood glucose concentrations, and milk production in primiparous (n=26) and multiparous (n=27) Holstein cows. Glucose was measured in coccygeal vein blood twice weekly using a hand-held glucometer (Abbott Precision Xtra) from May through July. Rectal temperatures were also collected. The days surrounding sample collections were categorized as having no HS (0 h above 68 Thermal Humidity Index [THI]), moderate HS (>0 to 8 h above 68 THI) or severe HS (>8 h above 68 THI). A subset of cows with ≥5 blood glucose measurements spanning different THI categories were categorized as hypoglycemic (n=8 primiparous; n=9 multiparous) or non-hypoglycemic (n=6 primiparous; n=5 multiparous) based upon blood glucose concentrations on no-HS days and severe-HS days (hypoglycemic if sustained reduction on severe-HS days). When analyzed together, milk production did not differ between those categorized as hypoglycemic and non-hypoglycemic. When separated based upon parity, there was a tendency for primiparous hypoglycemic cows to produce more milk than their non- hypoglycemic counterparts (P<0.15). Variability in milk production measured as standard deviation across no-, moderate- and severe-HS days did not differ based upon glycemic status but was greater in multiparous than primiparous cows (P<0.01). Rectal temperatures were greater on severe-HS days than on no- or moderate-HS days (P<0.01) and were positively correlated with the number of h of HS in a day (r=0.45; P<0.01). Neither milk nor glucose differed based upon the category of HS severity. Interestingly, however, glucose was negatively correlated with milk production on the day of measurement (r=-0.24; P<0.01) and day after measurement (r=-0.23; P<0.01). These results are an initial indication that hypoglycemia during HS is related to milk production, particularly in primiparous cows.
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    Effects of mid-gestational L-citrulline supplementation to twin-bearing ewes on umbilical blood flow, placental development, and lamb production traits
    Kott, Michelle Lynn (Virginia Tech, 2021-01-11)
    The interaction between the embryo and fetus with the maternal environment can have both short- and long-term consequences on health and development after birth. In some cases, these changes may be detrimental to the individual, but in other cases these developmental changes may be beneficial and manipulated to produce desired effects. Our interest is to use this concept of fetal programming to improve skeletal muscle development and meat production in ruminants. To achieve this, we targeted the period of gestation when fetal muscle fiber formation occurs. Primary muscle fibers form during embryonic development, and it is this small number of primary muscle fibers that will serve as templates for secondary fiber formation that occurs in the fetus during mid-gestation. Supplementing amino acids that influence blood flow within the reproductive tract is one potential way to provide fetuses with added nutrients during gestation, and this supplementation strategy may be especially useful when the maternal diet is compromised. L-citrulline was chosen for this work because of its long half-life in maternal circulation. This work utilized twin-bearing ewes with a moderate dietary energy restriction to assess the effects of mid-gestational L-citrulline supplementation on umbilical blood flow, placental function, neonatal lamb size, and lamb performance. We hypothesize that i.v. administration of L-citrulline will increase uterine and placental blood flow in gestating ewes and this will improve fetal growth, development, and overall postnatal performance. Blood flow parameters were not influenced by treatment (P>0.05). Circulating levels of progesterone and pregnancy-specific protein B (PSPB) were used as indicators of placental function and were unaffected by treatment administration (P>0.05). A treatment by time interaction was detected in both analyses, but no differences between treatments were detected within any time points. There was no effect of treatment on lamb weights or survival to weaning (P>0.05). Lamb sex effects are absent with the exception that body weights were greater in ewe lambs (P>0.05). There was no effect of treatment on any carcass traits or visceral organ weights assessed, though there was an effect of sex on dressing percentage and pancreas weight with wethers having a greater dressing percentage and heavier pancreases per kg body weight than that of ewes (P<0.05). In summary, contrary to our hypothesis L-citrulline supplementation to pregnant ewes under a minor to moderate metabolic challenge had no impact on blood flow and provided no programming benefit to the lambs.
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    Evaluating the impact of heat stress and altered glycemic state on plasma ɣ-Aminobutyric Acid (GABA) in lactating Holstein cows
    Arneson, Alicia Gest (Virginia Tech, 2021-06-28)
    Heat stress (HS) induces hyperinsulinemia and hypoglycemia in lactating dairy cows. We hypothesized that γ-aminobutyric acid (GABA) participates in the regulation of this altered glycemic state as it is produced by the pancreatic beta cells and has a stimulatory effect on pancreatic secretion of insulin. Multiparous lactating Holstein cows (n=6; 63.33±2.35 DIM, 3.17±0.40 lactations) were placed in environmentally controlled rooms for four experimental periods: 1) thermoneutral (TN; d 1-5; 18±4°C), 2) TN + hyperinsulinemic-hypoglycemic clamp (HHC; d 6-10), 3) heat stress (HS; d 16-20; 33±4°C), and 4) HS + euglycemic clamp (EC; d 21-25). Cows were milked twice daily, and blood samples were collected once daily via coccygeal venipuncture into heparinized evacuated tubes. Plasma GABA concentrations were determined using a competitive ELISA. The data were analyzed in two ways. The first analysis included data from all treatment periods and yielded no period-based differences in plasma GABA concentrations. In this analysis, plasma GABA was lowly correlated to plasma insulin concentrations (r = -0.29, P<0.01). The second excluded data from HHC and EC periods so that GABA concentrations during TN were directly compared to concentrations during HS. In this analysis, plasma GABA concentrations tended to be higher in TN than HS (16.31±2.14 vs 13.80±2.15 ng/ml, respectively, P = 0.06). Milk production was moderately correlated with plasma GABA (r=0.42, P<0.01) and the average plasma GABA during TN and HS was moderately correlated to baseline glucose levels for those periods (r=-0.57, P=0.05). Furthermore, the percent change in plasma GABA was strongly correlated with the percent change in plasma glucose from TN to HS (r=-0.95, P<0.01). Plasma GABA was again lowly correlated to plasma insulin concentrations (r = -0.35, P = 0.01). While these analyses are not indicative of causality, the results suggest that GABA is involved in the regulation of the altered glycemic state observed during HS. More research is needed to determine its precise role in heat-stressed lactating dairy cattle.
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    Exploring methods to understand bovine embryo competency in vitro
    Nix, Jada Lindsay (Virginia Tech, 2023-12-19)
    The development of a preimplantation embryo is a stepwise process consisting of morphological, biochemical, and genomic changes. Much remains unknown about the attainment of embryo competency to develop and establish pregnancy. To investigate this, we compared methods of selection at the oocyte or embryo level for improved blastocyst production. Brilliant cresyl blue staining was used to sort oocytes by their growth status (not fully grown vs. fully grown) and the timing of the first embryonic cell division to sort embryos. We found that an embryo's cleavage kinetics are more indicative of their competency than the growth status of the oocyte that gave rise to that embryo. We further investigated the cryopreservation survival of embryos with fast or slow cleavage kinetics and found no significant differences in their ability to hatch post-thawing. Next, we used the complete sequence of the cattle Y chromosome to identify oligonucleotides for efficient sexing of samples. These materials may be used to understand sexual dimorphism as a biological factor in future experiments. Finally, we designed a new method to induce targeted DNA sequence deletions and mRNA cleavage in zygotes using CRISPR-Cas. We targeted the gene OCT4, since the literature shows variable knockout outcomes. Our method improved deletion efficiency while accounting for preexisting or maternally inherited mRNA of the target gene. Our findings can be used to better understand early embryo development and biological drivers of quality, which can be leveraged to improve embryo production and transfer outcomes.
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    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.
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    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.
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    Immunological castration of boars temporarily reduces testosterone concentration, testis size and function, without long term effects on libido and sperm quality
    Lugar, Drew William (Virginia Tech, 2015-01-06)
    The objective was to determine the effects of immunocastration (Improvest) on reproductive characteristics in boars. Seventy-two boars were used in a randomized design with three treatments: single injection (SI) or double injection (DI) of Improvest, and intact controls (no Improvest; CNT) (n = 24/group). At wk 10, 15, 20, 25, and 40, blood was collected and serum harvested to evaluate testosterone concentrations via RIA. At wk 25, 18 pigs (n = 6/ group) were sacrificed and testes were removed, weighed, and measured. Libido was assessed at 32, 36, 47, 60, and 63+ wk of age (1 to 5; 1 = no libido; 5 = high libido) and semen collected beyond 60 wk of age. Testosterone concentrations were less for DI boars compared to CNT boars and SI boars at 20 and 25 wk of age (P < 0.001), but not different at 40 wk of age. All testicular measurements and weight were less for DI boars compared to SI and CNT boars (P < 0.001). There was no treatment effect on libido between 32 to 63+ wk of age. Semen volume, gel weight and total number of sperm cells were not different among treatments. Sperm concentration was greater for DI than SI (P = 0.011), and tended to be greater for DI compared to CNT (P = 0.102). 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.
  • 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.
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    Improving the Efficiency of Dairy Cattle Feeding
    McGill, Tyler R. (Virginia Tech, 2017-04-26)
    Biological functions that use amino acids (AA) are limited by AA supply. This concept was likened to staves in a barrel, where the shortest stave determines the barrel's ability to hold water (Mitchell and Block, 1946). Inaccuracies in models that predict nutrient supply and requirements of dairy cows result in inefficient feeding, as under-prediction of requirements results in deficiency, and over-prediction results in excess. To avoid limitations in production due to AA deficiencies, protein is fed in quantities that likely exceed requirements. Overfeeding of AA results in increased expenses for producers and increased N excretion to the environment, providing economic and environmental incentives to increase N-efficiency. Work presented in the following chapters evaluated the impact of AA supplementation on milk production in dairy cattle, and evaluated the PREP10 and NRC (2001) nutrient requirement model predictions. In two feeding trials (Chapter 2 and Chapter 3), low protein diets did not result in decreased milk production, indicating that protein requirements were overestimated. Although supplementation of AA did not increase milk production, low protein diets resulted in greater N-efficiency, especially when supplemented with Histidine. Evaluation of the PREP10 and NRC (2001) models (Chapter 4) used production data from the literature to identify deficiencies in prediction equations, and found that correction of model bias would considerably reduce prediction errors. Model inaccuracies affect the inefficiency of dairy cow feeding, and must be evaluated to improve feed efficiency. Such improvements could act synergistically with AA supplementation to more closely match nutrient supply to requirements.
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    Maintaining Proper Levels of DNA Methylation Marks Through the TET Family is Critical for Normal Embryo Development in Pigs
    Uh, Kyung-Jun (Virginia Tech, 2020-08-24)
    DNA methylation is one of the principal epigenetic modifications that plays an essential role in transcriptional regulation. After fertilization, mammalian embryos undergo dynamic changes in genome-wide DNA methylation patterns and the changes are essential for normal embryo development. Ten-eleven translocation (TET) methylcytosine dioxygenases are implicated in DNA demethylation by catalyzing the conversion of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC). The three members of TET protein family, TET1, TET2, and TET3, are highly expressed in preimplantation embryos in a stage-specific manner. Previous studies demonstrated that TET proteins are involved in diverse biological processes such as gene regulation, pluripotency maintenance, and cell differentiation by mediating 5mC oxidation. My dissertation research was conducted to elucidate the mechanistic roles of TET proteins in epigenetic reprogramming of mammalian embryos using porcine embryos as a model. The first set of studies focused on the relationship between TET proteins and pluripotency. To understand the role of TET proteins in establishing pluripotency in preimplantation embryos, CRISPR/Cas9 technology and TET-specific inhibitors were applied. TET1 depletion unexpectedly resulted in an increased expression of NANOG and ESRRB genes in blastocysts, although the DNA methylation levels of NANOG promoter were not changed. Interestingly, transcript abundance of TET3 was increased in blastocysts carrying inactivated TET1, which might have had an effect on the increase of NANOG and ESRRB. When the activity of TET enzymes was inhibited to eliminate the compensatory increase of TET3 under the absence of functional TET1, the expression levels of NANOG and ESRRB were decreased and methylation level of NANOG promoter was increased. In addition, ICM specification was impaired by the inhibition of TET enzymes. These results suggest that the TET family is a critical component of the pluripotency network of porcine embryos by regulating expression of genes involved in pluripotency and early lineage specification. In the next set of studies, the presence of TET3 isoforms in porcine oocytes and cumulus cells was investigated to dissect the gene structure of TET3 that could assist in understanding mechanistic actions of TET3 in the DNA demethylation process. Among the three TET3 isoforms identified in cumulus cells, only the pTET3L isoform, which contains CXXC domain that carry DNA binding property, was verified in mature porcine oocytes. Expression level of the pTET3L isoform was much higher in mature oocytes compared to that in somatic cells and tissues. In addition, the transcript level of this isoform was significantly increased during oocyte maturation. These results suggest that pTET3L isoform is predominantly present in mature porcine oocytes and that CXXC domain may play an important role in DNA demethylation in zygotes. In a follow-up study, the role of the TET3 CXXC domain in controlling post-fertilization demethylation in porcine embryos was investigated by injecting TET3 GFP-CXXC into mature porcine oocytes. The injected CXXC was exclusively localized in the pronuclei, indicating that the CXXC domain may localize TET3 to the nucleus. The CXXC overexpression reduced the 5mC level in zygotes and enhanced the DNA demethylation of the NANOG promoter in 2-cell stage embryos. Furthermore, the transcript abundance of NANOG and ESRRB was increased in blastocysts derived from GFP-CXXC overexpressing zygotes. These results provide an evidence that the CXXC domain of TET3 is critical for post-fertilization demethylation of porcine embryos and proper expression of pluripotency related genes in blastocysts. In the last set of studies, the impact of MBD proteins on porcine embryo development was examined under the hypothesis that competitive binding of MBD and TET proteins to 5mC contributes to the proper maintenance of DNA methylation levels in embryos. Cloning of porcine MBD1, MBD3, and MBD4 from mature oocytes indicates that the genes are highly conserved among different species, implying the involvement of porcine MBD proteins in the maintenance of DNA methylation. MBD1 overexpression in oocytes impaired preimplantation development of porcine embryos, suggesting that the MBD1 overexpression may have negatively affected porcine embryo development because proper DNA methylation levels were not preserved under the high level of MBD1. Collectively, the studies in my dissertation demonstrate that TET family proteins are important epigenetic players involved in the regulation of pluripotency and reprogramming of DNA methylation, and are thus crucial for normal embryo development. The findings in the dissertation will improve our understanding of epigenetic events occurring in mammalian embryos, and have the potential to overcome epigenetic defects that are observed in pluripotent stem cells and in-vitro derived embryos.
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    Manipulating Embryonic Development and Endometrial Function in Ruminants
    McCoski, Sarah R. (Virginia Tech, 2018-04-13)
    Early embryogenesis is highlighted by the emergence of several embryonic end extraembryonic lineages. One such lineage is the primitive endoderm, which will eventually give rise to the yolk sac. Once believed to be a vestigial structure, the yolk sac is now believed to play a more prominent role in embryogenesis as it provides nutrients to the preimplantation embryo. The endoderm may also interact with the trophectoderm lineage, as they develop in close contact within the embryo. The efficiency of developing primitive endoderm in vitro is considerably low, leading to a lapse in our understanding of its development and function in cattle and other ruminants. The goal of the first study was to establish a protocol for developing primitive endoderm cultures and characterizing these cells. Bovine embryos were produced in vitro, and primitive endoderm outgrowths were created with fibroblast growth factor 2 (FGF2) supplementation. These cells can be produced in culture with 80.3 5.6% efficiency. Furthermore, outgrowths can be maintained in culture for 6-8 weeks before reaching a quiescent state. A true bovine primitive endoderm cell line does not currently exist, however, these cells hold potential in improving the current understanding of early lineage specification in cattle. A second set of studies was performed to examine the effects of maternal obesity on the preimplantation conceptus and endometrium. Exposure to maternal obesity in utero affects offspring development at the postnatal, adolescent, and adult stages of development; however, its impacts on early embryogenesis are not well studied. This work utilized an obese ewe model. Once the obese phenotype was established, ewes were bred. Conceptus and endometrial tissue were collected at D 14 of pregnancy, and samples were processed for RNA-sequencing analysis. There were no differences in pregnancy rate, ovulation rate, or pregnancies/ovulation between obese and lean animals. At an RPKM threshold of 0.2, fold-change 2, and FDR 0.05, 669 and 21 differentially expressed genes (DEGs) were identified between obese- and lean-derived endometrial samples and conceptus samples, respectively. Likewise, 137 DEGs were identified between male and female conceptuses. The PANTHER GO-Slim Biological Process system identified several biological processes affected by obesity in both the endometrium and conceptus tissue. GO terms do not currently exist for "placenta" and "trophoblast", so a literature search was conducted to identify DEGs involved in implantation and placentation. This revealed 125 placentation DEGs in the endometrium, and 4 DEGs in conceptuses between obese and lean groups. A follow-up study was conducted to examine the abundance of transcripts with regulatory roles in embryogenesis. Conceptuses exhibited differential expression of DNA methyltransferase 1 (DNMT1) based on obesity exposure, fibroblast growth factor receptor 2 (FGFR2) in a sex*obesity interaction, and peroxisome proliferator-activated receptor gamma (PPARG) and prostaglandin-endoperoxide synthase 2 (PTGS2) in a sex-specific manner. Collectively these results identify the preimplantation period as a susceptible time to maternal obesity in both conceptus and endometrial tissue. Together, these studies aim to provide a better understanding of the events controlling early embryogenesis, and insight into the implication of insults during this time. These findings will prove to be beneficial in establishing the link between maternal health, endometrial function, and subsequent offspring outcomes, with the hope of promoting a more viable embryo and thus healthier offspring.
  • Milk production of Holstein dairy cattle that were conceived in winter or summer months
    Stallings, Jon; Clay, John S.; Rhoads, Michelle (Virginia Tech, 2016)
    This dataset contains the milk production records (mature equivalent milk, kg) of Holstein dairy cows in the United States during their first lactation. These records were obtained from dairy farms in three states (Georgia, Florida and Texas) from 1999 to 2011. For each production record, the season in which the cow calved is indicated. Winter encompasses the months of December, January and February; spring includes March, April and May; summer includes June, July and August; fall includes September, October and November. Cows included in this dataset were either conceived during summer (indicated as "1" in the HSCONCP column) or winter (indicated as "0" in the HSCONCP column). This dataset was anonymized to ensure privacy of the dairy producers. For a full dataset, interested parties should contact Dairy Records Management Systems, North Carolina State University, 313 Chapanoke Road, Suite 100, Raleigh, NC 27603 (Attn: Dr. John S. Clay, Director, john_clay@ncsu.edu).