Analysis of nonsynonymous SNPs in candidate genes that influence bovine temperament and evaluation of their effect in Brahman cattle.

BACKGROUND: Temperament is an important production trait in cattle and multiple strategies had been developed to generate molecular markers to assist animal selection. As nonsynonymous single nucleotide polymorphisms are markers with the potential to affect gene functions, they could be useful to predict phenotypic effects. Genetic selection of less stress-responsive, temperamental animals is desirable from an economic and welfare point of view. METHODS AND RESULTS: Two nonsynonymous single nucleotide polymorphisms identified in HTR1B and SLC18A2 candidate genes for temperament were analyzed in silico to determine their effects on protein structure. Those nsSNPs allowing changes in proteins were selected for a temperament association analysis in a Brahman population. Transversion effects on protein structure were evaluated in silico for each amino acid change model, revealing structural changes in the proteins of the HTR1B and SLC18A2 genes. The selected nsSNPs were genotyped in a Brahman population (n = 138), and their genotypic effects on three temperament traits were analyzed: exit velocity, pen score, and temperament score. Only the SNP rs209984404-HTR1B (C/A) showed a significant association (P = 0.0144) with pen score. The heterozygous genotype showed a pen score value 1.17 points lower than that of the homozygous CC genotype. CONCLUSION: The results showed that in silico analysis could direct the selection of nsSNPs with the potential to change the protein. Non-synonymous single nucleotide polymorphisms causing structural changes and reduced protein stability were identified. Only rs209984404-HTR1B shows that the allele affecting protein stability was associated with the genotype linked to docility in cattle.

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.

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.

Progesterone secretion by ovine granulosa cells: effects of nitric oxide and plane of nutrition.

The aim was to evaluate the effects of nutritional plane on in vitro progesterone (P4) secretion by granulosa (G) cells cultured in the presence or absence of effectors of the nitric oxide (NO) system. Ewes were randomly assigned into three nutritional groups: control (C), overfed (O; 2 × C), or underfed (U; 0.6 × C). Follicular development was induced by FSH injections. On day 15 of the estrous cycle, G cells were isolated and cultured with or without DETA-NONOate (NO donor), L-NAME (NO synthase [S] inhibitor), Arg and (or) LH for 8 h. DETA-NONOate decreased basal and LH-stimulated P4 secretion, and L-NAME increased basal P4 secretion in all groups. In U, Arg decreased LH-stimulated P4 secretion. These data demonstrate that (i) plane of nutrition affects basal P4 secretion by G cells, (ii) the NO donor decreases, NOS inhibitor increases but Arg does not affect basal P4 secretion, and (iii) effects of Arg on LH-stimulated P4 secretion are affected by plane of nutrition in FSH-treated sheep. Thus, plane of nutrition affects G cell function, and the NO system is involved in the regulation of basal and LH-stimulated P4 secretion. The mechanism of the NO system effects on secretory activity of G cells remains to be elucidated.

Considerations for Using Natural Service with Estrous Synchronization Programs.

Estrous synchronization, coupled with natural service, provides the benefit of female cows conceiving early, but there are an increased number of females expressing estrus in a short period of time. Thus, considerations need to be made for the bull. Select a protocol that will distribute estrus over a longer period of time and ensure bulls pass a breeding soundness examination. Mature bulls (3 years old or older) have increased efficiency in getting cows pregnant compared with younger bulls; therefore, a ratio of 1 mature bull to 25 cows is a good recommendation within an estrous synchronized herd.

The importance of developmental programming in the beef industry.

The beef industry relies on multiple focused segments (e.g., cow-calf, stocker/feeder, and meat packing) to supply the world with beef. Thus, the potential impact of developmental programming on the beef industry needs to be evaluated with regards to the different production traits that drive profitability within each segment. For example, when nutrient restriction of dams occurred early in gestation embryo survival was decreased and the ovarian reserve of heifer progeny was negatively affected. Restriction during mid- to late gestation negatively impacted first service conception rates and pregnancy success of daughters. Even non-nutrient stress has been reported to impact transgenerational embryo development through the male progeny. Primary and secondary muscle fibers form during months two to eight (Days 60-240) of gestation. Therefore, external stimuli (nutrition or environmental) during this window have the potential to decrease the postnatal number of muscle fibers; which has an irreversible impact on animal growth and performance. Nutrient restriction during the last third of gestation resulted in decreased weaning weights, and in some instances decreased dry mater intake, hot carcass weight, and marbling scores. Protein supplementation during late gestation; however, increased weaning weight and ADG to weaning, but progeny of dams restricted in protein in late gestation had greater ribeye area. The importance of developmental programming is recognized; however, its precise application depends on comprehension of its integrated effects across the multiple-focused segments of the beef industry.

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.

Plasma γ-Aminobutyric Acid (GABA) Concentrations in Lactating Holstein Cows during Thermoneutral and Heat Stress Conditions and Their Relationships with Circulating Glucose, Insulin and Progesterone Levels.

Heat-stressed lactating dairy cattle exhibit unique metabolic symptoms, many of which are undoubtedly involved in heat-induced subfertility. Because of its known systemic effects, we hypothesized that γ-aminobutyric acid (GABA) participates in the regulation of insulin and progesterone during heat stress. Multiparous lactating Holstein cows (n = 6) were studied during four experimental periods: (1) thermoneutral (TN; d 1-5), (2) TN + hyperinsulinemic-hypoglycemic clamp (d 6-10), (3) heat stress (HS; d 16-20), and (4) HS + euglycemic clamp (d 21-25). Blood samples were collected once daily via coccygeal venipuncture into heparinized evacuated tubes. Analysis of GABA concentrations from all four treatment periods yielded no differences. In direct comparison to TN concentrations, plasma GABA tended to decrease during the HS period (16.57 ± 2.64 vs. 13.87 ± 2.28 ng/mL, respectively, p = 0.06). Both milk production and plasma insulin were moderately correlated with plasma GABA (r = 0.35, p < 0.01; r = -0.32, p < 0.01). Plasma progesterone was correlated with plasma GABA concentrations during TN but not HS periods. These results are the first to indicate that peripheral GABA could be involved in the regulation of factors known to affect production and reproduction during heat stress. More research is needed to determine its precise role(s).

Evaluation of pregnancy associated glycoproteins assays for on farm determination of pregnancy status in beef cattle.

Transrectal ultrasonography is known as the gold standard for pregnancy detection, but requires costly equipment and technical skills; therefore, access to an inexpensive and more user-friendly method with similar accuracy could benefit cattle producers. Detection of pregnancy-associated glycoproteins can accurately determine pregnancy in ruminants; however, usually requires specialized equipment for the assay. Thus, the objectives of these studies were to 1) validate the IDEXX Alertys OnFarm Pregnancy Test (lateral flow) and compare the accuracy of all three commercial PAG assays to transrectal ultrasonography and 2) to determine the postpartum interval necessary for clearance of pregnancy-associated glycoproteins from the previous pregnancy to avoid false positives. In study 1, blood samples from previously identified pregnant Bos taurus females from six different herds (nulliparous n = 1,205 and multiparous n = 1,539; samples collected between d 27 to 285 of gestation over a three-year period) were utilized. In study 2, postpartum females (primiparous n = 48 and multiparous n = 66) from one herd were utilized: (n = 1,066; samples collected weekly for up to 12 weeks postpartum). In study 1, level of agreement between different methods of pregnancy detection was determined by Pearson's correlation and Kappa scores. In study 2, data were analyzed as a repeated measure using the MIXED procedure of SAS with main effects of parity, days postpartum (dpp), and parity by days postpartum, then data were analyzed further using the REG procedure of SAS. In study 1, transrectal ultrasonography and lateral flow were positively correlated (r = 0.77; P <0.01), with 92.4% agreement. In study 2, the abundance of absorbance of PAGs rapidly decreased from 0 to 50 days postpartum, then continued to gradually decrease (P <0.01; r = 0.90). Prior to 42 days postpartum, PAG concentrations were sufficiently elevated resulting in false positive readings in all assays. In conclusion, there is very good agreement between transrectal ultrasonography and PAG assays, but likelihood of false positive results are highif assays are performed fewer than 42 days postpartum.

The Effect of Copper and Zinc Sources on Liver Copper and Zinc Concentrations and Performance of Beef Cows and Suckling Calves.

To determine the influence of the source of gestational and postnatal Cu and Zn supplementation on cow and calf performance, cows (n = 287) were assigned to one of the following two treatments: (1) inorganic (INORG) treatment, in which cows were supplemented with 15 mg of Cu (as CuSO4) and 15 mg of Zn (as ZnSO4) per kg of diet DM, or (2) organic (ORG) treatment, in which cows were supplemented with 15 mg of Cu (as Cu proteinate; Bioplex Cu, Alltech, Inc., Nicholasville, KY, USA) and 15 mg of Zn (as Zn proteinate; Bioplex Zn, Alltech, Inc., Nicholasville, KY, USA) per kg of diet DM. The treatments were initiated prior to breeding and continued throughout gestation until weaning. Liver biopsies were collected for analysis of mineral content. Cow body condition score (BCS), body weight (BW), pregnancy data, calf weaning weight (WW), and antibody response of the calves were recorded. The cows receiving the INORG treatment had a greater BW (p < 0.05) and BCS (p < 0.01) at breeding in Year 2, while the cows on the ORG treatment had a greater (p < 0.05) BW at weaning in Year 2. The cows that received the ORG mineral had improved (p < 0.05) conception rates in Year 1. The calves receiving the ORG treatment had heavier (p < 0.05) 205-day adjusted WWs.

Genetic Variants and Their Putative Effects on microRNA-Seed Sites: Characterization of the 3' Untranslated Region of Genes Associated with Temperament.

The 3' untranslated region has an important role in gene regulation through microRNAs, and it has been estimated that microRNAs regulate up to 50% of coding genes in mammals. With the aim of allelic variant identification of 3' untranslated region microRNA seed sites, the 3' untranslated region was searched for seed sites of four temperament-associated genes (CACNG4, EXOC4, NRXN3, and SLC9A4). The microRNA seed sites were predicted in the four genes, and the CACNG4 gene had the greatest number with 12 predictions. To search for variants affecting the predicted microRNA seed sites, the four 3' untranslated regions were re-sequenced in a Brahman cattle population. Eleven single nucleotide polymorphisms were identified in the CACNG4, and eleven in the SLC9A4. Rs522648682:T>G of the CACNG4 gene was located at the predicted seed site for bta-miR-191. Rs522648682:T>G evidenced an association with both exit velocity (p = 0.0054) and temperament score (p = 0.0097). The genotype TT had a lower mean exit velocity (2.93 ± 0.4 m/s) compared with the TG and GG genotypes (3.91 ± 0.46 m/s and 3.67 ± 0.46 m/s, respectively). The allele associated with the temperamental phenotype antagonizes the seed site, disrupting the bta-miR-191 recognition. The G allele of CACNG4-rs522648682 has the potential to influence bovine temperament through a mechanism associated with unspecific recognition of bta-miR-191.

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.

Follicular determinants of pregnancy establishment and maintenance.

Synchronization of dominant follicle development and control of ovulation/oocyte retrieval are commonly used assisted reproductive technologies in both cattle and humans. The final maturation of the dominant follicle is intimately tied to the final maturation of the oocyte, preovulatory secretion of estradiol, preparation of follicular cells for luteinization, postovulatory secretion of progesterone and endocrine control of the oviductal and uterine environment for gamete and embryo development. The physiological maturity of a dominant/ovulatory follicle can affect the establishment and maintenance of pregnancy. Premature induction of the ovulatory process can reduce pregnancy rates and increase late embryonic/fetal mortality in cattle, which is likely mediated through inadequate oocyte competence and a compromised maternal environment. Oocyte competence increases with follicular maturity and is dependent upon acquisition of a complete complement of mRNA transcripts and establishment of the appropriate epigenetic marking of the oocyte genome before the preovulatory gonadotropin surge. Preovulatory secretion of estradiol is a reflection of follicular maturity and affects the oocyte, follicular cells, oviduct and uterus. The corpus luteum is a continuation of follicular maturation and rate of progesterone secretion following ovulation is linked to fertility. Advancements in our understanding of how the follicular microenvironment affects pregnancy establishment and maintenance will improve the efficiency of assisted reproductive technologies in all species. The purpose of this review is to discuss how follicular microenvironment, oocyte competence, preovulatory secretion of estradiol and postovulatory secretion of progesterone can affect pregnancy establishment and embryo/fetal survival, with an emphasis on cattle.

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.

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).

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.

Impact of preovulatory estradiol concentrations on subsequent luteal function in beef cattle.

It has been hypothesized that circulating concentrations of estradiol during the preovulatory period, can impact subsequent progesterone concentrations. Ovulation was synchronized in nonlactating beef cows (n = 53). Cows that exhibited estrus before gonadotrophin-releasing hormone (GnRH)-induced ovulation (d 0) had greater (p<.01) peak concentrations of estradiol compared with cows that did not express estrus (11.5 ± 0.8 vs. 6.2 ± 0.6 pg/mL), respectively, but there was no difference in ovulatory follicle size (p= .80) or interval from GnRH2 to ovulation (p=.23). Circulating concentrations of progesterone during luteal formation (d 3-7; p=.70 and p=.77) or mid-luteal phase (d 8-14; p=.39 and p=.12) were not affected by elevated periovulatory estradiol or an interaction with day. To investigate the direct influence of estradiol on luteal function, ovulation (d 0) was synchronized in nonlactating beef cows and cows were allocated to three groups (control, n = 5; vehicle injection, n = 4; or an estradiol antagonist (Fulvestrant; ICI 182,780), n = 4. Intrafollicular injection of vehicle (100 µL) or an estradiol antagonist (25 µg Fulvestrant in 100 µL) into the largest follicle occurred on d -2. Concentrations of estradiol increased (p<.0001) from d -2 to 0 but did not differ among groups (p>.50). Furthermore, plasma concentrations of progesterone on d 0 through 20 were not affected by treatment (p=.86). These results indicate that elevated preovulatory estradiol before ovulation was not required to prepare granulosa cells for luteinization or subsequent luteal progesterone secretion but did tend to impact luteal lifespan.

Greater numbers of antral follicles in the ovary are associated with increased concentrations of glucose in uterine luminal fluid of beef heifers.

Increased 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 on d16 after insemination would be greater in heifers with increased numbers of antral follicles. Heifers classified with either increased or diminished antral follicle counts were artificially inseminated following the CO-Synch protocol (d0). On d16 after insemination, reproductive tracts of heifers were collected at an abattoir to retrieve conceptuses to determine pregnancy. Uterine luminal fluid was collected, endometrium was biopsied, total RNA was extracted and glucose transporter transcript abundances were 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 in heifers with increased antral follicle numbers. Glucose ULF concentrations increased in pregnant heifers. Facilitated glucose transporter member 1 (SLC2A1) transcript abundance was increased in the endometrium of pregnant heifers but was not different due to antral follicle number or the interaction. Differences in uterine concentrations of glucose associated with antral follicle number could be due to another mechanism, since glucose transporters were not different between antral follicle numbers. Therefore, heifers with increased number of antral follicles have increased energy availability in the uterus to support trophoblast proliferation and function.

Modulation of expression of estrus, steroidogenesis and embryo development following peri-artificial insemination nutrient restriction in beef heifers.

Nutritional changes immediately after insemination cause increased embryonic mortality, but the mechanisms controlling this are not well known. Our objective was to evaluate the impact of nutritional change on estrus expression, steroid concentrations, peripheral and uterine luminal fluid metabolites, and embryo quality in beef heifers. Heifers (n = 139) were assigned to one of two pre-artificial insemination (AI) dietary treatments: LOW (≤ 90% NEm) or HIGH (≥ 139% NEm). Heifers were on treatment for 33-36 days before AI (d0) when half of the heifers in each treatment were randomly reassigned to generate four treatments; HIGH-HIGH, HIGH-LOW, LOW-HIGH, and LOW-LOW. Heifers remained on treatments until embryo collection (d 6-8). Negative energy balance was achieved among LOW heifers as demonstrated by body weight loss and increased NEFA concentrations (P < 0.05). Pre-AI treatment influenced expression of estrus (P = 0.05; HIGH 80.4 ± 4.0% vs. LOW 69.4 ± 4.2%). Estradiol concentrations and interval to estrus were not affected by treatment (P > 0.55); however, progesterone concentrations were reduced among LOW compared to HIGH (3.57 ± 0.27, 4.64 ± 0.26 ng/mL, respectively; P = 0.004), and heifers maintained on the HIGH pre-AI diet had consistently greater concentrations of progesterone from d 0 to d 8 (P = 0.014). Pre-AI treatment influenced embryo stage (P = 0.05; HIGH 3.61 ± 0.32 vs. LOW 2.72 ± 0.30). Post-AI treatment affected embryo grade (P = 0.02; HIGH 1.78 ± 0.23 vs. LOW 2.64 ± 0.27). In summary, pre-AI nutrient restriction caused decreased expression of estrus, reduced progesterone concentrations after AI, and negatively impacted embryo development, while post-AI restriction hindered embryo quality.

Prenatal transportation stress did not impact ovarian follicle count for three generations of female Brahman offspring.

As prenatal transportation stress altered behavior and adrenal glucocorticoid secretion of calves, we hypothesized that prenatal transportation stress would decrease ovarian reserve size and negatively impact female offspring fertility. The impact of prenatal transportation stress on ovarian follicle numbers in female offspring for three generations was studied. Brahman cows were transported for 2 h on day 60 ± 5, 80 ± 5, 100 ± 5, 120 ± 5, and 140 ± 5 of gestation. Ovaries were collected from offspring of transported or non-transported dams at multiple ages. Primordial, primary, secondary, and antral follicles were histologically analyzed. Antral follicle numbers were determined by ultrasound in a subset of offspring. Numbers of primordial, primary, secondary, and antral follicles were analyzed using the MIXED procedure, while the CORR procedure of SAS was used to determine the correlation between follicles observed by ultrasonography and histology. There were no differences (P > 0.05) in the number of primordial, primary, secondary, antral, or total follicles observed histologically due to treatment. Younger females had significantly greater numbers of follicles than older females (P < 0.0001). Antral follicles tended to be correlated with total histological ovarian follicles (P = 0.10). There was no difference in the number of antral follicles observed at ultrasound due to treatment (P = 0.3147), or generation (P = 0.6005) when controlling for age at observation. These results show that short-term transportation stress during early- to mid-gestation did not impact fertility as measured by ovarian follicle numbers in female Brahman offspring for three generations.