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In gilts, puberty is marked by standing estrus in the presence of a boar. Delayed puberty (DP; failure to display pubertal estrus) is a major reason for gilt removal. To investigate the physiological determinants underlying DP in gilts, transcriptomic data from tissues relevant to estrus and puberty, such as mediobasal hypothalamus, anterior pituitary gland, ovarian cortex, olfactory bulb, amygdala, and hippocampus, were obtained from age-matched DP (n= 8) and cyclic control gilts at follicular phase (n= 8) and luteal phase (n= 8) of the estrous cycle. A gene expression module analysis via three-way gene × individual × tissue clustering using tensor decomposition identified pituitary and ovary gene modules contributing to regulation of pubertal development. Analysis of gene expression in the hypothalamic–pituitary–ovary axis identified reduced expression of hypothalamic genes critical for stimulating gonadotropin secretion (KISS1and TAC3) and reduced expression of LHBin the anterior pituitary of DP gilts compared with their cyclic counterparts. Consequently, luteinizing hormone-induced genes in the ovary important for folliculogenesis (OXTR, RUNX2, and PTX3) were less expressed in DP gilts. Other intrafollicular genes (AHR, PTGS2, PTGFR, and IGFBP7) and genes in the steroidogenesis pathways (STARand CYP11A1) necessary to complete the ovulatory cascade were also less expressed in DP gilts. This is the first clustering of multi-tissue expression data from DP and cyclic gilts to identify genes differentially expressed in gilts of similar ages but at different levels of sexual development. A critical lack of gonadotropin support and reduced ovarian responsiveness underlie DP in gilts.Clustering of multi-tissue expression data and differential gene expression analysis of delayed puberty and normal cyclic gilts identified genes in the hypothalamic–pituitary–ovary axis associated with sexual maturity, development, and estrous cycle functions.Graphical Abstract

Embryo survival and pregnancy success is increased among animals that exhibit estrus prior to fixed time-artificial insemination, but there are no differences in conceptus survival to d16. The objective of this study was to determine effects of preovulatory estradiol on uterine transcriptomes, select trophectoderm (TE) transcripts, and uterine luminal fluid proteins. Beef cows/heifers were synchronized, artificially inseminated (d0), and grouped into either high (highE2) or low (lowE2) preovulatory estradiol. Uteri were flushed (d16); conceptuses and endometrial biopsies (n = 29) were collected. RNA sequencing was performed on endometrium. Real-time polymerase chain reaction (RT-PCR) was performed on TE (n = 21) RNA to measure relative abundance of IFNT, PTGS2, TM4SF1, C3, FGFR2, and GAPDH. Uterine fluid was analyzed using 2D Liquid Chromatography with tandem mass spectrometry-based Isobaric tags for relative and absolute quantitation (iTRAQ) method. RT-PCR data were analyzed using the MIXED procedure in SAS. There were no differences in messenger RNA (mRNA) abundances in TE, but there were 432 differentially expressed genes (253 downregulated, 179 upregulated) in highE2/conceptus versus lowE2/conceptus groups. There were also 48 differentially expressed proteins (19 upregulated, 29 downregulated); 6 of these were differentially expressed (FDR < 0.10) at the mRNA level. Similar pathways for mRNA and proteins included: calcium signaling, protein kinase A signaling, and corticotropin-releasing hormone signaling. These differences in uterine function may be preparing the conceptus for improved likelihood of survival after d16 among highE2 animals.

Varicocele, a condition of abnormally dilated veins within the pampiniform plexus, is associated with reduced male fertility in several species but its frequency and impact in rams is not well documented. Semen was collected and pampiniform plexus ultrasound performed in early December during breeding soundness examinations (BSE) of mature Composite-IV rams over a three-year study. A portion of rams were evaluated in multiple years and data from a total of 129 collections on 90 rams were available for analyses. Semen parameters were estimated by computer-assisted sperm analysis (CASA). Imaging software was used to quantify the cross-sectional area of five vessels per testis and average areas of the left testis (AAL), right testis (AAR), and both testes (AAT) were calculated. The correlation between AAL and AAR measured on the same ram within a year was moderate and positive (0.51; P< 0.001). However, correlations between AAL, AAR, and AAT measured on the same ram in consecutive years were not different from zero. Average vessel area was analyzed with fixed effects of testis side (AAL or AAR), year (2020, 2021, or 2022), ram age (2, 3, or ≥ 4 yr-old) and a random effect of ram. Across years, AAL was 23 to 25% greater than AAR and 16 to 18% greater in ≥ 4-yr-old than younger rams (P< 0.01). Unilateral varicocele (UV) was diagnosed when AAL/AAR or AAR/AAL ratio exceeded 1.5 in a ram and, for rams sampled in multiple years, those diagnosed in one year were considered affected for all subsequent years. Within year, prevalence of UV ranged from 16 to 36%. Scrotal circumference (SC) and CASA parameters were analyzed with fixed effects of year, ram age, and UV status and a random effect of ram. Within 2- and 3-yr-olds, sperm from UV rams had lower motility than unaffected rams (42.1 ± 6.7% vs 62.2 ± 4.1% and 47.8 ± 7.4% vs 70.1 ± 4.7%, respectively; P≤ 0.02) but no differences were observed within 4-yr-old rams. Across ages, sperm from UV rams had a lower percentage of normal than unaffected rams (91.5 ± 1.1% vs 94.3 ± 0.74%; P= 0.03). Sperm concentration, progressive motility, and SC were unaffected by UV status. Future studies will evaluate the effect UV status on siring capacity. Ultrasound methodologies previously refined to diagnose varicocele in boars helped identify sub-fertile rams that would not have been classified by routine BSE procedures. However, these methodologies would benefit from concurrent machine learning and software development to ensure imaging consistency across technicians and streamline procedures for real-time decision making. USDA is an equal opportunity provider and employer.

One-carbon metabolite supplementation during in vitro embryonic development improves blastocyst quality; however, our understanding of the effects of combining these metabolites during in vitro maturation (IVM) is limited. Supplementation with follistatin improved oocyte quality following IVM. Choline supplementation was beneficial during early embryonic development; however, the role of choline during oocyte maturation is unknown. The objective of this study was to investigate the combination of choline and follistatin during IVM on oocyte quality. We hypothesized that supplementation of choline with follistatin would synergistically improve oocyte quality. Follicles (2 to 7mm) were aspirated from slaughterhouse ovaries to obtain cumulus oocyte complexes for IVM that were treated with choline (0, 1.3, or 1.8 mM) and follistatin (0 or 10 ng/mL) in a 3 x 2 design for 23 hr and visually graded for maturation. Cumulus cells and oocytes were used for qPCR transcript abundance analysis (cumulus cells: CHPT1, CX43, EFGR1, FST and STAR; oocytes: BMP15, GDF9, CEPT1, CPBE4, MTHFR, NANOS, CX43 and COX2). Oocyte maturation was analyzed using the GLIMMIX procedure of SAS and transcript abundance was analyzed using the GLM procedure of SAS with the main effect of choline, follistatin and the interaction as fixed effects. There were no differences in the visual characteristics of the oocytes during maturation (P> 0.1). No differences in transcript abundance were observed with choline or follistatin in the cumulus cells (P> 0.1). Addition of 1.3 mM choline during IVM may increase CPEB4 (P= 0.1) in oocytes compared with controls. Transcript abundance of CEPT1 tended to be reduced in oocytes supplemented with 1.8 mM choline and follistatin compared with control oocytes (P= 0.07). Incorporation of follistatin with 1.8 mM choline supplementation during IVM tended (P= 0.08) to reduce CPEB4 in oocytes compared with controls. Other markers of oocyte quality (BMP15, GDF9, NANOS, CX43 and COX2) were not different between choline or follistatin. In contrast to the hypothesis, the combination of choline and follistatin appeared to reduce overall in vitro oocyte quality. While no statistical differences were observed for the other markers, it appears that either 1.3 mM choline or follistatin supplementation may provide a beneficial impact on oocyte quality. Choline 1.3 mM supplementation may improve cell cycle progression from MI to MII phase through the up-regulation of CBEP4. This further supports the conclusion that choline is beneficial for in vitro oocyte development. Although follistatin appears to antagonize the actions of choline during oocyte maturation, they target different mechanisms (TGFβ signaling and DNA methylation, respectively) and both provide long-term benefits during in-vitro embryonic development. Further research is, therefore, warranted to investigate the antagonizing relationship between choline and follistatin during IVM and long-term impacts on blastocyst development. USDA is an equal opportunity provider and employer.

Interest is growing in methods to apply precision nutritional supplements strategically during the peri-conceptual period to enhance progeny performance of beef females. Studies where supplemental methionine (MET) was provided to bovine females in the peri-conceptual period demonstrated epigenetic modifications in muscle of the progeny. Guanidinoacetic acid (GAA) has been fed directly to livestock to increase creatine synthesis, but has not been evaluated for use in fetal programming. We fed MET and GAA to cross-bred heifers during the peri-conceptual period as a 2 x 2 factorial design to influence carcass characteristics of their steer calves. It was, therefore, important to determine if the interactions of dietary MET and GAA in the peri-conceptual period would alter reproductive efficiency. The null hypothesis was that these supplements would not alter reproductive efficiency of beef heifers when fed in the peri-conceptual period. Heifers were divided into four breeding groups for management purposes. On day -63, heifers (n = 20/diet, age = 384.1 ± 1.1 d) were assigned HeatTime collars and individually fed MET (10 g/d), GAA (40 g/d), the combination (MET + GAA), or ground corn carrier (CON) for 126 days. On day -9, heifers received 100 mg of gonadotropin releasing hormone and a CIDR. On day -2, the CIDR was removed and luteolysis was initiated with 25 mg of prostaglandin F2a. All heifers were inseminated with male sexed semen from a single sire and administered a second 100 mg injection of gonadotropin releasing hormone on day 0. On day 63, a blood sample was collected from all pregnant heifers (CON n = 10; MET n = 8; GAA n = 7; MET + GAA n =10) to evaluate circulating progesterone concentrations. Reproductive tracts were collected to determine CL weight, gravid uterine weight, and placentome number. Data were analyzed using the MIXED procedure of SAS with group, MET, GAA, and the interaction of MET and GAA as fixed effects. Days to first estrus were greater and number of estrus events prior to synchronization were fewer in CON heifers compared with supplemented heifers (P< 0.05). Luteal weight tended to be greater in heifers supplemented with MET compared with CON and GAA heifers (P= 0.09), but peak intensity of estrus, concentration of progesterone at day 63 of pregnancy, gravid uterine weight, and placentome number did not differ due to diet (P> 0.10). Supplementation with MET or GAA may improve onset of puberty but does not negatively alter reproductive performance in cross-bred beef heifers. These supplements can be used for fetal programming of muscle development in steer progeny without negatively impacting reproductive performance in the breeding herd. USDA is an equal opportunity provider and employer. Funded in part by USDA-NIFA-AFRI # 2022-67015-36196.

Use of growth promoting implants in beef heifers in Southwestern rangelands has been reported to improve growth, however, the effect on ovarian parameters is unknown. Therefore, the objective of this study was to investigate the influence of level of supplementation when heifers were administered a second Synovex-C implant at weaning on ovarian parameters. At two months of age all heifers (n = 17) were administered a Synovex- C implant. At weaning (8-months old), heifers were stratified by body weight and antral follicle counts and assigned to receive one of two levels of supplementation (High or Low) and Implant (No Implant or Implant). Heifers assigned to Implant were administered a second Synovex-C implant at weaning, thereby creating four treatment groups: High Implant (n = 4), High No Implant (n = 4), Low Implant (n = 5), and Low No Implant (n = 4). Heifers were utilized in a complete randomized block design with a 2x2 factorial. Heifers were individually fed supplement three times a week for 26 weeks with refusals being weighed after each feeding to determine intake. For the first 4 weeks of the study, High and Low heifers were offered 4.54 kg/h/d and 2.72 kg/h/d, respectively, of 20% CP cube. After acclimation High and Low heifers were offered 2.95 kg/h/d and 1.59 kg/h/d, respectively, of 32% CP cube for the remainder of the study. At the end of the supplementation period heifers underwent bilateral ovariectomies. Weight, height, and length of each ovary was recorded. Surface antral follicles were counted on both ovaries and recorded. Height and length of the largest preovulatory follicle was recorded and follicular fluid was collected for analysis of estradiol and progesterone concentrations. Ovarian and follicular measurements were analyzed using MIXED procedures in SAS. Ovarian weight (P≥ 0.16), ovarian length (P≥ 0.46), or ovarian height (P≥ 0.32) were not influenced by implant, supplement level, or their interaction. There was no difference among total surface antral follicle count between implant (No Implant: 34.2 ± 4.3 vs Implant: 40.0 ± 4.6), supplement level (High: 32.75 ± 4.6 vs Low: 41.43 ± 4.3), and their interaction (P≥ 0.19). Preovulatory follicle diameter was not impacted by implant (No Implant: 10.82 ± 1.27 mm vs Implant: 9.42 ± 1.27 mm), supplement level (High: 10.48 ± 1.27 mm vs Low: 9.77 ± 1.27 mm), or their interaction (P≥ 0.45). Implant, supplement level, or their interaction did not influence progesterone (P≥ 0.43), estradiol (P≥ 0.42), or the estradiol:progesterone ratio (P≥ 0.41) in follicular fluid. In conclusion, administration of a second Synovex-C implant at weaning did not impact ovarian parameters or the composition of follicular fluid. USDA is an equal opportunity provider and employer.

Recent research reported changes in fetal growth due to increased methyl donor availability through periconceptual supplementation of the dam. We hypothesized that maternal supplementation of methionine (MET) or guanidinoacetic acid (GAA) during the periconceptual period will result in a methyl donor surplus or deficiency in maternal serum and ultimately influence fetal body and organ weight at d +63 gestation. The objectives of this study were to evaluate effects of methyl donor surplus or deficiency during the periconceptual period of gestation on metabolite concentrations in the heifer and the effects on early fetal development. Eighty MARC II heifers (n = 20 per treatment, initial BW = 346 ± 8 kg) were stratified by age and weight to one of four treatment groups to receive 100 g of supplement: ground corn carrier as control (CON), MET (10 g/d) in ground corn, GAA (40 g/d) in ground corn, and MET + GAA (10 g/d MET + 40 g/d GAA) in ground corn. Supplementation began 63 d before breeding (d –63) until d 63 of gestation (d +63), and serum samples were collected before feeding on d -63, at breeding, and d +63. Heifers were bred using male sexed semen from the same sire, and 35 heifers were confirmed pregnant (CON, n = 10; MET, n = 8; GAA, n = 7; MET + GAA, n =10) and harvested at d +63 of gestation to collect maternal and fetal samples. Data were analyzed as a 2 × 2 factorial design with 2 levels of MET and 2 levels of GAA. Methionine concentrations in maternal serum were greater (P= 0.05) in MET and MET + GAA supplemented heifers at d 0 and d +63 compared with CON and GAA at d -63, d 0, and d +63. There were no differences (P≥ 0.15) in the concentration of GAA or creatine due to supplementation; however, both were decreased at breeding (P <0.01) compared with d -63 and d +63. Fetuses from MET supplemented heifers had greater (P= 0.01) brain weight and Longissimus muscle than dams not receiving MET. We conclude that MET supplementation during the periconceptual period can alter methyl donor availability and increase fetal muscle development. USDA is an equal opportunity provider and employer.

The biological role of kisspeptin in the reproduction of swine has been far less studied than in other species. In the porcine hypothalamus, kisspeptin is coexpressed with neurokinin B and dynorphin in KNDy neurons. Although kisspeptin-stimulated secretion of gonadotropin hormones in gilts was established early, increased expression of hypothalamic KISS1 was only recently confirmed to be associated with puberty and cyclicity in pigs. Gene knockout (KO) models to study kisspeptin in livestock did not exist until TALEN gene editing technology was used to knockout KISS1R in pigs, resulting in a hypogonadotropic-hypogonadal (HH) phenotype that caused pubertal failure. Little is known about the reproductive physiology of KISS1R KO pigs, except that exogenous follicle-stimulating hormone and gonadotropin-releasing hormone only partially restored gonadal development in boars. To further explore the biological function of kisspeptin and its receptor in swine, we recently used CRISPR/cas9-ribonucleoproteins to knockout KISS1 in pigs (10.3389/fgene.2022.1078991). A total of 684 zygotes were injected resulting in 69 piglets with 58% bearing KISS1 KO alleles (KISS1-/-). The proportion of KISS1-/-alleles did not affect BW or ADG in these mosaic founder pigs. Two boars and 2 gilts with complete KISS1 KO exhibited HH and failed to become pubertal. Germ and Sertoli cells were disorganized in small seminiferous tubules. Mating mosaic F0 pigs (frequency of KISS1-/-, 35 to 88%) resulted in 170 F1 piglets with KISS1-/-alleles observed in a Mendelian frequency. These KISS1-/-pigs displayed normal BW growth during development but were also HH. Ovarian development of KISS1-/-gilts was arrested at a juvenile state, although antral follicles were present. The KISS1-/-boars lacked testicular development and had dramatically reduced concentrations of androgens and skatole, suggesting the lack of boar taint in their meat. Importantly, KISS1 and KISS1R are expressed in ovarian, uterine and testicular tissue; however, the direct role of kisspeptin in these reproductive organs remains unknown in livestock. These KISS1-/-pigs provide a unique animal model that opens new avenues for understanding the role of kisspeptin and gonadotropin hormones in regulation of reproductive development and function in pigs as well as livestock in general. These animals facilitate our ability to explore questions such as: 1) how KNDy neurons function and control gonadotropin pulses or 2) what role does KISS1 play in the development and function of the ovary, uterus and testis in domestic animals. Some of these questions have heretofore only been addressable with difficult surgical modifications, and therefore, KISS1-/-pigs represent an alternative to painful procedures in livestock research. Pursuing these fundamental studies is expected to lead to methods that restore fertility in KISS1-/-pigs. This, in turn, could provide new possibilities for the elimination of surgical castration from pork production. The USDA is an equal opportunity provider and employer.

Recent research has demonstrated that feeding rumen protected methionine (MET) products increases circulating methionine while feeding guanidinoacetic acid (GAA) may result in a methyl deficiency. Therefore, we hypothesized that maternal MET or GAA supplementation from 63 days before breeding until d 63 of gestation would increase fetal hepatic methyl donors or result in a fetal hepatic methyl donor deficiency, respectively. Eighty MARC II heifers (n = 20 per treatment; initial BW = 344 ± 8 kg; day -63) were weighed and assigned to one of four breeding groups and four treatments stratified by age and starting weight. Heifers were fed a total mixed ration to gain 0.68 kg/d and received 100 g of one of four treatments top dressed: control supplement of ground corn (CON), MET (10 g/d) in a ground corn carrier, GAA (40 g/d) in a ground corn carrier, or MET + GAA (10 g/d MET + 40 g/d GAA) in a ground corn carrier. At breeding (d 0), all heifers were bred to a single sire using male sexed semen. At d 63 of gestation, 35 heifers (CON n = 10, MET n = 8, GAA n = 7, MET + GAA, n = 10) confirmed pregnant with bull calves were humanely slaughtered and fetal liver tissues were collected and sent to Metabolon Inc. for global metabolomics analysis. Data were analyzed as a 2 x 2 factorial with 2 levels of GAA and 2 levels of MET with breeding group included in the model. For this abstract, only metabolites from the methionine cycle and adjacent pathways will be reported and only metabolites with significant P-values will be discussed (P≤ 0.05). There were no main effects or interactions for the abundance of methionine, GAA, creatine, SAM, SAH or homocysteine. Glycine was decreased and sarcosine increased in fetuses from MET supplemented dams. Serine was affected by a MET x GAA interaction being decreased in livers of CON, MET, and MET + GAA fetuses compared with GAA. Cystathione, S-methylcysteine, s-methylcysteine sulfoxide, and hypotaurine were greater in fetuses of MET supplemented heifers and pyridoxal phosphate was reduced in fetal liver of MET supplemented heifers. The abundance of urea was greater in GAA supplemented fetuses. These results indicate that fetuses from dams fed methionine may be able to maintain balance in methionine supply by shunting methionine through adjacent pathways including the transsulfuration pathway. We also interpret these data to imply that excess methionine is not utilized by the fetus and is metabolized to products that can be excreted. Lastly, GAA did not alter the fetal methyl supply. USDA is an equal opportunity provider and employer. Funded by (USDA-NIFA-AFRI # 2022-67015-36196).

Follicular progression during peripuberty is affected by diet. Vascular endothelial growth factor A (VEGFA) induces follicle progression in many species; however, there are limited studies to determine if diet may alter the effects of angiogenic VEGFA165-stimulated follicle progression or antiangiogenic VEGFA165b follicle arrest. We hypothesized that diet affects the magnitude of angiogenic and antiangiogenic VEGFA isoform actions on follicular development through diverse signal transduction pathways. To test this hypothesis, beef heifers in our first trial received Stair-Step (restricted and refeeding) or control diets from 8 to 13 months of age. Ovaries were collected to determine follicle stages, measure vascular gene expression and conduct ovarian cortical cultures. Ovarian cortical cultures were treated with phosphate-buffered saline (control), 50 ng/ml VEGFA165, VEGFA165b, or VEGFA165 + VEGFA165b. The Stair-Step heifers had more primordial follicles (P < 0.0001), greater messenger RNA abundance of vascular markers VE-cadherin (P < 0.0001) and NRP-1 (P < 0.0051) than controls at 13 months of age prior to culture. After culture, VEGFA isoforms had similar effects, independent of diet, where VEGFA165 stimulated and VEGFA165b inhibited VEGFA165-stimulated follicle progression from early primary to antral follicle stages. In vitro cultures were treated with VEGFA isoforms and signal transduction array plates were evaluated. VEGFA165 stimulated expression of genes related to cell cycle, cell proliferation, and growth while VEGFA165b inhibited expression of those genes. Thus, VEGFA isoforms can act independently of diet to alter follicle progression or arrest. Furthermore, follicle progression can be stimulated by VEGFA165 and inhibited by VEGFA165b through diverse signal transduction pathways.

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 ± 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 B12injection. 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.This study determined that disrupted one carbon metabolism negatively affects multiple physiological systems; however, supplementation strategies that do not negatively impact one-carbon metabolism will not negatively impact the heifer.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.

Developmental programming became an area of interest to understand negative environmental impacts on progeny performance. Recently, the concept that we may be able to harness developmental programming to target animals to their niche in the production system has gained recognition. Female fertility is an area where developmental programming has been moderately successful; however, the mechanisms remain unclear. Although some studies have demonstrated differences in gonadal development and attainment of puberty in response to developmental programming, these have not translated to improved fertility. To improve response to developmental programming, it is critical to identify factors that contribute to inconsistencies across studies.

Preovulatory estradiol is known to impact embryo quality and survival. The objective of this study was to determine the effects of preovulatory estradiol on the uterine environment and conceptus survival through maternal recognition of pregnancy. Beef cows/heifers were AIed following induced ovulation. Cows were grouped into high and low preovulatory estradiol. Conceptuses were collected on day 16 nonsurgically (Rep 1; n = 20), or following slaughter (Rep 2; n = 29). Blood was collected to determine plasma glucose concentrations, and uterine luminal fluid (ULF) was analyzed for protein, glucose, and interferon tau (IFNT) concentrations. Total cellular RNA was extracted from caruncular (CAR) and intercaruncular (INCAR) endometrial tissue. There was no effect of preovulatory estradiol on conceptus recovery rate (P= 0.38) or on apoptosis rate in the trophectoderm (P= 0.64). Cows in which a conceptus was recovered had greater concentrations of protein in the ULF (P= 0.04). Animals with elevated preovulatory estradiol had greater endometrial abundance of SLC2A1 (P= 0.05) and SLC5A1 (P= 0.04) in both INCAR and CAR tissue. Presence of a conceptus also tended to increase (P= 0.10) abundance of SLC5A1 in INCAR. In CAR tissue, cows with a conceptus had decreased SLC2A4 abundance (P= 0.05). In summary, conceptus recovery rates, apoptosis in the trophectoderm, IFNT, glucose, and protein concentration in ULF did not differ between cows that did or did not have an increase in preovulatory estradiol concentrations. Thus, there is no indication of increased conceptus survival to day 16 of pregnancy based on estradiol concentrations.Conceptus survival to d 16 was similar among cows with and without elevated preovulatory estradiol; however, glucose transporter expression, and glucose and protein concentration in uterine fluids were influenced by estradiol and conceptus presence.

There are differences between cows and ewes in ovarian reserve parameters. No major genes influencing follicle numbers have been identified in cows, whereas several genes have variants reported to influence follicle numbers in ewes. In contrast, antral follicle count is associated with fertility in cows, and we reported that beef cows that failed to conceive in 2 consecutive years had fewer antral follicles and smaller reproductive tracts than cows that had never failed to conceive. In the current study, we tested the hypothesis that ewes that had failed to lamb (BARREN) in 4 consecutive annual breeding seasons of 21– 35 d with rams that had passed a breeding soundness exam have fewer follicles and smaller reproductive tracts than ewes that consistently produced lambs (FERTILE) under the same breeding protocols. BARREN ewes (n = 19) were paired by breed, age, and sire to a FERTILE control ewe (n = 19) and reproductive tracts were recovered at necropsy. All visible antral follicles in both ovaries were counted and a representative cross section of one ovary was embedded for histological evaluation of primordial follicle numbers. The reproductive tract was weighed, and ovulatory status was evaluated. Paired t-tests indicated that BARREN ewes had 25.6 ± 5.0 fewer antral follicles per pair of ovaries, 17.3 ± 4.4 fewer primordial follicles per histological section, and 54.5 ± 18.7 g lighter reproductive tracts than FERTILE ewes (P≤ 0.01). The odds of FERTILE ewes being ovulatory at necropsy were 2.25 that of BARREN ewes, but this did not differ from expectation by McNemar’s test (P= 0.13). The results indicate that size of the ovarian reserve influences fertility in ewes as it does in cows. USDA is an equal opportunity provider and employer.

Mammalian females are born with a finite supply of follicles within their ovaries that may be related to fertility and reproductive longevity. The number of microscopic follicles in the ovarian cortex is positively correlated to the number of antral follicles. In cattle, the number of antral follicles can be determined by ultrasonographic examination of the ovaries, is repeatable within a female, and has a moderate heritability. Studies in rodents suggest that nutritional status early in life can alter structure and function of the ovarian reserve, resulting in changes in follicle numbers, estrous cycle parameters, and reproductive longevity. This raises questions about how early life events might influence the ovarian reserve and reproductive performance in bovine females. A central hypothesis of our research is that the number of follicles that a heifer has when entering her first breeding season is influenced by external factors such as maternal nutrition during pregnancy, maternal stress during pregnancy, peri-pubertal nutrition, and health events. Understanding how these factors influence the structure and function of the ovarian reserve in replacement heifers may allow us to translate this knowledge into management practices that developmentally program the bovine ovary prior to the first breeding season to improve reproductive performance in the cow herd. Accomplishing this is made difficult because our understanding of the mechanisms controlling pre-antral follicle development in cattle is limited and growth of pre-antral follicles is occurring at the microscopic level within the ovarian cortex where it is difficult to monitor changes in real time. There has been some success, but variation in response across studies indicates that further research to understand the causes of this variation is required before we will be able to apply developmental programming in beef production systems to improve ovarian function in replacement heifers. USDA is an equal opportunity provider and employer.

The objective of this study was to determine the dose of folate and vitamin B12in beef heifers fed rumen protected methionine and choline required to maintain increased B12levels and intermediates of the methionine-folate cycle in circulation. Angus heifers (n = 30; BW = 392.6 ± 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 B12concentrations 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 MAT2Aand MAT2Bwere 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 B12and 320 mg folate once weekly to maintain increased concentrations of B12and 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.This study determined the optimal doses for vitamin B12(20 mg injected weekly) and folate (320 mg injected weekly) injection to beef heifers during the periconceptual period when rumen protected methionine and choline were fed at 0.08% of dry matter and 60 g/d, respectively. These doses set the basis upon which future studies can be based when feeding and injecting one-carbon metabolites.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 B12and folate at 20 mg and 320 mg once per week, respectively resulted in sustained elevated concentrations of one-carbon metabolites.

Transcript abundance of two forms of GnRH and its receptor have been characterized in bovine ovaries. The objective was to investigate the relationship of GnRH-1, GnRH-2, GnRH-R, intrafollicular estradiol (E2) and progesterone (P4) during follicular development. Ovaries were collected from beef cows at specific stages of follicular development [pre-selection (PRE, n = 9), post-selection (POST, n = 9), and post-selection 24h after luteal regression (PG, n = 9)]. The largest follicle per stage was aspirated to obtain granulosa cells (GC) and follicular fluid (FF). Total cellular RNA was extracted from GC and RT-PCR was performed for GnRH-1, GnRH-2, GnRH-R and GAPDH. Radioimmunoassays were performed to determine FF concentrations of E2 and P4. Data were analyzed using the MIXED and REG procedures in SAS. There was no difference (P ≥ 0.23) in mRNA abundance of GnRH-2, GnRH-R or FF concentrations of P4 across follicular stages. There was an effect of stage on FF E2 (PRE: 17,925±20,273, POST: 28,458±21,503, and PG: 252,616±21,503 pg/mL; P < 0.01). Stage affected GnRH-1 mRNA abundance (PRE: 2.28±0.55, POST: 0.92±0.55, and PG: 0.11±0.55; P = 0.03). There was no relationship of GnRH-1 and GnRH-2 mRNA abundance or effect of FF P4 on GnRH-R mRNA abundance (P ≥ 0.23). There was no effect of FF P4 on GnRH-1 mRNA abundance (P = 0.68). There was no effect of FF E2 on GnRH-2 mRNA abundance (P = 0.66). As FF E2 increased GnRH-R mRNA abundance tended to increase (P = 0.10;r2=0.13). As FF P4 concentrations increased GnRH-2 mRNA abundance tended to decrease (P = 0.09;r2=0.12). As FF E2 increased GnRH-1 mRNA abundance decreased (P = 0.02;r2=0.20). In conclusion, there were differences in peptide and receptor mRNA abundance of GnRH in relation to FF E2 and P4 at specific stages of follicular development. This is supportive of a regulatory relationship between ovarian GnRH and steroidogenesis. This work is supported by AFRI Grant No.2018-67016-27578 from USDA. USDA is an equal opportunity provider and employer.

Management strategies utilized during pre-breeding development of replacement heifers can impact fertility and the ovarian reserve. Angus-Hereford crossbred heifers (n= 233) were utilized over a 3-yr period to determine the effects of administration of a growth promoting implant at either branding or weaning on growth, reproduction, and ovarian development. Heifer calves were randomly assigned to one of three treatments: 1) nonimplanted controls (CON; n= 79), 2) implanted at approximately 2 mo of age (average calf age = 58 d) with Synovex-C (BIMP, n= 82), or 3) implanted at approximately 7 mo of age (average calf age = 210 d) with Synovex-C (WIMP; n= 72). In years 2 and 3, a subset of heifers (year 2 n= 16; year 3 n= 14) were unilaterally ovariectomized. Heifers implanted at 2 mo of age were heavier at weaning, yearling (mid-February; average calf age = 332 d), and at the beginning of the breeding season (P< 0.01) compared to CON and WIMP heifers. Average daily gain (ADG) was similar among treatments from weaning to yearling and weaning to the start of the breeding season (P≥ 0.61); however, WIMP heifers had increased (P= 0.05) ADG from yearling to the start of the breeding season compared to BIMP heifers. Antral follicle count and reproductive tract scores were not influenced by implant treatment (P≥ 0.18). Response to synchronization of estrus was increased (P= 0.02) in WIMP compared to CON heifers, with BIMP heifers similar to all other treatments. First service conception rates tended to be increased (P= 0.09) in CON heifers compared to WIMP heifers, with BIMP heifers similar to CON and WIMP. Final pregnancy rates were similar (P= 0.54) among treatments. A treatment × yr interaction was detected (P= 0.01) for the number of primordial follicles/section with increased primordial follicles in WIMP heifers in year 3 compared to BIMP and WIMP heifers in year 2 and CON heifers in year 3, as well as in BIMP compared to WIMP heifers in year 2. Utilization of growth promoting implants did not negatively impact postweaning reproductive development or compromise pregnancy rates in beef heifers. Based on these results, administration of a growth promoting Synovex-C implant at 2 mo of age may allow for increased body weight at weaning, without hindering reproductive performance.Management of beef females during the first year of life can impact fertility and reproductive longevity. Cattle producers can improve calf weight gains by using growth promoting implants; however, to be applicable, they must not negatively impact heifer reproductive performance or development. Understanding the impact of growth promoting implants on growth, fertility, and reproductive development is important to determine if they can be utilized as an effective management strategy in heifers intended to be retained in the breeding herd. To determine if growth promoting implants influence fertility, 233 heifer calves either received no implant, a Synovex-C implant at 2 mo of age, or a Synovex-C implant at 7 mo of age. Implanting heifers at 2 mo of age increased body weight at weaning. Implanting heifers at 7 mo of age did not improve body weight gains. Implanting heifers at 2 or 7 mo of age resulted in similar pregnancy rates. By using a growth promoting implant at 2 mo of age in beef heifers, producers may be able to increase heifer weaning weight without negatively affecting reproductive development or pregnancy rates. Additional body weight at weaning may provide a profit advantage for heifers not retained as replacements.Growth promoting implants may be used to improve weight gain in replacement heifers; however, there may be an influence of timing of implant administration on reproductive performance. Utilization of a Synovex-C implant at approximately 2 mo of age can effectively increase average daily gain without negatively impacting fertility or reproductive tract development, providing producers additional management strategies and the opportunity to market heifers not retained as replacements at an increased body weight.

Colostrum intake by neonatal piglets can be measured using the immunoglobulin immunocrit assay (iCrit). Lactocrine effects occur when maternally derived, milk-borne bioactive factors are transferred to the neonatal circulation with consumption of colostrum during nursing and affect development of somatic tissues, which can have long-term consequences in adulthood. Lactocrine deficiency, indicated by low neonatal iCrit, altered uterine gene expression and reduced fecundity in adult, neonatally lactocrine-deficient gilts. Litter size in pigs is dependent on both ovarian and uterine function. It was hypothesized that lactocrine deficiency affects development of ovarian follicles in gilts. The objective was to determine the number of primordial, primary, and secondary follicles in ovaries of gilts with high (12% ± 0.5; n = 10) or low (1.9% ± 0.4; n = 10) iCrit, determined on postnatal day (PND) 1 after birth. Paired high- and low-iCrit gilts were chosen from the same litters (birth weight; 3.1 ± 0.2 lbs). On PND 14, ovaries were collected and histological sections prepared (3 sections per animal; 30–150 µm apart). Ovarian follicles in each section were staged and the number of follicles in each category were quantified and subjected to ANOVA. Total number of ovarian follicles did not differ with iCrit (P = 0.55; 1,370.6 ± 147.8 follicles per section). The proportion of primordial, primary, and secondary follicles was 89.6 ± 1.15%, 7.7 ± 0.87%, 2.7 ± 0.51%, respectively. The number of primordial (P = 0.55), primary (P = 0.64), and secondary (P = 0.93) follicles did not differ with iCrit. Results indicate that ovarian follicular development of neonatal gilts is not sensitive to immunocrit status. Although lactocrine deficiency did not influence the ovarian follicular profile at PND 14, it remains unknown whether lactocrine programming alters ovarian follicular dynamics in neonatally lactocrine-deficient adults. USDA is an equal opportunity provider and employer.

The hypothesis was that long-term treatment of cattle with estradiol (E2) and bovine somatotropin (bST) would alter the earliest stages of folliculogenesis. Nonlactating Holstein cows (n = 26) were treated in a 2 × 2 arrangement with E2(2 × 24 mg implants, 67.1 ± 1.4 days) and bST (Posilac, 63.6 ± 1.5 days). At Day 67 ± 1.3, one ovary was removed for morphometric and immunohistochemical analysis. For each ovary, 388 ± 38 microscopic fields (2 × 2 mm) were examined and follicles within each field were classified by histological stage. Fields that contained no follicles were classified as empty. Empty fields (n = 100 per ovary) were further classified as containing no evidence of follicles or containing atretic remnants of follicles. Approximately 30 4-μm sections per ovary were stained for proliferating cell nuclear antigen (PCNA), and 150 fields per ovary were evaluated. Additional sections (n = 10 per ovary) were assessed immunohistochemically for apoptosis, and fluorescence intensity was determined for each follicle. Treatment with bST significantly decreased percentage of empty fields containing atretic remnants. Treatment with E2induced activation of follicles as shown by a decrease in percentage of primordial follicles and an increase in percentage of primary follicles as determined by PCNA staining. At the primary follicle stage the combination of bST + E2decreased apoptosis as shown by decreased fluorescence intensity. Thus, E2induced activation of follicles, bST enhanced survival, and the combination lowered atresia.

Increased numbers of antral follicles are associated with greater fertility and a uterine environment that is more supportive of early embryonic development in beef heifers. Glucose is a primary energy source for embryos and glucose concentrations are elevated in uterine luminal fluid (ULF) of pregnant heifers. We hypothesized that ULF glucose concentrations and endometrial transcript abundance for glucose transporters at d16 after insemination would be greater in heifers with increased numbers of antral follicles. Heifers classified with either increased (32 ± 1.1) or diminished (14.7 ± 1.1) antral follicle counts were selected and artificially inseminated following the Select Synch protocol (d0). At d16 after insemination, heifers were sent to the abattoir and reproductive tracts were collected to retrieve conceptuses to determine pregnancy. Uterine luminal fluid was collected, the endometrium was biopsied, total RNA was extracted and glucose transporter transcript abundance was determined. Data were analyzed using the MIXED procedure of SAS with antral follicle group, pregnancy status, and the interaction as fixed effects. Glucose concentrations in ULF were greater (P < 0.05) in heifers with increased antral follicle numbers compared to heifers with diminished numbers (122.65 ± 11.91 vs 84.12 ± 12.42 mg/dL). Glucose ULF concentrations were increased (P < 0.05) in pregnant vs. non-pregnant heifers (124.84 ± 12.81 vs 81.93 ± 11.50 mg/dL). Endometrial glucose transporter 1 (GLUT1) transcript abundance was increased in pregnant heifers (P < 0.01) but was not different due to antral follicle number or the interaction. Therefore, differences in glucose concentrations associated with antral follicle number may be due to differences in GLUT1 transcription before d16 or due to differences in protein abundance or functionality. Taken together, heifers with increased number of antral follicles may have increased energy availability in the uterus for trophoblast proliferation and function. USDA is an equal opportunity provider and employer.

Calves from transported dams had greater concentrations of plasma cortisol when restrained and cleared plasma cortisol at a slower rate than calves from non-transported dams. Considering this hypothalamic-pituitary-adrenal axis effect, investigation of other parameters influencing reproduction is warranted in offspring exposed to prenatal transportation stress. The purpose was to determine impact of prenatal transportation stress on offspring ovarian follicle count. Brahman cows were transported for 2 h on d 60, 80, 120, and 140 (± 5 d) of gestation. Offspring from transported (Stressed, n = 19) or non-transported (Control, n = 15) dams were slaughtered at 5 yr (Replication 1, n = 14) or ovariectomized at 8 yr (Replication 2, n = 20). A cross-section of ovary was collected, serially sectioned, and stained. Numbers of total, primordial, primary, secondary, and antral follicles were determined per section. Total ovarian follicle count for each stage was calculated using ovary dimensions. The MIXED procedure of SAS was used to analyze ovarian follicle count with treatment, replicate, and the interaction as fixed effects. Total ovarian follicle count decreased with age (P < 0.01; R1 = 383,663, R2 = 154,560); however, there was no difference in total ovarian follicle count between offspring due to treatment (P = 0.17; S = 221,813, C = 316,409). Similarly, there was no difference in primordial (P = 0.22; S = 127,300, C = 188,304), primary (P = 0.28; S = 58,610, C = 77,237), or antral (P = 0.48; S = 23,202, C = 28,695) follicle count between offspring due to treatment. Fewer secondary follicles were observed in Stressed offspring compared to Control offspring (P = 0.03). These results suggest that the ovarian follicular reserve, AFC, and potential fertility of cows may not be impacted by exposure to prenatal transportation stress. USDA is an equal opportunity provider and employer.

The relationship of ovarian reserve parameters to fertility in mammalian females continues to be debated. We demonstrated improved uterine function, creating an environment that is more supportive of early embryonic development in beef heifers with increased numbers of follicles. In the present study we hypothesized that timing of luteolysis differs between heifers with increased compared to heifers with diminished numbers of follicles. Angus heifers (n = 20/ group) were classified as low (14.7 ± 1.1) or high (32 ± 1.1) antral follicle number based on ovarian ultrasonography, and artificially inseminated (d0) following a Select Synch protocol. At d16 after insemination, heifers were slaughtered, and reproductive tracts collected. Tracts were flushed with 20 mL of physiological saline to determine pregnancy status by presence of a conceptus and endometrial samples from the horn ipsilateral to the CL were frozen for determination of transcript abundance. Real-time RT-PCR was performed to determine relative transcript abundance of oxytocin receptor (OXTR) and interferon-stimulated gene-15 (ISG15) with glyceraldehyde-3-phospate dehydrogenase as the endogenous reference gene. Transcript abundance was analyzed using the MIXED procedure of SAS with follicle group, pregnancy status and the interaction as fixed effects. Transcript abundance of ISG15 was greater in pregnant endometrium than non-pregnant endometrium (2.75 ± 0.25 vs 0.16 ± 0.22-fold; P < 0.0001) with no influence of follicle group or the interaction. The interaction of follicle group and pregnancy status influenced OXTR transcript abundance (P = 0.06). Luteolytic mechanisms were activated in non-pregnant heifers with diminished numbers of follicles (1.46 ± 0.25-fold) and were not activated in pregnant heifers (0.06 ± 0.22-fold) or heifers with increased numbers of follicles that were not pregnant (0.32 ± 0.30-fold). These data indicate a wider window of recognition of pregnancy in beef heifers with increased numbers of ovarian follicles. USDA is an equal opportunity provider and employer.

The climate simulations from atmospheric general circulation models (GCMs) are often used to analyze the potential effects of climate change on environmental resources. It has been demonstrated that there are differences among the simulations from various GCMs, on spatial scales ranging from global to regional. This paper quantifies the differences in temperature and precipitation simulated by three major GCMs for four specific regions: an agricultural region (the North American winter wheat belt), a hydrologic region (the Great Basin), a demographic region (the high-density population corridor of the northeast United States), and a political region (the state of Texas). Both the current (control) climate and the climatic response to a doubling of atmospheric carbon dioxide (CO2) are considered

Rapid changes in flow below hydroelectric facilities result from peaking operations, where water is typically stored in a reservoir at night and released through turbines to satisfy increased electrical demand during the day. Potential impacts of these short-term, recurring disturbances of aquatic systems below dams are important considerations in hydropower development. Reduced biotic productivity in tailwaters may be due directly to flow variations or indirectly to a variety of factors related to flow variations, such as changes in water depth or temperature, or scouring of sediments. Many riverine fish and invertebrate species have a limited range of conditions to which they are adapted. The relatively recent pattern of daily fluctuations in flow is not one to which most species are adapted; thus, such conditions can reduce the abundance, diversity, and productivity of these riverine organisms. Information needs for site-specific evaluations of potential impacts at hydroelectric peaking projects are outlined, along with management and mitigation options to reduce anticipated adverse effects.

In acute toxicity tests, green algae Selenastrum capricornutum, diatoms Nitzschia palea, adult snails Physa gyrina, juvenile cladocerans Daphnia magna, larval midges Chironomus tentans, adult amphipods Gammarus minus, juvenile fathead minnows Pimephales promelas, and embryo-larva stages of rainbow trout Salmo gairdneri and largemouth bass Micropterus salmoides were exposed for 4 hours (algae), 48 hours (arthropods and snails), 96 hours (fathead minnows), 7 days (largemouth bass), and 27 days (rainbow trout) to two phenols (phenol and ß-naphthol), two azaarenes (quinoline and acridine), and two polycyclic aromatic hydrocarbons (naphthalene and phenanthrene) present in coal-derived oils. Median lethal or median effective concentrations (LC50s or EC50s) ranged from 0.03 mg/liter for phenanthrene and rainbow trout to 286.54 mg/liter for phenol and the green alga. The rainbow trout embryo-larva assay was the most sensitive of the test systems to all the chemicals except quinoline. For this last compound, systems with juvenile fathead minnows and largemouth bass embryos were the most sensitive. As test systems, fish embryos and larvae were the most sensitive, juvenile fathead minnows and arthropods had intermediate sensitivity, and algae and snails were the most resistant to the test compounds under the test conditions. Within each chemical class (phenols, azaarenes, and polycyclic aromatic hydrocarbons), toxicity increased with increased ring number except for the reversed relationship with the azaarenes and fathead minnows. Thus, ß-naphthol (two rings) was 2 to 45 times more toxic than phenol (one ring); acridine (three rings) was 7 to 27 times more toxic than quinoline (two rings); and phenanthrene (three rings) was 3 to 9 times more toxic than naphthalene (two rings). There was also a relationship between increases in toxicity and increases in the calculated octanol-water partition coefficients of the compounds. Received January 26, 1983 Accepted November 15, 1983

Eight years ago when I examined the corporate planning process at the Norton Company, I had one major complaint—the wrong people were doing the job. Possibly no one else sees the same problem, but ever since then, I have asked managers in other companies how their planning is organized and how it operates. At this point, I must conclude there are some very wellknown companies that have ineffective systems, for the same reason that I felt ours was ineffective then.

Features broadway director Hal Prince's new Toronto production `Show Boat'. Racism issue; Performance history; Controversial issues regarding his musicals.

While sequence variation can be informative to associate regions of the genome with specific traits and improve genetic selection, the epigenome may provide a more powerful tool to manage cattle. Identifying practices that are producer friendly and effectively control epigenetic function within animals is crucial to translating developmental programming to a production setting. Initial studies of developmental programming investigated how environmental or nutritional stresses during fetal and peri-natal development impacted performance of animals later in life. These studies demonstrated changes in methylation, alterations in transcript abundance, and negative impacts on physiology, but they also suggested that we may be able to beneficially impact the epigenome and developmentally program animals to excel in their niche in the production system. Maternal nutritional status during the third trimester influenced date of conception of female progeny in several studies but failed to do so in other studies. Transcriptomic analyses provided evidence that nutritional treatments alter mRNA abundance in brain, liver, muscle, and ovary, but does not conclusively demonstrate that this is due to functional changes in the epigenome. If developmental programming is to be applied in production systems, responses must be consistent and beneficial. Reducing nutrient intake in heifers during peri-pubertal development increased number of primordial follicles in the ovaries and reproductive longevity. While nutritional programming of the ovarian reserve in peri-pubertal heifers appears to occur consistently across locations and studies, it does not ensure that subsequent environmental stressors will not induce changes in the ovarian reserve that will negate beneficial effects. These studies demonstrate that it is possible to developmentally program the epigenome in cattle in ways that will improve production traits; however, there remains a need for studies to improve the consistency of response and to determine best practices that fit into production systems. USDA is an equal opportunity provider and employer.

Placental separation is a complex physiological event in reproductive physiology and the underlying molecular mechanisms remain unclear. When comparing different experiments the timing of tissue collections is a significant consideration due to the variability in time between fetal expulsion and expulsion of the placenta (30 min to >24 h). This makes comparison of tissues samples across animals difficult and supports the need for serial tissue collections within animal. Additionally, the instrument most commonly used, a modified Richter-Resinsinger effeminator, for placentome collections is difficult to obtain and there are no data in the literature record regarding subsequent reproductive performance of animals subjected placentome collections. To facilitate continued research into the physiology behind placental separation, we designed an instrument from readily available components and performed serial transvaginal placentome collections in cattle. Three placentomes at 2-h intervals were collected after expulsion of the calf in 18 multiparous cows. There was no incidence of mortality and all cows resumed estrous after the procedure. Neither time from placentome collection nor age had a significant effect on pregnancy status at diagnosis (P > 0.05). These results demonstrate the viability of and utility of this device for collecting multiple placentomes in cattle.

Focuses on the popularity of large-scale musicals in Canada. Salient features.

Features Frank Sinatra and his music. 80th birthday of Sinatra on December 12, 1995; Singing style; `Frank Sinatra: An American Legend,' by his daughter Nancy Sinatra as a chronicle of his personal history; `The Frank Sinatra Reader,' by Steven Petkov and Leonard Mustazza as an anthology of essays, reviews and news stories; `Sinatra! The Song Is You,' by Will Friedwald as a study of his music.

Features classical theaters found in rural Canada. Wolfville, Nova Scotia-based Acadia University's Atlantic Theatre Festival; Productions staged; Outstanding stage players; Art festivities in Stratford, Ontario; Summer festivals held in Stratford.

Reproductive failure is the primary reason that a beef cow leaves the production herd; however, the low heritability of reproductive traits makes identification of genetic markers for fertility difficult. Postpartum interval is a moderately heritable trait that influences fertility because cows that ovulate earlier postpartum may have higher first service conception rates. However, in most production settings, the time of first ovulation is unknown. The objective of the current study was to identify single measurement factors that could be indicator traits of fertility in beef cows prior to the start of breeding. The hypothesis tested was that cows that become pregnant earlier in the breeding season have longer intervals from calving to the start of breeding (CSBI), more follicles detectable by ultrasound, greater circulating concentrations of progesterone, and decreased Period1 mRNA expression in the leukocytes. Second (n = 20) and fifth (n = 20) parity crossbred beef cows were subjected to an ovarian ultrasound scan six days prior to the start of a 55-day breeding season. Blood samples were collected at ovarian ultrasound and at approximately 2-week intervals throughout the breeding season. Nine days after the bulls were removed, pregnancy was diagnosed by ultrasound and fetal age was estimated. Parity did not affect any of the endpoints examined. On the basis of CL detected by ultrasound and serum progesterone concentrations, 36 of 40 cows had ovulated prior to the start of breeding. There was no difference in CSBI between cows that were bred in the first 21 days (n = 17), the second 21 days (n = 18), or the last 13 days of the breeding season (n = 5; CSBI = 73 ± 1.6 days). Cows that conceived in the first 21 days of the breeding season had greater serum progesterone concentrations at ovarian ultrasound prior to breeding than cows that conceived in the second 21 days or cows conceiving in the last 13 days of the breeding season (7.0 ± 0.7 vs. 2.8 ± 0.6 and 3.6 ± 1.6 ng/ml, P = 0.0005). By 42 days of breeding, serum progesterone concentrations in cows conceiving during the first 42 days of breeding were greater than in cows conceiving in the last 13 days of breeding (7.9 ± 0.3 and 7.3 ± 0.6 vs. 2.5 ± 1.1 ng/ml, P < 0.0001). The number of antral follicles prior to the start of breeding did not differ among the three groups, and there was no relationship between CSBI, number of antral follicles, or serum progesterone concentrations prior to breeding. Relative levels of Period1 mRNA measured in the leukocytes by real-time RT-PCR and corrected for GAPDH were lower in cows that conceived in the first 42 days of the breeding season as compared to cows that conceived in the last 13 days of breeding (6.6 ± 3.5 and 3.9 ± 3.4 vs. 26.8 ± 6.2 arbitrary units; P = 0.009). Sub-functional corpora lutea in the postpartum period are associated with decreased fertility; therefore, greater serum concentrations of progesterone in cows that conceived early may indicate that these cows had ovulated earlier postpartum, and that their reproductive axis was better primed for maintenance of pregnancy. Lower relative amounts of Period1 mRNA in cows that conceived in the first 42 days of breeding indicates that the biological clock may be set higher in cows that conceive earlier in the season. (platform)

In ruminants, development of the female reproductive tract starts before birth, and primordial follicles in the ovaries activate during the third trimester of gestation. Lambs born as twins have lower birth weights and smaller ovarian reserves than lambs born as singletons. We hypothesized that ewe lambs that were twins in uterowould have smaller reproductive tracts and differential expression of genes involved in early follicular development. Crossbred ewes (n = 112) were sacrificed at the USMARC abattoir on days 75, 95, or 135 of gestation. Gravid uteri were harvested and fetuses were removed and weighed. The reproductive tract was removed from each ewe fetus (n = 47). The left ovary was fixed for histological evaluation and the right ovary was frozen in liquid nitrogen for gene expression analysis. The uterus was weighed and frozen. Data were analyzed using the MIXED procedure of SAS with litter size, gestational day, and the interaction as the fixed effects. Fetal weights and uterine weights increased between days 75 and 135 of gestation (P< 0.0001). Ewe fetuses that were twins weighed less on day 135 of gestation than ewe fetuses that were singletons (P= 0.008); however, there was no difference in uterine weight (P= 0.43) between twins and singletons. There were very few primordial follicles at day 75, but the number of primordial, primary, and secondary follicles per section increased at day 95 and 135, as the process of follicle formation completed and follicles began to activate (P< 0.008). At day 135, twins had fewer primordial follicles per histological section than singletons (P= 0.03). Anti-Müllerian Hormone mRNA was not detectable by real-time RT-PCR, and levels of myelocytomatosis viral oncogene homolog (v-MYC) and Indian Hedgehog (IHH) mRNA did not differ between twins and singletons (P> 0.05). There was an increase in v-MYCgene expression from day 75 to day 135 as primordial follicles activated and granulosa cells began to proliferate (P= 0.01). Levels of IHHmRNA increased (P= 0.001) between day 95 and day 135 as follicles transitioned to the early secondary stage. Gestating as a twin decreased fetal weights and decreased the number of primordial follicles, but did not alter development of the reproductive tract or expression of developmental genes in the ovaries of ewe fetuses. These results suggest that intrauterine growth restriction due to crowding increases the rate of loss of germ cells during the last trimester. USDA is an equal opportunity provider and employer.

The pulsatile uterine secretion of prostaglandin F2 alpha (PGF) triggers the regression of the corpus luteum (CL). Recent studies have explored global changes in gene expression in response to PGF that may contribute to structural and functional regression of the CL. Activating transcription factor 3 (ATF3) is a stress-adaptive gene that regulates proliferation or apoptosis under stress conditions. It is a member of the mammalian activation transcription factor/cAMP responsive element-binding (CREB) protein family that represses transcription from promoters with ATF sites. It is believed that ATF3 may repress transcription by stabilizing the binding of inhibitory cofactors at the promoter. The present study investigated induction and possible role of ATF3 during CL regression. Postpubertal cattle of composite breeding were treated at midcycle with saline (n=3) or PGF (Lutalyse, n=12). Ovariectomies were performed after 0, 0.5, 1, 2, 4h and corpora lutea were processed for immunohistochemistry, protein or RNA isolation. Ovaries were also collected at a local slaughterhouse from first trimester pregnant cows. Luteal cells were prepared and sorted by centrifugal elutriation to isolate small luteal cells (SLC) and large luteal cells (LLC). Western blot analysis showed that PGF treatmentin vivoincreased activity of several members of the MAPK family including phosphorylated ERK, and the stress kinases JNK and p38 MAPK and their downstream targets JUN and HSP27. Real-time PCR analysis and in situhybridization showed that ATF3mRNA increased within 1h of PGF treatment in vivo. Western blot and immunohistochemistry confirmed elevation of ATF3 protein within 1h of PGF treatment in vivo; ATF3 was prominently expressed in the nuclei of LLC within 1h and was maintained for at least 4h. In vitrotreatment with PGF stimulated concentration- and time-dependent increases in ERK, JNK and p38 MAPK phosphorylation. Western blot demonstrated that PGF treatment increased ATF3 expression in LLC. The SLC were much less responsive to PGF compared to LLC. The cytokine tumor necrosis factor alpha (TNF) robustly increased JNK and p38 MAPK phosphorylation and ATF3 expression in luteal cells. In order to identify a role for ATF3 in the regressing CL, we transduced SLC and LLC with an adenovirus to express ATF3 and a control adenovirus expressing galactosidase GLB1. Expression of GLB1 had little effect on progesterone synthesis. Expression of ATF3 reduced basal and LH-stimulated progesterone production in LLC (28.1±8.2% and 42.6±12.0% inhibition, respectively; mean±SEM), and in SLC (34.7±4.9% and 67.0±16.7% inhibition, respectively; mean±SEM), but it did not change the expression of STAR, CYP11A1 and HSD3B proteins in 24h incubations. In conclusion, the action of PGF in LLC is associated with the rapid activation of stress activated protein kinases and the induction of ATF3, which may contribute to the reduction in steroid synthesis during CL regression. Because ATF3 did not affect expression of key regulators of progesterone synthesis, it may increase cholesterol metabolism or export, thus diverting cholesterol from the steroidogenic pathway. Supported by the Department of Veterans Affairs, USDA NIFA-AFRI 2011-67015-20067 and the Olson Center for Women's Health, UNMC. USDA is an equal opportunity provider and employer.