Clustering of multi-tissue transcriptomes in gilts with normal cyclicity or delayed puberty reveals genes related to pubertal development†.

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 (KISS1 and TAC3) and reduced expression of LHB in 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 (STAR and 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.

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.

Influence of estradiol on bovine trophectoderm and uterine gene transcripts around maternal recognition of pregnancy†.

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.

The ovarian follicle of ruminants: the path from conceptus to adult.

This review resulted from an international workshop and presents a consensus view of critical advances over the past decade in our understanding of follicle function in ruminants. The major concepts covered include: (1) the value of major genes; (2) the dynamics of fetal ovarian development and its sensitivity to nutritional and environmental influences; (3) the concept of an ovarian follicle reserve, aligned with the rise of anti-Müllerian hormone as a controller of ovarian processes; (4) renewed recognition of the diverse and important roles of theca cells; (5) the importance of follicular fluid as a microenvironment that determines oocyte quality; (6) the 'adipokinome' as a key concept linking metabolic inputs with follicle development; and (7) the contribution of follicle development to the success of conception. These concepts are important because, in sheep and cattle, ovulation rate is tightly regulated and, as the primary determinant of litter size, it is a major component of reproductive efficiency and therefore productivity. Nowadays, reproductive efficiency is also a target for improving the 'methane efficiency' of livestock enterprises, increasing the need to understand the processes of ovarian development and folliculogenesis, while avoiding detrimental trade-offs as greater performance is sought.

Vascular endothelial growth factor A isoforms modulate follicle development in peripubertal heifers independent of diet through diverse signal transduction pathways.

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.

Effects of preovulatory estradiol on uterine environment and conceptus survival from fertilization to maternal recognition of pregnancy.

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.

Effects of IL8 and immune cells on the regulation of luteal progesterone secretion.

Recent studies have suggested that chemokines may mediate the luteolytic action of prostaglandin F2α (PGF). Our objective was to identify chemokines induced by PGF in vivo and to determine the effects of interleukin 8 (IL8) on specific luteal cell types in vitro. Mid-cycle cows were injected with saline or PGF, ovaries were removed after 0.5-4 h, and expression of chemokine was analyzed by qPCR. In vitro expression of IL8 was analyzed after PGF administration and with cell signaling inhibitors to determine the mechanism of PGF-induced chemokine expression. Purified neutrophils were analyzed for migration and activation in response to IL8 and PGF. Purified luteal cell types (steroidogenic, endothelial, and fibroblast cells) were used to identify which cells respond to chemokines. Neutrophils and peripheral blood mononuclear cells (PBMCs) were cocultured with steroidogenic cells to determine their effect on progesterone production. IL8, CXCL2, CCL2, and CCL8 transcripts were rapidly increased following PGF treatment in vivo. The stimulatory action of PGF on IL8 mRNA expression in vitro was prevented by inhibition of p38 and JNK signaling. IL8, but not PGF, TNF, or TGFB1, stimulated neutrophil migration. IL8 had no apparent action in purified luteal steroidogenic, endothelial, or fibroblast cells, but stimulated ERK phosphorylation in neutrophils. In coculture experiments neither IL8 nor activated neutrophils altered basal or LH-stimulated luteal cell progesterone synthesis. In contrast, activated PBMCs inhibited LH-stimulated progesterone synthesis from cultured luteal cells. These data implicate a complex cascade of events during luteolysis, involving chemokine signaling, neutrophil recruitment, and immune cell action within the corpus luteum.

Incorporation of genetic technologies associated with applied reproductive technologies to enhance world food production.

Animal breeding and reproductive physiology have been closely related throughout the history of animal production science. Artificial insemination provides the best method of increasing the influence of sires with superior genetics to improve production traits. Multiple ovulation embryo transfer (MOET) provides some ability to increase the genetic influence of the maternal line as well. The addition of genetic technologies to this paradigm allows for improved methods of selecting sires and dams carrying the best genes for production and yield of edible products and resistance to diseases and parasites. However, decreasing the number of influential parents within a population also increases the risk of propagating a recessive gene that could negatively impact the species (Reprod Domest Anim 44:792-796, 2009; BMC Genomics 11:337, 2010). Furthermore, antagonistic genotypic relationships between production traits and fertility (Anim Prod Sci 49:399-412, 2009; Anim Genet 43:442-446, 2012) suggest that care must be taken to ensure that increasing the frequency of genes with a positive influence on production does not negatively impact the fertility of the replacement females entering the herd.

Developmental programming of reproduction in the female animal.

Successful reproduction is a cornerstone in food animal industry in order to sustain food production for human. Therefore, various methods focusing on genetics and postnatal environment have been identified and applied to improve fertility in livestock. Yet there is evidence indicating that environmental factors during prenatal and/or neonatal life can also impact the function of reproductive system and fertility in the animals during adulthood, which is called the developmental programming of reproduction. The current review summarizes data associated with the developmental origins of reproduction in the female animals. In this regard, this review focuses on the effect of plane of nutrition, maternal body condition, hypoxia, litter size, maternal age, parity, level of milk production and milk components, lactocrine signaling, stress, thermal stress, exposure to androgens, endocrine disrupting chemicals, mycotoxins and pollutants, affliction with infection and inflammation, and maternal gut microbiota during prenatal and neonatal periods on the neuroendocrine system, puberty, health of reproductive organs and fertility in the female offspring. It is noteworthy that these prenatal and neonatal factors do not always exert their effects on the reproductive performance of the female by compromising the development of organs directly related to reproductive function such as hypothalamus, pituitary, ovary, oviduct and uterus. Since they can impair the development of non-reproductive organs and systems modulating reproductive function as well (e.g., metabolic system and level of milk yield in dairy animals). Furthermore, when these factors affect the epigenetics of the offspring, their adverse effects will not be limited to one generation and can transfer transgenerationally. Hence, pinpointing the factors influencing developmental programming of reproduction and considering them in management of livestock operations could be a potential strategy to help improve fertility in food animals.

Developmental programming of the ovarian reserve in livestock.

Mammalian females are born with a finite number of follicles in their ovaries that is referred to as the ovarian reserve. There is a large amount of variation between females in the number of antral follicles that they are born with, but this number is positively correlated to size of the ovarian reserve, has a strong repeatability within a female, and a moderate heritability. Although the heritability is moderate, numerous external factors including health, nutrition, ambient temperature, and litter size influence the size and function of the ovarian reserve throughout life. Depletion of the ovarian reserve contributes to reproductive senescence, and genetic and epigenetic factors can lead to a more rapid decline in follicle numbers in some females than others. The relationship of the size of the ovarian reserve to development of the reproductive tract and fertility is generally positive, although some studies report antagonistic associations of these traits. It seems likely that management decisions and environmental factors that result in epigenetic modifications to the genome throughout life may cause variability in the function of ovarian genes that influence fecundity and fertility, leading to differences in reproductive longevity among females born with ovarian reserves of similar size. This review summarizes our current understanding of factors influencing size of the ovarian reserve in cattle, sheep, and pigs and the relationship of the ovarian reserve to reproductive tract development and fertility. It provides strategies to apply this knowledge to improve diagnostics for better assessment of fertility and reproductive longevity in female livestock.

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.

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.

The plasminogen activator system in the ovine placentome during late gestation and stage-two of parturition.

The process of placental separation is not completely understood. In domestic animals, especially cattle, it is important that expulsion of the fetal membranes takes place in a timely manner in order to achieve maximal reproductive efficiency. The activity of the matrix-metalloprotease (MMP) family of proteases is known to be reduced in placentomes from cases of retained placenta. Members of the MMP family are known to be activated by the plasminogen activator (PA) family of proteases. We hypothesized that the expression and activity of the PA family increase in the cotyledon and/or caruncle as parturition approaches, with maximal expression and activity at parturition. To test this hypothesis, we performed reverse-transcriptase quantitative PCR and plasminogen-casein zymography to detect the presence and activity of PA family members in the placentome leading up to and during parturition in spontaneous and dexamethasone-induced parturient ewes. The results from our experiments indicated that serine proteases inhibitor E1 (SERPINE1) mRNA abundance in the cotyledon was different between treatment groups (P = 0.0002). In the caruncle, gene expression for plasminogen activator urokinase-type (PLAU) was different (P = 0.0154), and there was a strong trend for differences in SERPINE1 expression (P = 0.0565). These results demonstrate that expression of the PA system in the placentome changes from late pregnancy to parturition, and the presence or activity of these enzymes may occur after fetal expulsion.

An extreme model of fertility in sheep demonstrates the basis of controversies surrounding antral follicle count and circulating concentrations of anti-Müllerian hormone as predictors of fertility in ruminants.

Ovarian ultrasonography and measurement of circulating concentrations of anti-Müllerian hormone (AMH) have been used to estimate follicle number and predict fertility in mammalian females, but no study has evaluated follicle number and circulating concentrations of AMH in ewes known to differ in fertility. We tested the hypothesis that ewes that had failed to lamb (BARREN) in four consecutive annual breeding seasons of 21-35 d have fewer follicles and diminished circulating concentrations of AMH compared to closely matched ewes that consistently produced lambs (FERTILE) under the same breeding protocols. Once identified, BARREN ewes (n = 19) were paired by breed and sire to a FERTILE control ewe (n = 19) and reproductive tracts were recovered at necropsy. Visible antral follicles in both ovaries were counted and a representative cross section of one ovary was embedded for histological evaluation of pre-antral follicle numbers. Paired t-tests indicated that BARREN ewes had fewer antral follicles, fewer primordial follicles, and decreased circulating concentrations of AMH compared to FERTILE ewes (P ≤ 0.01), but there were ewes in each fertility classification that had ovarian phenotypes like the opposite fertility classification. The best technologies we have currently for estimating follicle numbers are ultrasonography and measurement of circulating concentrations of AMH, but no single technique is perfect for predicting fertility. A better understanding of the under-lying biological mechanisms linking AMH, folliculogenesis, and fertility is required to improve the use of measurements of follicle number for predicting fertility in livestock.

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.

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.

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.

Proteomics dataset of epididymal fluid, seminal plasma, and proteins loosely attached to epididymal and ejaculated sperm from Angus bulls.

Peptides and proteins were identified by liquid chromatography with tandem mass spectrometry analysis (LCMS-MS) on an Orbitrap Velos mass spectrometer to further understand biological mechanisms that regulate increased longevity in epididymis compared to ejaculated sperm. Semen from sexually mature bulls were collected and then bulls were slaughtered to collect epididymal samples from the cauda epididymis. All samples were centrifuged to separate spermatozoa from fluids. A high ionic solution was used to remove surface proteins from spermatozoa. Four unique samples were generated: (1) epididymal fluid, (2) seminal plasma (ejaculated fluid), and proteins stripped from (3) epididymal sperm, and (4) ejaculated sperm. Samples were analyzed by LCMS-MS, and data were interpreted with Protein Pilot 5. False discovery rate (FDR) was set at 1%. Unique proteins (n = 458) were identified in ejaculated samples, 178 proteins in seminal plasma and 298 proteins stripped from ejaculated sperm. In epididymal samples, 311 proteins were identified in the fluid, and 334 were identified among proteins stripped from epididymal sperm. This dataset can be useful for further understand of biological mechanisms that control sperm longevity. This dataset is related to the article 'Proteomic analyses identify differences between bovine epididymal and ejaculated spermatozoa that contribute to longevity' by (Zoca et al., 2022).

Proteomic analyses identify differences between bovine epididymal and ejaculated spermatozoa that contribute to longevity.

Sperm are stored for extended periods of time in the epididymis, but upon ejaculation motility is increased and lifespan is decreased. The objective of this study was to identify differences in proteins between epididymis and ejaculated samples that are associated with longevity. Ejaculated semen was collected from mature Angus bulls (n = 9); bulls were slaughtered and epididymal semen was collected. Epididymal and ejaculated semen were centrifuged to separate sperm and fluid. Fluids were removed and sperm pellets were resuspended in a high ionic solution and vortexed to remove loosely attached proteins. Sperm samples were centrifuged, and the supernatant was removed; both fluid and sperm samples were snap frozen in liquid nitrogen and stored at -80 °C. Protein analysis was performed by LCMS/MS. A different group of yearling Angus cross bulls (n = 40) were used for sperm cultures. Ejaculated (n = 20) and epididymal (n = 20) semen were diluted and cultured in a commercial media at pH 5.8, 6.8 and 7.3, at 4 °C. Sperm were evaluated for motility and viability every 24 h until motility was lower than 20%. There was an effect of pH, time and pH by time interaction for motility and viability for both ejaculated and epididymal sperm (P ≤ 0.05). At 216 h of incubation epididymal sperm at pH 7.3 and ejaculated sperm at pH 6.8 reached motility below 20%. A total of 458 unique proteins were identified; 178, 298, 311, and 344 proteins were identified in ejaculated fluid, ejaculated sperm, epididymal fluid and epididymal sperm, respectively. There were 8, 24, 10, and 18 significant KEGG pathways (FDR <0.05) for ejaculated fluid, epididymal fluid, ejaculated sperm, and epididymal sperm, respectively. The metabolic pathway was identified as the most important KEGG pathway; glycolysis/gluconeogenesis, pentose phosphate, and glutathione metabolism pathways were significant among proteins only present in epididymal samples within the metabolic pathway. Other proteins identified that may be related to epididymal sperm's increased longevity were peroxidases and glutathione peroxidases for their antioxidant properties. In summary, energy metabolism in the epididymis appears to be more glycolytic compared to ejaculated and epididymis sperm have a larger number of antioxidants available which may be helping to maintain sperm in a quiescent state. Epididymal sperm remained viable (membrane integrity) longer than ejaculated sperm when cultured at the same pH.

Influence of conceptus presence and preovulatory estradiol exposure on uterine gene transcripts and proteins around maternal recognition of pregnancy in beef cattle.

The uterine environment must provide sufficient endocrine conditions and nutrients for pregnancy maintenance and conceptus survival. The objective of this study was to determine the effects of preovulatory estradiol and conceptus presence on uterine transcripts and uterine luminal fluid (ULF) proteins. Beef cows/heifers were synchronized and artificially inseminated (d 0). Uteri were flushed (d 16); conceptuses and endometrial biopsies were collected. Total cellular RNA was extracted from endometrium for RNA sequencing and RT-PCR validation. There were two independent ULF pools made for each of the following groups: highE2/conceptus, highE2/noconceptus, lowE2/conceptus, and lowE2/noconceptus that were analyzed using the 2D LC-MS/MS based iTRAQ method. There were 64 differentially expressed genes (DEGs) and 77 differentially expressed proteins (DEPs) in common among the highE2/conceptus vs highE2/noconceptus and lowE2/conceptus vs lowE2/noconceptus groups. In summary, the interaction between preovulatory estradiol and the conceptus induces the expression of genes, proteins, and pathways necessary for pregnancy.