Identification of Lipids and Cytokines in Plasma and Follicular Fluid before and after Follicle-Stimulating Hormone Stimulation as Potential Markers for Follicular Maturation in Cattle.

The process of follicle maturation leading to ovulation is a key milestone in female fertility. It is known that circulating lipids and cytokines play a role in the follicle's ability to go through follicular maturation and the ovulatory processes. However, the specific mechanisms are not well understood. We posit that dysregulation of granulosa cells influences the ovarian environment, which tries to adapt by changing released lipids and cytokines to achieve follicular maturation. Eleven non-lactating adult females underwent estrus synchronization with two injections of PGF2α 14 days apart. Daily blood samples were collected for 28 days to monitor steroid hormone production after the second injection. To understand the potential impacts of lipids and cytokines during ovulation, a low-dose FSH stimulation (FSHLow) was performed after resynchronization of cows, and daily blood samples were collected for 14 days to monitor steroid hormone production until ovariectomies. The lipidomic analysis demonstrated increased circulating diacylglycerides and triacylglycerides during the mid-luteal phase and after FSHLow treatment. Cholesteryl esters decreased in circulation but increased in follicular fluid (FF) after FSHLow. Increased circulating concentrations of TNFα and reduced CXCL9 were observed in response to FSHLow. Therefore, specific circulating lipids and cytokines may serve as markers of normal follicle maturation.

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.

Effects of administration of a growth promoting implant during the suckling phase or at weaning on growth, reproduction, and ovarian development in replacement heifers grazing native range.

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.

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.

VEGFA165 can rescue excess steroid secretion, inflammatory markers, and follicle arrest in the ovarian cortex of High A4 cows†.

A population of cows with excess androstenedione (A4; High A4) in follicular fluid, with follicular arrest, granulosa cell dysfunction, and a 17% reduction in calving rate was previously identified. We hypothesized that excess A4 in the ovarian microenvironment caused the follicular arrest in High A4 cows and that vascular endothelial growth factor A would rescue the High A4 phenotype. In trial 1, prior to culture, High A4 ovarian cortex (n = 9) had greater numbers of early stage follicles (primordial) and fewer later-stage follicles compared to controls (n = 11). Culture for 7 days did not relieve this follicular arrest; instead, High A4 ovarian cortex had increased indicators of inflammation, anti-Mullerian hormone, and A4 secretion compared to controls. In trial 2, we tested if vascular endothelial growth factor A isoforms could rescue the High A4 phenotype. High A4 (n = 5) and control (n = 5) ovarian cortex was cultured with (1) PBS, (2) VEGFA165 (50 ng/mL), (3) VEGFA165B (50 ng/mL), or (4) VEGFA165 + VEGFA165B (50 ng/mL each) for 7 days. Follicular progression increased with VEGFA165 in High A4 cows with greater early primary, primary, and secondary follicles than controls. Similar to trial 1, High A4 ovarian cortex secreted greater concentrations of A4 and other steroids and had greater indicators of inflammation compared to controls. However, VEGFA165 rescued steroidogenesis, oxidative stress, and fibrosis. The VEGFA165 and VEGFA165b both reduced IL-13, INFα, and INFß secretion in High A4 cows to control levels. Thus, VEGFA165 may be a potential therapeutic to restore the ovarian steroidogenic microenvironment and may promote folliculogenesis.

Transcriptomic data of bovine ovarian granulosa cells of control and High A4 cows.

Microarray analysis using Affymetrix Bovine GeneChip 1.0 ST Array to determine RNA expression analysis was performed on somatic granulosa cells from two different groups of cows classified based on androstenedione concentration within the follicular fluid (Control vs High A4) of estrogen-active dominant follicles. The normalized linear microarray data was deposited to the NCBI GEO repository (GSE97017 - RNA Expression Data from Bovine Ovarian Granulosa Cells from High or Low Androgen-Content Follicles). Subsequent ANOVA determined genes that were enriched (≥ 1.5 fold more) or decreased (≤ 1.5 fold less) in the High A4 granulosa cells compared to Control granulosa cells and analyzed filtered datasets of these differentially expressed genes are presented as tables. MicroRNAs that are differentially expressed in Control and High A4 granulosa cells are also reported in tables. The standard deviation of the analyzed array data in relation to the log of the expression values are shown as a figure. Ingenuity Pathway Analysis determined upstream regulators of differently expressed genes as presented in a table. These data have been further analyzed and interpreted in the companion article "A High-Androgen Microenvironment Inhibits Granulosa Cell Proliferation and Alters Cell Identity" (McFee et. al., 2021 [1].

A high-androgen microenvironment inhibits granulosa cell proliferation and alters cell identity.

A naturally occurring bovine model with excess follicular fluid androstenedione (High A4), reduced fertility, and polycystic ovary syndrome (PCOS)-like characteristics has been identified. We hypothesized High A4 granulosa cells (GCs) would exhibit altered cell proliferation and/or steroidogenesis. Microarrays of Control and High A4 GCs combined with Ingenuity Pathway Analysis indicated that High A4 GCs had cell cycle inhibition and increased expression of microRNAs that inhibit cell cycle genes. Granulosa cell culture confirmed that A4 treatment decreased GC proliferation, increased anti-Müllerian hormone, and increased mRNA for CTNNBIP1. Increased CTNNBIP1 prevents CTNNB1 from interacting with members of the WNT signaling pathway thereby inhibiting the cell cycle. Expression of CYP17A1 was upregulated in High A4 GCs presumably due to reduced FOS mRNA expression compared to Control granulosa cells. Furthermore, comparisons of High A4 GC with thecal and luteal cell transcriptomes indicated an altered cellular identity and function contributing to a PCOS-like phenotype.

Attainment and maintenance of pubertal cyclicity may predict reproductive longevity in beef heifers†.

We hypothesized the manner that heifers achieve puberty may indicate their future reproductive longevity. Heifers with discontinued or delayed cyclicity during puberty attainment may have irregular reproductive cycles, anovulation, and infertility in their first breeding season contributing to a shorter reproductive lifespan. Therefore, plasma progesterone (P4) was measured from weaning to breeding on 611 heifers born 2012-2017 and four pubertal classifications were identified: (1) Early; P4 ≥ 1 ng/ml < March 12 with continued cyclicity, (2) Typical; P4 ≥ 1 ng/ml ≥ March 12 with continued cyclicity, (3) Start-Stop; P4 ≥ 1 ng/ml but discontinued cyclicity, and (4) Non-Cycling; no P4 ≥ 1 ng/ml. Historical herd records indicated that 25% of heifers achieved puberty prior to March 12th in the 10 years prior to the study. Start-Stop and Non-Cycling yearling heifers were lighter indicating reduced growth and reproductive maturity traits compared with Early/Typical heifers. In addition, Non-Cycling/Start-Stop heifers were less responsive to prostaglandin F2 alpha (PGF2α) to initiate estrous behavior and ovulation to be artificially inseminated. Non-Cycling heifers had fewer reproductive tract score-5 and reduced numbers of calves born in the first 21-days-of-calving during their first breeding season. Within the Start-Stop classification, 50% of heifers reinitiated cyclicity with growth traits and reproductive parameters that were similar to heifers in the Early/Typical classification while those that remained non-cyclic were more similar to heifers in the Non-Cycling group. Thus, heifers with discontinued cyclicity or no cyclicity during puberty attainment had delayed reproductive maturity resulting in subfertility and potentially a shorter reproductive lifespan.

Evaluation of estrous synchronization protocols on ewe reproductive efficiency and profitability.

A 2-year experiment was conducted to determine the effect of a single injection of prostaglandin after initiation of the breeding season on ewe estrous synchronization. Rambouillet ewes (n = 101; Year 1 = 52; Year 2 = 49) assigned to one of three treatments: untreated (CON); 12-d CIDR insert (CIDR); or 1 injection of prostaglandin at d 2.5 (1 PG) after rams were placed with ewes. Rams were placed with ewes at the time of CIDR removal (d 0) and remained with ewes during a 35-d breeding season. Both the CIDR- (94%) and 1 PG (73.5%) treatment groups had a larger number (P ≤ 0.01) of ewes bred in the first 5 d of the breeding season compared to ewes of the control (33%) group. As expected, CIDR-treated ewes had a shorter time to mating, than 1 PG-treated ewes and ewes of the control group had a longer interval to mating than both CIDR- and 1 PG-treated ewes (P ≤ 0.01). The number of lambs born per ewe and kg of lamb weaned per ewe was not different (P ≥ 0.31) among treatment groups. Additionally, there was no difference (P =  0.78) in net profit per ewe among treatment groups. Based on these data, utilizing a single injection of PG 2.5 d after initiation of the breeding season resulted in similar pregnancy rates at d 5 of the breeding season when compared with CIDR-treated ewes indicating the potential utilization of the 1PG protocol in a confinement setting as a viable method for estrous synchronization.

Brangus cows have ovarian reserve parameters more like Brahman than Angus cows.

Bos indicus females have more surface antral follicles than Bos taurus females; however, histological studies demonstrated no difference in total number of primordial follicles between these two biological types of cattle. Primordial follicle density in the ovary was less in Nelore ovaries compared to Angus ovaries, but no studies have examined the primordial follicle density in Bos indicus cross-bred females. It, therefore, was hypothesized that primordial follicle density in the ovary would decrease as percentage Bos indicus increased. Ovaries were collected from cross-bred Angus (n = 32, no Bos indicus influence), Brangus (n = 15), or Brahman (n = 9) cows and prepared for histological evaluation. There was no difference in total number of primordial follicles per ovary between breeds (P > 0.10). When numbers of primordial follicles were expressed on a per gram of ovarian tissue basis, there were fewer primordial follicles per gram of ovarian tissue in Brangus and Brahman cows than in Angus cows (P < 0.05). Brangus cows did not differ from Brahman cows in primordial follicle density (P > 0.10). Differences in primordial follicle density could indicate differences in capacity of ovarian stroma to produce factors necessary for oogonial proliferation and primordial follicle formation among breeds. Identifying these factors could improve the aprroach for culturing pre-antral follicles of cattle. Furthermore, these results explain why ultrasonographic antral follicle counts may need to be adjusted to a greater threshold to predict size of the ovarian reserve and determine ovarian reserve related reproductive traits in Bos indicus females.

Developmental Resiliency: In Utero Adaption to Environmental Stimuli.

Stimuli experienced in utero can have a lasting impact on livestock growth, reproduction, and performance. Variations in environment, production system, and management strategies lead to discrepancies in the literature regarding how specific treatments influence animal performance. Studies comparing the influence of maternal undernutrition to well-fed counterparts typically result in decreased productivity of offspring. Via adaptation to nutritional or environmental stressors, dams may develop mechanisms to ensure proper nutrient supply to the fetus. It appears nutrient deprivation must be severe for consistent results. Potential mechanisms for altered performance in grazing systems and overnutrition settings are discussed.

The effect of precipitation received during gestation on progeny performance in Bos indicus influenced beef cattle.

The objective of this retrospective study was to determine the effect of precipitation level during key fetal development periods on beef progeny performance. The hypothesis that was precipitation level during different periods of gestation would program subsequent calves for an environment similar to that experienced in utero resulting in altered growth and reproductive performance. Data were collected on Brangus cows (n = 2,429) over a 46-yr span at the Chihuahuan Desert Rangeland Research Center. Recorded precipitation values were used to calculate average precipitation associated with total gestation (April-March), early gestation, (July-September), and late gestation (December-February). These values were used to classify treatments: low (z value ≤ -1.00), average (z value -0.99 to +0.99), and high (z value ≥ +1.00) for each time period. Calves experiencing high precipitation throughout gestation had heavier body weight (BW) at birth (P = 0.02), weaning (P = 0.05), and adjusted 205-d BW (P = 0.04) than those experiencing low precipitation . Female progeny gestated during low precipitation throughout gestation were more likely to remain (P < 0.0001) in the herd and calve after the age of 8 yr when compared to heifers experiencing high precipitation levels in utero (38% vs. 16% ± 5%, respectively). In addition, a greater percentage (P < 0.0001) of heifers experiencing low precipitation levels during the early gestation period produced a calf within the herd after 8 yr of age. Similarly, calves experiencing low precipitation during those same time points also had a greater number of calves while in production (P < 0.0001) when compared to the average and high precipitation groups. These results indicate that selection of heifers exposed to lower than average precipitation levels in utero may result in increased herd retention and productivity.

BEEF SPECIES-RUMINANT NUTRITION CACTUS BEEF SYMPOSIUM: Influence of management decisions during heifer development on enhancing reproductive success and cow longevity1.

Profitability of beef cattle producers can be directly tied to the productive lifespan of a cow. Management decisions influencing heifer nutrition and reproduction play a key role in establishing heifer fertility and longevity. Altering feeding strategies to utilize compensatory growth has been reported to have a positive influence on fertility during the first breeding season; however, there are discrepancies in the literature as to the impact this strategy may have on the proportion of heifers attaining puberty prior to entering their first breeding season. Ultimately, this may affect lifetime productivity as heifers born early in the calving season produce more kilograms of weaned calf over their first 6 calves, as well as, remain in the herd longer than those animals born later in the calving season. Thus, incorporation of reproductive technologies to identify fertile animals or aid in improving conception earlier in the breeding season may improve heifer reproductive performance and longevity. Reproductive tract scores have been utilized to identify animals which are more reproductively mature, increasing the likelihood of successful artificial insemination. Antral follicle counts (AFC), which predict the number of follicles in the ovarian reserve, have not only been reported to have a moderate to high heritability, but have also been used to identify animals with greater reproductive potential. Beef heifers classified as high AFC have greater pregnancy rates and longevity than low AFC heifers. Additionally, maternal diet may play a role in influencing reproductive success and ultimately cow longevity. Improving maternal plane of nutrition has been reported to increase female offspring pregnancy rates. Overall, recent research has provided producers with a variety of methods to optimize animal performance while enhancing reproductive success and longevity to ensure profitability.

Growth and Reproductive Performance of Rangeland Beef-Cattle as Influenced by Controlled and Uncontrolled Populations of Horn Flies (Diptera: Muscidae).

A 4-yr study was conducted on native rangeland to assess the growth and reproductive performance of cows (Bos taurus) infested with naturally occurring seasonal populations of horn flies (Haematobia irritans). One hundred five Angus × Hereford cow-calf pairs were evaluated as a randomized complete block that was replicated across 4 yr. Cows were approximately 39 d postpartum at the beginning of each yearly trial and were randomly allocated to either an untreated control (UTC) or an insecticide-treated (TRT) herd. Horn fly populations were monitored throughout each yearly replication and blood serum progesterone levels were used to estimate postpartum interval lengths and days to pregnancy. Initial body weights of cows were collected in May with final body weights and calf weaning weights acquired in October of each year. Monthly horn fly control ranged from 85.55 to 99.57% throughout the 4 yr. Cows within UTC herds maintained on average 530.10 ± 94.74 more (P = 0.0015) flies per animal than TRT. However, no differences were detected between treatment groups for any of the reproductive parameters evaluated (P > 0.05). Despite a lack of difference in the reproductive parameters measured, TRT cows gained more (P = 0.0492) weight throughout the fly seasons when compared to UTC cattle. Furthermore, calves paired with insecticide-treated cows tended (P = 0.0680) to wean 16.28 ± 8.04 kg heavier than calves paired with cows exposed to naturally occurring horn fly populations.

Maternal age influences the number of primordial follicles in the ovaries of yearling Angus heifers.

The number of antral follicles detectable by ultrasonography in heifers is influenced by age of the dam, because daughters of primiparous cows have fewer antral follicles than daughters of mature cows. We, therefore, hypothesized that heifers with primiparous dams would have fewer primordial follicles in their ovaries than heifers born to mature (4+ y) cows. Angus heifers (n = 464) were submitted for ultrasonographic evaluation of antral follicle number at 325, 355, and 385 d of age. Ovaries were collected from a random subset of heifers (n = 79) and processed for histological evaluation to determine number of primordial follicles. A greater percentage of heifers with primiparous dams had a corpus luteum at first ultrasonographic examination; however, a greater percentage of heifers with multiparous dams had ovulated by the start of breeding (P < 0.01). Heifers with primiparous dams had fewer antral follicles detectable by ultrasonography (P < 0.01). Heifers with primparous dams had fewer surface antral follicles on their ovaries (P < 0.01), and the number of primordial follicles per histological section was less for heifers with primiparous dams (P = 0.02). These data indicate that the lesser number of antral follicles detectable by ultrasonography in heifers with primparous dams is due to less ovarian follicle reserves. Selecting replacement heifers from mature dams may result in daughters with greater fertility and reproductive longevity; however, further research is necessary to determine if interactions between size of the ovarian follicle reserve and age at puberty influence fertility and reproductive longevity in replacement heifers.

Transcriptomes of bovine ovarian follicular and luteal cells.

Affymetrix Bovine GeneChip® Gene 1.0 ST Array RNA expression analysis was performed on four somatic ovarian cell types: the granulosa cells (GCs) and theca cells (TCs) of the dominant follicle and the large luteal cells (LLCs) and small luteal cells (SLCs) of the corpus luteum. The normalized linear microarray data was deposited to the NCBI GEO repository (GSE83524). Subsequent ANOVA determined genes that were enriched (≥2 fold more) or decreased (≤-2 fold less) in one cell type compared to all three other cell types, and these analyzed and filtered datasets are presented as tables. Genes that were shared in enriched expression in both follicular cell types (GCs and TCs) or in both luteal cells types (LLCs and SLCs) are also reported in tables. The standard deviation of the analyzed array data in relation to the log of the expression values is shown as a figure. These data have been further analyzed and interpreted in the companion article "Gene expression profiling of ovarian follicular and luteal cells provides insight into cellular identities and functions" (Romereim et al., 2017) [1].