Delayed endometrial preparation for the induction of luteolysis as a potential factor for improved reproductive performance in Angus beef heifers with high antral follicle counts.

Antral Follicle Count (AFC) and anti-Müllerian hormone (AMH) concentrations are reflective for ovarian reserve and have been associated with improved reproductive performance in cattle. Key events for regulation of uterine receptivity are orchestrated by progesterone. As progesterone concentrations are greater in animals with high than low AFC, we tested the hypothesis, if the resulting improved uterine environment will lead to improved conceptus elongation and endometrial response to interferon tau. For four years, 10 heifers with lowest and highest AFC, respectively, were selected from 120 heifers. Reproductive tracts and blood samples for progesterone and AMH analysis were collected after synchronization and insemination. For a recovered conceptus, length was determined, and interferon tau (IFNT) transcript abundance was analyzed. Endometrial transcript abundance of interferon-stimulated gene 15 (ISG15) and oxytocin receptor (OXTR) were analyzed. Progesterone concentrations did not differ between Low and High AFC Group (P = 0.1). A difference in conceptus length was not observed. Endometrial abundance of ISG15 did not differ between Pregnant Low and High AFC heifers. Abundance of OXTR was greater in Open Low AFC than Open High AFC heifers (P < 0.01). Interaction of AMH and Pregnancy Status was determined, with greater AMH in Pregnant than Open High AFC heifers (P < 0.05). Improved uterine environment in High vs. Low AFC heifers did not result in longer conceptuses or improved endometrial response. As the increase in OXTR transcript abundance was only detected in Low AFC heifers, reported differences in reproductive performance might be associated with earlier initiation of luteolysis.

Maternal nutrition and developmental programming of offspring.

Developmental programming is the concept that 'stressors' during development (i.e. pregnancy, the perinatal period and infancy) can cause long-term changes in gene expression, leading to altered organ structure and function. Such long-term changes are associated with an increased risk of a host of chronic pathologies, or non-communicable diseases including abnormal growth and body composition, behavioural or cognitive dysfunction, metabolic abnormalities, and cardiovascular, gastro-intestinal, immune, musculoskeletal and reproductive dysfunction. Maternal nutrition during the periconceptual period, pregnancy and postnatally can have profound influences on the developmental program. Animal models, including domestic livestock species, have been important for defining the mechanisms and consequences of developmental programming. One of the important observations is that maternal nutritional status and other maternal stressors (e.g. environmental temperature, high altitude, maternal age and breed, multiple fetuses, etc.) early in pregnancy and even periconceptually can affect not only embryonic/fetal development but also placental development. Indeed, altered placental function may underlie the effects of many maternal stressors on fetal growth and development. We suggest that future directions should focus on the consequences of developmental programming during the offspring's life course and for subsequent generations. Other important future directions include evaluating interventions, such as strategic dietary supplementation, and also determining how we can take advantage of the positive, adaptive aspects of developmental programming.

Yearling bulls have reduced sperm concentration and increased seminal plasma interleukin-8 after a 28-day breeding season.

We hypothesized that yearling bulls selected for a 28-d breeding season would have reduced sperm concentrations and morphology, and have increased seminal plasma concentrations of pro-inflammatory cytokine interleukin-8 (IL-8). Yearling bulls were selected based on a breeding soundness examination (BSE) at approximately 415 d of age and contained at least 750 million sperm in the ejaculate, with 12 bulls randomly selected for breeding (BREEDERS) and 12 bulls not selected for breeding (NON-BREEDERS). After a 28-d breeding period, all bulls underwent a BSE. Plasma and seminal plasma were collected at each time point for analysis. Data were analysed utilizing either the MIXED or GLIMMIX procedures with repeated measures in SAS with breeding group, age and the interaction as fixed effects. Sperm concentration per ml of ejaculate was reduced (p < .05) in yearling bulls used for breeding compared with those not used for breeding at the end of the breeding season. Seminal plasma IL-8 concentrations in yearling bulls used for breeding were increased (p < .05) after the breeding season compared with bulls not used for breeding. Taken together, yearling bulls selected for a 28-d breeding season have reduced sperm production per ml of an ejaculate and increased inflammatory response in the seminal plasma that can lead to impaired breeding response if they are to be used for more than 30 d of breeding.

Relationship of length of the estrous cycle to antral follicle number in crossbred beef heifers.

Length of the menstrual cycle was positively associated with antral follicle number in women. If this pattern is consistent in cattle, a value-added benefit to using automated activity monitors to determine estrous status could be the ability to predict antral follicle count (AFC). We, therefore, hypothesized that as inter-estrous interval increased ultrasonographic AFC would be greater in crossbred beef heifers. Over 3 yr, crossbred beef heifers (n = 1,394) were fitted with automated activity monitors for 81 d. From days 42 to 46, heifers were submitted for ultrasonographic examination to determine AFC. From days 60 to 81, heifers were visually observed twice daily for 45 min for signs of behavioral estrus. Heifers that had a behavioral estrus that coincided with a sensor-based estrus and had a previous sensor-based estrus between 15 and 26 d earlier were used for the analysis (n = 850). A combination of regression analyses and correlation analyses were applied to understand the association between data collected by sensors and follicle number determined by ultrasonographic examination. Antral follicle count was analyzed using the GLM procedure of SAS with estrous cycle length (15 to 26 d) as a fixed effect. Estrus was more likely to initiate in the early morning hours and peak activity was greater (P < 0.0001) when estrus initiated between 0200 and 0800 hours then when estrus initiated at other times of the day. Antral follicle count did not differ due to length of the estrous cycle (P = 0.87). Thus, length of the estrous cycle obtained from three-axis accelerometers cannot be used to predict follicle number in crossbred beef heifers; however, machine learning approaches that combine multiple features could be used to integrate parameters of activity with other relevant environmental and management data to quantify AFC and improve reproductive management in beef cows.

One-carbon metabolites supplementation and nutrient restriction alter the fetal liver metabolomic profile during early gestation in beef heifers.

Maternal nutrition is pivotal for proper fetal development, with one-carbon metabolites (OCM) playing a key role in fetal epigenetic programming through DNA and histone methylation. The study aimed to investigate the effects of nutrient restriction and OCM supplementation on fetal liver metabolomics in pregnant beef-heifers, focusing on metabolites and pathways associated with amino acid, vitamin and cofactor, carbohydrate, and energy metabolism at day 63 of gestation. Thirty-one crossbred Angus heifers were artificially inseminated and allocated to 4 nutritional treatments in a 2 × 2 factorial arrangement of treatments, with the 2 factors being dietary intake/rate of gain (control-diet [CON]; 0.60 kg/d ADG, vs. restricted-diet [RES]; -0.23 kg/d ADG) and OCM supplementation (supplemented [+OCM] vs. not supplemented [-OCM]). The resulting treatment groups-CON - OCM, CON + OCM, RES - OCM, and RES + OCM were maintained for 63 day post-breeding. Following this period, fetal liver tissues were collected and subjected to metabolomic analysis using UPLC-tandem mass-spectrometry. We identified 288 metabolites, with the majority (n = 54) being significantly influenced by the main effect of gain (P ≤ 0.05). Moreover, RES showed decreased abundances of most metabolites in pathways such as lysine metabolism; leucine, isoleucine, and valine metabolism; and tryptophan metabolism, compared to CON. Supplementation with OCM vs. no OCM supplementation, resulted in greater abundance of metabolites (P ≤ 0.05) affecting pathways associated with methionine, cysteine, S-adenosylmethionine and taurine metabolism; guanidino and acetamido metabolism; and nicotinate and nicotinamide metabolism. Notably, OCM supplementation with a moderate rate of gain increased the concentrations of ophthalmate, N-acetylglucosamine, and ascorbic-acid 3-sulfate, which are important for proper fetal development (P ≤ 0.05). Nutrient restriction reduced the majority of liver metabolites, while OCM supplementation increased a smaller number of metabolites. Thus, OCM supplementation may be protective of metabolite concentrations in key developmental pathways, which could potentially enhance fetal development under nutrient-restricted conditions.

Maternal nutrition is crucial for pregnancy outcomes, influencing offspring health and productivity. Poor nutrition during pregnancy can lead to fetal growth restrictions, impacting liver development. Such changes can increase the risk of metabolic syndromes and predispose them to impaired immune function. In cattle, optimal nutrition during early pregnancy is essential for reproductive efficiency and herd health. This period is critical for developmental programming through epigenetic changes triggered by environmental or genetic factors. These modifications are heritable which are influenced by maternal diet and play a critical role in determining health outcomes post-birth, relying significantly on the availability of one-carbon metabolites (OCM) like methionine, choline, folate, and vitamin B12. Supplementing these nutrients during early gestation may counteract the negative effects of poor nutrition. This study explores the impact of OCM supplementation and dietary restrictions on the fetal liver metabolism in beef heifers during early gestation. Our findings showed that dietary restrictions decrease fetal liver metabolites, whereas OCM supplementation increases certain metabolites, indicating a compensatory effect to support fetal development under nutrient-restricted conditions. Highlighting the importance of maternal nutrition, our findings provide valuable insights for developing nutritional strategies to enhance livestock efficiency and inform dietary guidelines during pregnancy for better health outcomes.

Influence of Maternal Nutrition and One-Carbon Metabolites Supplementation during Early Pregnancy on Bovine Fetal Small Intestine Vascularity and Cell Proliferation.

To investigate the effects of nutrient restriction and one-carbon metabolite (OCM) supplementation (folate, vitamin B12, methionine, and choline) on fetal small intestine weight, vascularity, and cell proliferation, 29 (n = 7 ± 1 per treatment) crossbred Angus beef heifers (436 ± 42 kg) were estrous synchronized and conceived by artificial insemination with female sexed semen from a single sire. Then, they were allotted randomly to one of four treatments in a 2 × 2 factorial arrangement with the main factors of nutritional plane [control (CON) vs. restricted feed intake (RES)] and OCM supplementation [without OCM (-OCM) or with OCM (+OCM)]. Heifers receiving the CON level of intake were fed to target an average daily gain of 0.45 kg/day, which would allow them to reach 80% of mature BW by calving. Heifers receiving the RES level of intake were fed to lose 0.23 kg/heifer daily, which mimics observed production responses in heifers that experience a diet and environment change during early gestation. Targeted heifer gain and OCM treatments were administered from d 0 to 63 of gestation, and then all heifers were fed a common diet targeting 0.45 kg/d gain until d 161 of gestation, when heifers were slaughtered, and fetal jejunum was collected. Gain had no effect (p = 0.17) on the fetal small intestinal weight. However, OCM treatments (p = 0.02) displayed less weight compared to the -OCM groups. Capillary area density was increased in fetal jejunal villi of RES - OCM (p = 0.02). Vascular endothelial growth factor receptor 2 (VEGFR2) positivity ratio tended to be greater (p = 0.08) in villi and was less in the crypts (p = 0.02) of the RES + OCM group. Cell proliferation decreased (p = 0.02) in villi and crypts of fetal jejunal tissue from heifers fed the RES + OCM treatment compared with all groups and CON - OCM, respectively. Spatial cell density increased in RES - OCM compared with CON + OCM (p = 0.05). Combined, these data show OCM supplementation can increase expression of VEGFR2 in jejunal villi, which will promote maintenance of the microvascular beds, while at the same time decreasing small intestine weight and crypt cell proliferation.

One-carbon metabolite supplementation to nutrient-restricted beef heifers affects placental vascularity during early pregnancy.

We hypothesized that restricted maternal nutrition and supplementation of one-carbon metabolites (OCM; methionine, folate, choline, and vitamin B12) would affect placental vascular development during early pregnancy. A total of 43 cows were bred, and 32 heifers successfully became pregnant with female calves, leading to the formation of four treatment groups: CON - OCM (n = 8), CON + OCM (n = 7), RES - OCM (n = 9), and RES + OCM (n = 8). The experimental design was a 2 × 2 factorial, with main factors of dietary intake affecting average daily gain: control (CON; 0.6 kg/d ADG) and restricted (RES; -0.23 kg/d ADG); and OCM supplementation (+OCM) in which the heifers were supplemented with rumen-protected methionine (7.4 g/d) and choline (44.4 g/d) and received weekly injections of 320 mg of folate and 20 mg of vitamin B12, or received no supplementation (-OCM; corn carrier and saline injections). Heifers were individually fed and randomly assigned to treatment at breeding (day 0). Placentomes were collected on day 63 of gestation (0.225 of gestation). Fluorescent staining with CD31 and CD34 combined with image analysis was used to determine the vascularity of the placenta. Images were analyzed for capillary area density (CAD) and capillary number density (CND). Areas evaluated included fetal placental cotyledon (COT), maternal placental caruncle (CAR), whole placentome (CAR + COT), intercotyledonary fetal membranes (ICOT, or chorioallantois), intercaruncular endometrium (ICAR), and endometrial glands (EG). Data were analyzed with the GLM procedure of SAS, with heifer as the experimental unit and significance at P ≤ 0.05 and a tendency at P > 0.05 and P < 0.10. Though no gain × OCM interactions existed (P ≥ 0.10), OCM supplementation increased (P = 0.01) CAD of EG, whereas nutrient restriction tended (P < 0.10) to increase CAD of ICOT and CND of COT. Additionally, there was a gain × OCM interaction (P < 0.05) for CAD within the placentome and ICAR, such that RES reduced and supplementation of RES with OCM restored CAD. These results indicate that maternal rate of gain and OCM supplementation affected placental vascularization (capillary area and number density), which could affect placental function and thus the efficiency of nutrient transfer to the fetus during early gestation.

In cow­calf production, periods of poor forage availability or quality can result in nutrient restriction during pregnancy. Previous studies have shown that even moderate maternal feed restriction during pregnancy, including very early in pregnancy, has profound effects on fetal and placental development, potentially having lasting impacts on calf growth and body composition later in life. One-carbon metabolites (OCM) in the diet are biomolecules required for methylation reactions and participate in the regulation of gene expression. Our objective was to evaluate the effects of nutrient restriction and OCM supplementation (specifically methionine, choline, folate, and vitamin B12) on placental vascular development during early pregnancy. Proper placental vascular development is necessary for healthy pregnancy outcomes, reflected by normal birth weight and healthy offspring. Our results indicated that maternal rate of gain and OCM supplementation affect placental vascularization, which could affect placental function and thereby fetal development throughout gestation. In the context of beef cattle production, our study sheds light on strategies that could enhance placental vascular development during early pregnancy. However, it is essential to recognize the nuances in our data, highlighting the need for further research to fully comprehend these intricate processes.

Effects of dietary restriction and one-carbon metabolite supplementation during the first 63 days of gestation on the maternal gut, vaginal, and blood microbiota in cattle.

BACKGROUND: Maternal diet quality and quantity have significant impacts on both maternal and fetal health and development. The composition and function of the maternal gut microbiome is also significantly influenced by diet; however, little is known about the impact of gestational nutrient restriction on the bovine maternal microbiome during early gestation, which is a critical stage for maternal microbiome-mediated fetal programming to take place. The objective of the present study was to evaluate the impacts of diet restriction and one-carbon metabolite (OCM) supplementation during early gestation on maternal ruminal, vaginal, and blood microbiota in cattle. Thirty-three beef heifers (approx. 14 months old) were used in a 2 × 2 factorial experiment with main factors of target gain (control [CON]; targeted 0.45 kg/d gain vs restricted [RES]; targeted - 0.23 kg/d gain), and OCM supplementation (+ OCM vs - OCM; n = 8/treatment; except n = 9 for RES-OCM). Heifers were individually fed, starting treatment at breeding (d 0) and concluding at d 63 of gestation. Ruminal fluid and vaginal swabs were collected on d - 2, d 35, and d 63 (at necropsy) and whole blood was collected on d 63 (necropsy). Bacterial microbiota was assessed using 16S rRNA gene (V3-V4) sequencing. RESULTS: Overall ruminal microbiota structure was affected by gain, OCM, time, and their interactions. The RES heifers had greater microbial richness (observed ASVs) but neither Shannon nor Inverse Simpson diversity was significantly influenced by gain or OCM supplementation; however, on d 63, 34 bacterial genera showed differential abundance in the ruminal fluid, with 25 genera enriched in RES heifers as compared to CON heifers. In addition, the overall interaction network structure of the ruminal microbiota changed due to diet restriction. The vaginal microbiota community structure was influenced by gain and time. Overall microbial richness and diversity of the vaginal microbiota steadily increased as pregnancy progressed. The vaginal ecological network structure was distinctive between RES and CON heifers with genera-genera interactions being intensified in RES heifers. A relatively diverse bacterial community was detected in blood samples, and the composition of the blood microbiota differed from that of ruminal and vaginal microbiota. CONCLUSION: Restricted dietary intake during early gestation induced significant alterations in the ruminal microbiota which also extended to the vaginal microbiota. The composition of these two microbial communities was largely unaffected by OCM supplementation. Blood associated microbiota was largely distinctive from the ruminal and vaginal microbiota.

One-carbon metabolite supplementation increases vitamin B12, folate, and methionine cycle metabolites in beef heifers and fetuses in an energy dependent manner at day 63 of gestation.

One-carbon metabolites (OCM) are metabolites and cofactors which include folate, vitamin B12, methionine, and choline that support methylation reactions. The objectives of this study were to investigate the effects of moderate changes in maternal body weight gain in combination with OCM supplementation during the first 63 d of gestation in beef cattle on (1) B12 and folate concentrations in maternal serum (2) folate cycle intermediates in maternal and fetal liver, allantoic fluid (ALF), and amniotic fluid (AMF) and (3) metabolites involved in one-carbon metabolism and related metabolic pathways in maternal and fetal liver. Heifers were either intake restricted (RES) and fed to lose 0.23 kg/d, or fed to gain 0.60 kg/d (CON). Supplemented (+ OCM) heifers were given B12 and folate injections weekly and fed rumen-protected methionine and choline daily, while non-supplemented (-OCM) heifers were given weekly saline injections. These two treatments were combined in a 2 × 2 factorial arrangement resulting in 4 treatments: CON-OCM, CON + OCM, RES-OCM, and RES + OCM. Samples of maternal serum, maternal and fetal liver, ALF, and AMF were collected at slaughter on day 63 of gestation. Restricted maternal nutrition most notably increased (./ ≤ 0.05) the concentration of vitamin B12 in maternal serum, 5,10-methylenetetrahydrofolate and 5,10-methenyltetrahydrofolate in maternal liver, and cystathionine in the fetal liver; conversely, maternal restriction decreased (P = 0.05) 5,10-methylenetetrahydrofolate concentration in fetal liver. Supplementing OCM increased (P ≤ 0.05) the concentrations of maternal serum B12, folate, and folate intermediates, ALF and AMF 5-methyltetrahydrofolate concentration, and altered (P ≤ 0.02) other maternal liver intermediates including S-adenosylmethionine, dimethylglycine, cystathionine Glutathione reduced, glutathione oxidized, taurine, serine, sarcosine, and pyridoxine. These data demonstrate that OCM supplementation was effective at increasing maternal OCM status. Furthermore, these data are similar to previously published literature where restricted maternal nutrition also affected maternal OCM status. Altering OCM status in both the dam and fetus could impact fetal developmental outcomes and production efficiencies. Lastly, these data demonstrate that fetal metabolite abundance is highly regulated, although the changes required to maintain homeostasis may program altered metabolism postnatally.

Maternal stresses that occur during pregnancy, such as restricted nutrition, can impact the developmental outcomes of the offspring in a process known as developmental programming. This programming can occur through epigenetics, which involves changes in fetal gene expression and can occur through the addition of methyl groups to DNA. These changes regulate gene transcription in the offspring and can alter offspring health, efficiency, and life-long outcomes. One-carbon metabolites (OCM), which are nutrients like the amino acid methionine and the vitamins B12, folate, and choline, act as intermediates or cofactors for the donation of methyl groups to DNA. This study investigated the effects of differing maternal rates of gain along with OCM supplementation during early gestation on OCM and related metabolite concentrations in the dam and fetus. We found that supplementing OCM to beef heifers increased maternal OCM and related metabolite concentrations and fetal fluid OCM concentrations. We also found that low maternal gain increased maternal serum and liver OCM concentrations. We can conclude from these findings that both maternal rate of gain and OCM supplementation can impact maternal OCM concentrations at day 63 of gestation and further research is needed to see if those maternal impacts will affect the developing fetus or calf later in its life.

Differing planes of nutrition alter serum and histotroph amino acid composition in nonpregnant, multiparous beef cows on day 3 of the estrous cycle.

Endometrial-derived uterine histotroph is a critical component of nutrient supply to a growing conceptus throughout gestation; however, the effect of nutritional plane on histotroph nutrient composition remains unknown in multiparous cows. We hypothesized that differing planes of nutrition would alter histotroph and serum nutrient composition in beef cattle. Thus, we evaluated serum and histotroph amino acid and glucose composition, and serum non-esterified fatty acids (NEFA) and blood urea nitrogen (BUN) in cows individually fed to maintain body weight (BW; 0 kd/d, n = 9; CON) compared with those losing moderate BW (-0.7 kg/d, n = 9; NEG). After 49 d of differing nutritional planes, cows were subjected to the 7-d CoSynch + controlled internal drug release device estrus synchronization protocol and then slaughtered on day 62. Blood serum (days 0 and 62) and uterine histotroph [day 62; from uterine horns ipsilateral and contralateral to the corpus luteum (CL)] were collected and analyzed for concentrations of amino acids, glucose, and NEFA. Performance characteristics, body composition via ultrasound (days 0 and 62), and carcass characteristics were collected. Body condition score, change in BW, average daily gain, dry matter intake, and gain:feed were decreased (P ≤ 0.05) in NEG vs. CON cows. There were no differences in body composition or carcass characteristics, except an increase (P ≤ 0.05) in dressing percentage in NEG cows due to differences in gut fill, consistent with study design. Serum NEFA increased (P ≤ 0.05) in the NEG group, but there were no differences between NEG vs. CON in glucose or BUN. Serum histidine increased (P ≤ 0.05) and alanine, isoleucine, and tryptophan decreased (P ≤ 0.05) in NEG vs. CON cows. Compared with that of the uterine horn ipsilateral to the CL, histotroph from the uterine horn contralateral to the CL had increased (P ≤ 0.05) isoleucine, asparagine, and proline concentrations in NEG cows, and decreased (P ≤ 0.05) tryptophan as a proportion of essential and total amino acids. There were no differences in glucose concentrations of histotroph contralateral or ipsilateral to the CL. Cow nutritional plane does alter serum and histotroph amino acid composition, although the presence of an embryo may be necessary to fully elucidate these changes. Differences in serum and histotroph tryptophan should be given consideration in future studies due to its importance as an essential amino acid in protein synthesis and bioactive affects.

Amino acids are important in protein synthesis and bioactive affects. Maternal diet could impact histotroph amino acid composition which serves as a nutrient supply to the conceptus throughout pregnancy and is especially critical during early pregnancy, before the placenta is fully functional. Cows were subjected to their diets for 62 d, resulting in decreased body condition, average daily gain, dry matter intake, G:F, and a greater change in body weight (BW) among moderate loss cows. These data demonstrate our model for moderate BW loss was successful. Moderate BW loss cows exhibited alterations in serum and histotroph amino acid composition in the uterine horn contralateral to the corpus luteum (CL). However, in the present study, histotroph amino acid alterations were in the uterine horn contralateral to the CL, which would be opposite of the developing conceptus. Nevertheless, because the 2 uterine horns communicate via the common uterine body, the pre-implantation conceptus should have access to the histotroph from the contralateral uterine horn. Thus, future studies are needed to fully elucidate effects of nutritional plane on histotroph nutrient composition, and its potential impact on pregnancy.

Disrupted one-carbon metabolism in heifers negatively affects their health and physiology.

The objective of this study was to determine the dose-dependent response of one-carbon metabolite (OCM: methionine, choline, folate, and vitamin B12) supplementation on heifer dry matter intake on fixed gain, organ mass, hematology, cytokine concentration, pancreatic and jejunal enzyme activity, and muscle hydrogen peroxide production. Angus heifers (n = 30; body weight [BW] = 392.6 ±â€…12.6 kg) were individually fed and assigned to one of five treatments: 0XNEG: total mixed ration (TMR) and saline injections at days 0 and 7 of the estrous cycle, 0XPOS: TMR, rumen-protected methionine (MET) fed at 0.08% of the diet dry matter, rumen-protected choline (CHOL) fed at 60 g/d, and saline injections at days 0 and 7, 0.5X: TMR, MET, CHOL, 5-mg B12, and 80-mg folate injections at days 0 and 7, 1X: TMR, MET CHOL, 10-mg vitamin B12, and 160-mg folate at days 0 and 7, and 2X: TMR, MET, CHOL, 20-mg vitamin B12, and 320-mg folate at days 0 and 7. All heifers were estrus synchronized but not bred, and blood samples were collected on days 0, 7, and at slaughter (day 14) during which tissues were collected. By design, heifer ADG did not differ (P = 0.96). Spleen weight and uterine weight were affected cubically (P = 0.03) decreasing from 0XPOS to 0.5X. Ovarian weight decreased linearly (P < 0.01) with increasing folate and B12 injection. Hemoglobin and hematocrit percentage were decreased (P < 0.01) in the 0.5X treatment compared with all other treatments. Plasma glucose, histotroph protein, and pancreatic α-amylase were decreased (P ≤ 0.04) in the 0.5X treatment. Heifers on the 2X treatment had greater pancreatic α-amylase compared with 0XNEG and 0.5X treatment. Interleukin-6 in plasma tended (P = 0.08) to be greater in the 0XPOS heifers compared with all other treatments. Lastly, 0XPOS-treated heifers had reduced (P ≤ 0.07) hydrogen peroxide production in muscle compared with 0XNEG heifers. These data imply that while certain doses of OCM do not improve whole animal physiology, OCM supplementation doses that disrupt one-carbon metabolism, such as that of the 0.5X treatment, can induce a negative systemic response that results in negative effects in both the dam and the conceptus during early gestation. Therefore, it is necessary to simultaneously establish an optimal OCM dose that increases circulating concentrations for use by the dam and the conceptus, while avoiding potential negative side effects of a disruptive OCM, to evaluate the long-term impacts of OCM supplementation of offspring programming.

The feeding of one-carbon metabolites (including methionine and B vitamins) has been shown to improve fetal growth and milk production in species such as mice, sheep, and dairy cattle. Extending this to beef cattle around the time of breeding is a growing area of research. Our group previously determined that one-carbon metabolite supplementation to beef heifers altered the abundance of circulating methionine-folate cycle intermediates in a dose-dependent manner. Therefore, we aimed to determine a whole-body response to one-carbon metabolite supplementation in heifers by measuring the effects on specific physiological systems as well as a total systemic response. We determined that treatments that negatively altered the methionine-folate cycle yielded a fundamental negative whole-body response to supplementation.

Paternal effects on fetal programming.

Paternal programming is the concept that the environmental signals from the sire's experiences leading up to mating can alter semen and ultimately affect the phenotype of resulting offspring. Potential mechanisms carrying the paternal effects to offspring can be associated with epigenetic signatures (DNA methylation, histone modification and non-coding RNAs), oxidative stress, cytokines, and the seminal microbiome. Several opportunities exist for sperm/semen to be influenced during development; these opportunities are within the testicle, the epididymis, or accessory sex glands. Epigenetic signatures of sperm can be impacted during the pre-natal and pre-pubertal periods, during sexual maturity and with advancing sire age. Sperm are susceptible to alterations as dictated by their developmental stage at the time of the perturbation, and sperm and seminal plasma likely have both dependent and independent effects on offspring. Research using rodent models has revealed that many factors including over/under nutrition, dietary fat, protein, and ingredient composition (e.g., macro- or micronutrients), stress, exercise, and exposure to drugs, alcohol, and endocrine disruptors all elicit paternal programming responses that are evident in offspring phenotype. Research using livestock species has also revealed that sire age, fertility level, plane of nutrition, and heat stress can induce alterations in the epigenetic, oxidative stress, cytokine, and microbiome profiles of sperm and/or seminal plasma. In addition, recent findings in pigs, sheep, and cattle have indicated programming effects in blastocysts post-fertilization with some continuing into post-natal life of the offspring. Our research group is focused on understanding the effects of common management scenarios of plane of nutrition and growth rates in bulls and rams on mechanisms resulting in paternal programming and subsequent offspring outcomes. Understanding the implication of paternal programming is imperative as short-term feeding and management decisions have the potential to impact productivity and profitability of our herds for generations to come.

A Longitudinal Characterization of the Seminal Microbiota and Antibiotic Resistance in Yearling Beef Bulls Subjected to Different Rates of Gain.

In this study, we evaluated the seminal and fecal microbiota in yearling beef bulls fed a common diet to achieve moderate (1.13 kg/day) or high (1.80 kg/day) rates of weight gain. Semen samples were collected on days 0 and 112 of dietary intervention (n = 19/group) as well as postbreeding (n = 6/group) using electroejaculation, and the microbiota was assessed using 16S rRNA gene sequencing, quantitative PCR (qPCR), and culturing. The fecal microbiota was also evaluated, and its similarity with seminal microbiota was assessed. A subset of seminal bacterial isolates (n = 33) was screened for resistance against 28 antibiotics. A complex and dynamic microbiota was detected in bovine semen, and the community structure was affected by sampling time (R2 = 0.16, P < 0.001). Microbial richness increased significantly from day 0 to day 112, and diversity increased after breeding (P > 0.05). Seminal microbiota remained unaffected by the differential rates of gain, and its overall composition was distinct from fecal microbiota, with only 6% of the taxa shared between them. A total of 364 isolates from 49 different genera were recovered under aerobic and anaerobic culturing. Among these seminal isolates were pathogenic species and those resistant to several antibiotics. Overall, our results suggest that bovine semen harbors a rich and complex microbiota which changes over time and during the breeding season but appears to be resilient to differential gains achieved via a common diet. Seminal microbiota is distinct from the fecal microbiota and harbors potentially pathogenic and antibiotic-resistant bacterial species. IMPORTANCE Increasing evidence from human and other animal species supports the existence of a commensal microbiota in semen and that this seminal microbiota may influence not only sperm quality and fertility but also female reproduction. Seminal microbiota in bulls and its evolution and factors shaping this community, however, remain largely underexplored. In this study, we characterized the seminal microbiota of yearling beef bulls and its response to the bull age, different weight gains, and mating activity. We compared bacterial composition between seminal and fecal microbiota and evaluated the diversity of culturable seminal bacteria and their antimicrobial resistance. Our results obtained from sequencing, culturing, and antibiotic susceptibility testing provide novel information on the taxonomic composition, evolution, and factors shaping the seminal microbiota of yearling beef bulls. This information will serve as an important basis for further understanding of the seminal microbiome and its involvement in reproductive health and fertility in cattle.

Concentrations of vitamin B12 and folate in maternal serum and fetal fluids, metabolite interrelationships, and hepatic transcript abundance of key folate and methionine cycle genes: the impacts of maternal nutrition during the first 50 d of gestation.

Adequate maternal nutrition is key for proper fetal development and epigenetic programming. One-carbon metabolites (OCM), including vitamin B12, folate, choline, and methionine, play a role in epigenetic mechanisms associated with developmental programming. This study investigated the presence of B12 and folate in maternal serum, allantoic fluid (ALF), and amniotic fluid (AMF), as well as how those concentrations in all three fluids correlate to the concentrations of methionine-folate cycle intermediates in heifers receiving either a control (CON) or restricted (RES) diet for the first 50 d of gestation and fetal hepatic gene expression for methionine-folate cycle enzymes. Angus cross heifers (n = 43) were estrus synchronized, bred via artificial insemination with semen from a single sire, and randomly assigned to one of two nutrition treatments (CON = 20, RES = 23). Heifers were ovariohysterectomized on either day 16 (n = 14), 34 (n = 15), or 50 of gestation (n = 14), where samples of maternal serum (n = 42), ALF (n = 29), and AMF (n = 11) were collected and analyzed for concentrations of folate and B12. Concentrations of B12 and folate in ALF were greater (P < 0.05) in RES compared to CON. For ALF, folate concentrations were also greater (P < 0.01) on day 34 compared to day 50. There was a significant (P = 0.04) nutrition × fluid interaction for B12 concentrations where concentrations were greatest in restricted ALF, intermediate in control ALF, and lowest in CON and RES serum and AMF. Folate concentrations were greatest (P < 0.01) in ALF, intermediate in serum, and lowest in AMF. Additionally, positive correlations (P < 0.05) were found between ALF and AMF folate concentrations and AMF concentrations of methionine, serine, and glycine. Negative correlations (P < 0.05) between AMF folate and serum homocysteine were also observed. Both positive and negative correlations (P < 0.05) depending on the fluid evaluated were found between B12 and methionine, serine, and glycine concentrations. There was a downregulation (P = 0.05) of dihydrofolate reductase and upregulation (P = 0.03) of arginine methyltransferase 7 gene expression in RES fetal liver samples compared with CON fetal liver on day 50. Combined, these data show restricted maternal nutrition results in increased B12 and folate concentrations present in fetal fluids, and increased expression of genes for enzymes within one-carbon metabolism.

When pregnant cattle have restricted access to feed or specific nutrients, calf development can be affected, and the degree of impairment depends, at least partially, on timing, duration, and severity of the limitations. A biochemical pathway present in cells that can be affected by limited nutrition is one-carbon metabolism. This pathway is related to epigenetics, which regulates gene expression or the turning on and off of genes. Two important vitamins in one-carbon metabolism are vitamins B12 and folate. By understanding the amounts of those vitamins available to the developing calf, we can gain better insight into the regulation and potential avenues of improvement of calf growth and development. In this study, we found a nutrient restricted maternal diet increased the amount of B12 and folate in calf allantoic and amniotic fluids. We also found that folate and B12 were correlated to the presence of other nutrients in serum, allantoic fluid, and amniotic fluid. In addition, we found that a protein methylating gene in one-carbon metabolism had increased expression in calves from heifers receiving limited nutrition. This study is an important step in understanding how the nutrients available to a pregnant heifer during gestation affects nutrients available to the conceptus.

Weaning transition, but not the administration of probiotic candidate Kazachstania slooffiae, shaped the gastrointestinal bacterial and fungal communities in nursery piglets.

As in-feed antibiotics are phased out of swine production, producers are seeking alternatives to facilitate improvements in growth typically seen from this previously common feed additive. Kazachstania slooffiae is a prominent commensal fungus in the swine gut that peaks in relative abundance shortly after weaning and has beneficial interactions with other bacteriome members important for piglet health. In this study, piglets were supplemented with K. slooffiae to characterize responses in piglet health as well as fungal and bacterial components of the microbiome both spatially (along the entire gastrointestinal tract and feces) and temporally (before, during, and after weaning). Litters were assigned to one of four treatments: no K. slooffiae (CONT); one dose of K. slooffiae 7 days before weaning (day 14; PRE); one dose of K. slooffiae at weaning (day 21; POST); or one dose of K. slooffiae 7 days before weaning and one dose at weaning (PREPOST). The bacteriome and mycobiome were analyzed from fecal samples collected from all piglets at day 14, day 21, and day 49, and from organ samples along the gastrointestinal (GI) tract at day 21 and day 49. Blood samples were taken at day 14 and day 49 for cytokine analysis, and fecal samples were assayed for antimicrobial resistance. While some regional shifts were seen in response to K. slooffiae administration in the mycobiome of the GI tract, no remarkable changes in weight gain or health of the animals were observed, and changes were more likely due to sow and the environment. Ultimately, the combined microbiome changed most considerably following the transition from suckling to nursery diets. This work describes the mycobiome along the piglet GI tract through the weaning transition for the first time. Based on these findings, K. slooffiae administered at this concentration may not be an effective tool to hasten colonization of K. slooffiae in the piglet GI tract around the weaning transition nor support piglet growth, microbial gut health, or immunity. However, diet and environment greatly influence microbial community development.

One-carbon metabolite supplementation to heifers for the first 14 d of the estrous cycle alters the plasma and hepatic one-carbon metabolite pool and methionine-folate cycle enzyme transcript abundance in a dose-dependent manner.

The objective of this study was to determine the dose of folate and vitamin B12 in beef heifers fed rumen protected methionine and choline required to maintain increased B12 levels and intermediates of the methionine-folate cycle in circulation. Angus heifers (n = 30; BW = 392.6 ±â€…12.6 kg) were individually fed and assigned to one of five treatments: 0XNEG: Total mixed ration (TMR) and saline injections at day 0 and 7 of the estrous cycle, 0XPOS: TMR, rumen protected methionine (MET) fed at 0.08% of the diet DM, rumen protected choline (CHOL) fed at 60 g/d, and saline injections at day 0 and 7, 0.5X: TMR, MET, CHOL, 5 mg B12, and 80 mg folate at day 0 and 7, 1X: TMR, MET CHOL, 10 mg vitamin B12, and 160 mg folate at day 0 and 7, and 2X: TMR, MET, CHOL, 20 mg B12, and 320 mg folate at day 0 and 7. All heifers were estrus synchronized but not bred, and blood was collected on day 0, 2, 5, 7, 9, 12, and 14 of a synchronized estrous cycle. Heifers were slaughtered on day 14 of the estrous cycle for liver collection. Serum B12 concentrations were greater in the 0.5X, 1X, and 2X, compared with 0XNEG and 0XPOS on all days after treatment initiation (P < 0.0001). Serum folate concentrations were greater for the 2X treatment at day 5, 7, and 9 of the cycle compared with all other treatments (P ≤ 0.05). There were no differences (P ≥ 0.19) in hepatic methionine-cycle or choline analyte concentrations by treatment. Concentrations of hepatic folate cycle intermediates were always greater (P ≤ 0.04) in the 2X treatment compared with the 0XNEG and 0XPOS heifers. Serum methionine was greater (P = 0.04) in the 0.5X and 2X heifers compared with 0XNEG, and S-adenosylhomocysteine (SAH) tended (P = 0.06) to be greater in the 0.5X heifers and the S-adenosylmethionine (SAM):SAH ratio was decreased (P = 0.05) in the 0.5X treatment compared with the 0XNEG, 0XPOS, and 2X heifers. The hepatic transcript abundance of MAT2A and MAT2B were decreased (P ≤ 0.02) in the 0.5X heifers compared with the 0XNEG, 0XPOS, and 2X heifers. These data support that beef heifers fed rumen protected methionine and choline require 20 mg B12 and 320 mg folate once weekly to maintain increased concentrations of B12 and folate in serum. Furthermore, these data demonstrate that not all supplementation levels are equal in providing positive responses, and that some levels, such as the 0.5X, may result in a stoichiometric imbalance in the one-carbon metabolism pathway that results in a decreased SAM:SAH ratio.

The strategic inclusion of one-carbon metabolites, which include vitamins and minerals that are found in human prenatal vitamins, to beef cattle feeding and management protocols during the periconceptual period (the time around breeding) is a novel concept. Therefore, this study aimed to identify the feeding and injection doses of one-carbon metabolites in beef heifers to maintain increased circulating concentrations of one-carbon metabolites for use as a model from which other studies could base their treatments on. We determined that daily feeding of methionine and choline at 0.08% of dry matter and 60 g/d, respectively, and administration of vitamin B12 and folate at 20 mg and 320 mg once per week, respectively resulted in sustained elevated concentrations of one-carbon metabolites.

Fetal Hepatic Lipidome Is More Greatly Affected by Maternal Rate of Gain Compared with Vitamin and Mineral Supplementation at day 83 of Gestation.

Herein, we evaluated the hepatic lipid metabolic profiles of bovine fetuses in response to maternal vitamin and mineral supplementation (VMSUP; supplemented (VTM) or not (NoVTM)) and two different rates of gain (GAIN; low gain (LG), 0.28 kg/d, or moderate gain (MG), 0.79 kg/d). Crossbred Angus heifers (n = 35; initial BW = 359.5 ± 7.1 kg) were randomly assigned to a 2 × 2 factorial arrangement, resulting in the following treatment combinations: NoVTM-LG (n = 9), NoVTM-MG (n = 9), VTM-LG (n = 9), and VTM-MG (n = 8). Heifers received their treatments until d 83 of gestation, when they were ovariohysterectomized. Fetuses were harvested and liver samples were analyzed via ultrahigh-performance liquid chromatography-tandem mass spectroscopy to characterize lipid profiles and abundances. We identified 374 biochemicals/metabolites belonging to 57 sub-pathways of the lipid metabolism super-pathway. The majority of the biochemicals/metabolites (n = 152) were significantly affected by the main effect of GAIN. Maternal moderate rates of gain resulted in greater abundances (p ≤ 0.0001) of ω-3 fatty acids (eicosapentaenoate, docosapentaenoate, and docosahexaenoate) and lower abundances (p ≤ 0.0001) of ω-6 fatty acids. Further, MG resulted in the accumulation of several diacylglycerols and depletion of the majority of the monoacylglycerols. Concentrations of nearly all acylcarnitines (p ≤ 0.03) were decreased in VTM-LG fetal livers compared to all other treatment combinations, indicating a greater rate of complete oxidation of fatty acids. Levels of secondary bile acids were impacted by VMSUP, being greater (p ≤ 0.0048) in NoVTM than in VTM fetal livers. Moreover, NoVTM combined with lower rate of gain resulted in greater concentrations of most secondary bile acid biochemicals/metabolites. These data indicate that maternal diet influenced and altered fetal hepatic lipid composition in the first trimester of gestation. Maternal body weight gain exerted a greater influence on fetal lipid profiles than vitamin and mineral supplementation. Specifically, lower rate of gain (0.28 kg/d) resulted in an increased abundance of the majority of the biochemicals/metabolites identified in this study.

Polymorphism of the follicle stimulating hormone receptor does not impact reproductive performance or in-vitro embryo production in beef heifers.

Assisted reproductive technologies are used to propagate desirable genetics in a shortened timeframe. Selected females undergo ovarian stimulation with the use of follicle stimulating hormone (FSH) to increase embryo recovery for subsequent transfer programs. The FSH receptor (FSHR) c.337 C > G variant was reported to have a reduction in viable embryo numbers in an ovarian stimulation protocol. We, therefore, hypothesized that FSHR c.337 C > G would result in reduced in-vitro blastocyst development. Beef heifers were genotyped and selected based on the c.337 C > G FSHR genotype (CC, CG, GG; n = 15-16/genotype). Estrus was synchronized with a Select Synch protocol and heifers were slaughtered 5 days after induced ovulation. Anterior pituitaries, serum and reproductive tracts were collected at slaughter for analysis. Cumulus oocyte complexes (COCs) were collected and pooled within genotype for in-vitro fertilization (IVF) and subsequent blastocyst development. No differences were observed in carcass weights, anterior pituitary weights, serum progesterone, corpus lutea weight, surface follicle counts, histological follicle counts or follicular fluid estradiol concentration (P > 0.1) due to FSHR genotype. Differences were observed for ovulation rates in the GG FSHR genotype group (P < 0.01). However, cleavage and blastocyst rates were not affected due to FSHR genotype (P > 0.1), following standard IVF protocols. The FSHR variant does not influence antral follicle counts, estradiol production, or in-vitro blastocyst development in beef heifers. The GG FSHR genotype had an increased ovulation rate, which may indicate a greater potential for twinning, but research with a larger population is warranted to support this hypothesis.

DNA methylation dataset of bovine embryonic fibroblast cells treated with epigenetic modifiers and divergent energy supply.

Fetal programming is established early in life, likely through epigenetic mechanisms that control gene expression. Micronutrients can act as epigenetic modifiers (EM) by modulating the genome through mechanisms that include DNA methylation and post-translational modification of chromatin. Among the EM, methionine, choline, folate, and vitamin B12 have been suggested as key players of DNA methylation. However, the effects of supplementing these four EM, involved in the methionine folate cycle on DNA methylation, are still under investigation. This manuscript provides the genome-wide DNA methylation dataset (GSE180362) of bovine embryonic fibroblast cells exposed to different supplementation levels of glucose and methionine, choline, folate, and vitamin B12 (collectively named as Epigenetic Modifiers - EM). The DNA methylation was measured using MSP-I digestion and Reduced Representation Bisulfite Sequencing. Bioinformatics analyses included data quality control, read mapping, methylation calling, and differential methylation analyses. Supplementary file S1 and data analysis codes are within this article. To our knowledge, this is the first dataset investigating the effects of four EM in bovine embryonic fibroblast DNA methylation profiles. Furthermore, this data and its findings provide information on putative candidate genes responsive to DNA methylation due to EM supplementation.

Epigenetic Modifier Supplementation Improves Mitochondrial Respiration and Growth Rates and Alters DNA Methylation of Bovine Embryonic Fibroblast Cells Cultured in Divergent Energy Supply.

Epigenetic modifiers (EM; methionine, choline, folate, and vitamin B12) are important for early embryonic development due to their roles as methyl donors or cofactors in methylation reactions. Additionally, they are essential for the synthesis of nucleotides, polyamines, redox equivalents, and energy metabolites. Despite their importance, investigation into the supplementation of EM in ruminants has been limited to one or two epigenetic modifiers. Like all biochemical pathways, one-carbon metabolism needs to be stoichiometrically balanced. Thus, we investigated the effects of supplementing four EM encompassing the methionine-folate cycle on bovine embryonic fibroblast growth, mitochondrial function, and DNA methylation. We hypothesized that EM supplemented to embryonic fibroblasts cultured in divergent glucose media would increase mitochondrial respiration and cell growth rate and alter DNA methylation as reflected by changes in the gene expression of enzymes involved in methylation reactions, thereby improving the growth parameters beyond Control treated cells. Bovine embryonic fibroblast cells were cultured in Eagle's minimum essential medium with 1 g/L glucose (Low) or 4.5 g/L glucose (High). The control medium contained no additional OCM, whereas the treated media contained supplemented EM at 2.5, 5, and 10 times (×2.5, ×5, and ×10, respectively) the control media, except for methionine (limited to ×2). Therefore, the experimental design was a 2 (levels of glucose) × 4 (levels of EM) factorial arrangement of treatments. Cells were passaged three times in their respective treatment media before analysis for growth rate, cell proliferation, mitochondrial respiration, transcript abundance of methionine-folate cycle enzymes, and DNA methylation by reduced-representation bisulfite sequencing. Total cell growth was greatest in High ×10 and mitochondrial maximal respiration, and reserve capacity was greatest (p < 0.01) for High ×2.5 and ×10 compared with all other treatments. In Low cells, the total growth rate, mitochondrial maximal respiration, and reserve capacity increased quadratically to 2.5 and ×5 and decreased to control levels at ×10. The biological processes identified due to differential methylation included the positive regulation of GTPase activity, molecular function, protein modification processes, phosphorylation, and metabolic processes. These data are interpreted to imply that EM increased the growth rate and mitochondrial function beyond Control treated cells in both Low and High cells, which may be due to changes in the methylation of genes involved with growth and energy metabolism.