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.

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.

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.

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.

Effect of progesterone supplementation in a resynchronization protocol on follicular dynamics and pregnancy success.

The objective of this study was to evaluate the necessity of a controlled internal drug releasing device (CIDR) in a fixed-time AI resynchronization protocol as well as to compare a commercially available blood pregnancy test with transrectal ultrasonography for Day 28 pregnancy detection. Over a two-year period, beef cows and heifers from twelve herds were inseminated using the 7-day CO-Synch + CIDR protocol. On Day 21 following the first insemination, the protocol was repeated, with animals receiving either a CIDR or no CIDR. Pregnancy status (AI1) was determined on Day 28 by both transrectal ultrasonography and the IDEXX Rapid Visual Pregnancy Test. Non-pregnant animals by both methods (CIDR: n = 190 cows, n = 228 heifers; no CIDR: n = 185 cows, n = 223 heifers) received an injection of Prostaglandin F2alpha and were inseminated at the appropriate time or bred following detection of estrus. Corpora lutea (CL) number and largest follicle diameter were recorded on a subset of non-pregnant animals (CIDR: n = 66 cows, n = 46 heifers; no CIDR: n = 76 cows, n = 41 heifers) at time of pregnancy diagnosis on Day 28. Final pregnancy status was determined a minimum of 31 days following the second AI (AI2). The GLIMMIX procedure of SAS was utilized for estrus and pregnancy data; while the MIXED procedure was utilized for analyses of CL number and largest follicle diameter. There was no effect (P ≥ 0.55) of treatment on AI1 pregnancy, AI2 pregnancy, or overall pregnancy rates. The presence of a CIDR during the resynchronization increased (P < 0.001) estrus expression prior to AI2. There was an effect of treatment by age on AI2 pregnancy (P < 0.01); heifers that received a CIDR had greater AI2 pregnancy rates than heifers that did not receive a CIDR (P = 0.04), but there was no difference between cows with and without a CIDR. Treatment had no effect (P > 0.10) on embryonic loss (between the first and second pregnancy diagnosis), CL number, or follicle diameter. Although, there was a tendency for the interaction of treatment by age on follicle size (P = 0.07), with cows having larger follicles than heifers in the no CIDR group but not the CIDR group. In conclusion, use of a CIDR in this resynchronization protocol increased estrus expression, increased AI2 pregnancy for heifers, but did not improve pregnancies in cows, and did not influence overall pregnancy or embryonic loss.

Role of preovulatory concentrations of estradiol on timing of conception and regulation of the uterine environment in beef cattle.

The ability to induce ovulation with an injection of GnRH facilitated the development of fixed-time artificial insemination (AI) protocols. However, animals that exhibit estrus prior to fixed-time AI have greater pregnancy success. Thus, the objectives of the present experiments were to determine the impact of estrus expression prior to fixed-time AI on timing of conception and to characterize the role of preovulatory estradiol in regulating changes in expression of uterine genes. In experiment 1, data were collected from 4,499 beef cows inseminated by fixed-time AI in 31 different herds. Animals that did not conceive to AI but exhibited estrus before timed-AI were more likely to conceive during cycle 1 after AI, and overall conceived earlier in the breeding season compared to animals that did not exhibit estrus. In experiment 2, beef cows were synchronized using a fixed-time AI protocol. Uterine horn biopsies and blood samples were collected on Day 0, 5, 10, or 16. Concentrations of estradiol on Day 0 did not influence expression of progesterone receptor, ER beta, or oxytocin receptor. Increased concentrations of estradiol on Day 0 increased expression of ER alpha from Days 0 to 5 of the estrous cycle. Furthermore, cows with increased concentrations of estradiol on Day 0 had increased expression of inhibin beta A, and uterine milk protein precursor. Thus, animals that do not exhibit estrus prior to fixed-time AI had decreased breeding season pregnancy success and conceived later in the breeding season, and preovulatory concentrations of estradiol likely play a major role in this establishment of pregnancy, not only directly by regulating uterine gene expression, but also indirectly throughout the subsequent estrous cycle.

Influence of Sexcel™ (gender ablation technology) gender-ablated semen in fixed-time artificial insemination of beef cows and heifers.

Previous research has indicated that gender-sorted semen has decreased conception rates compared to conventional semen. A new method to skew the gender ratio of semen has been developed that does not use traditional sorting technology. Thus, the objective of this study was to evaluate the use of gender-ablated semen in a fixed-time AI protocol. Beef heifers and cows (n = 878) from six herds were synchronized with the 7-d CO-Synch plus CIDR protocol, and artificially inseminated (AI) after CIDR removal (cows 60-66 h; heifers 52-56 h). Estrus detection aids were applied at CIDR removal and estrus activity was determined at time of AI. Animals remained separated from bulls for at least 10 d after AI. Pregnancy success and fetal age were determined between d 28 and 70 after AI. Semen from five sires was utilized in this study, with two sires used in each herd. Sire 1 was used in all herds and the use of the second sire varied by herd (both conventional and gender-ablated semen from each sire was used in each herd). Data were analyzed using the GLIMMIX procedure in SAS and included the influence of semen type, estrus expression, semen type by estrus expression, dam age, and sire in the model. Herd was included as a random variable. Overall, conventional semen had greater conception rates compared to gender-ablated semen (P < 0.01; 67% vs 52%), and cows that had fully activated patches and partially activated patches had greater conception rates compared to animals that had not exhibited estrus (P < 0.01; 69%, 65%, and 45%). There was no semen type by estrus expression interaction (P = 0.24). Conception rates tended to be greater for conventional semen among animals that had activated patches (P = 0.06; 73% vs 65%) and partially activated patches (P = 0.06; 72% vs 59%). Conception rates were greater for conventional semen (P < 0.01; 56% vs 33%) among animals that did not exhibit estrus. There was no effect of dam age (P = 0.40) or sire (P = 0.92) on conception rates. In conclusion, Sexcel™ gender-ablated semen can successfully be used in fixed-time AI protocols among animals that exhibit estrus, but caution should be used among animals that do not exhibit estrus.

Oxylipin concentrations in bovine corpora lutea during maternal recognition of pregnancy.

The objective was to determine the effects of pregnancy status on oxylipin profiles and eicosanoid metabolizing enzymes and in corpora lutea (CL) or endometrial (caruncle; CAR and intercaruncle; IC) tissues. Angus crossed cattle were synchronized with the CO-Synch protocol and artificially inseminated (AI). Sixteen days after AI, cattle were euthanized, and reproductive tracts collected from 6 non-pregnant and 6 pregnant cows. Oxylipin profiles and concentrations of progesterone (P4) were obtained from CL tissues. The activity of cytochrome P450 1A (CYP1A) and UDP-glucuronosyltransferase (UGT) enzymes were determined using specific luminogenic substrates. Data were analyzed using the MIXED procedure of SAS, and the model included pregnancy status. Corpora lutea of pregnant cattle contained greater (P < 0.05) concentrations of 9,10-DiHODE, 15,16-DiHODE, and 9,10-DiHOME. These oxylipins have been observed to increase cellular proliferation and vasodilation. Activity of CYP1A in the CL and UGT in CAR and IC was not different (P > 0.05) between pregnant and non-pregnant cattle. In the CL, activity of UGT was decreased (P < 0.05) in pregnant vs. non-pregnant cattle. The decrease in CL UGT activity during pregnancy indicates alterations in local hormone metabolism, while no differences in CL weight nor amount of P4 in CL were different between pregnant and non-pregnant cattle. Moreover, the increase in specific concentrations of oxylipins in the CL may indicate a novel pathway of steroid and eicosanoid metabolism during maternal recognition of pregnancy.

Influence of GnRH supplementation at CIDR removal on estrus expression and interval to estrus in beef cattle.

Previous research has indicated that multiple small doses of GnRH following CIDR removal increased circulating concentrations of estradiol. Therefore, our objective was to determine if a single small dose of GnRH (5 µg or 10 µg) at CIDR removal would impact expression of estrus and/or interval to estrus. Beef cows and heifers (n = 1620; n = 1057 cows, n = 563 heifers) were synchronized using the 7-day CO-Synch + CIDR protocol, and randomly assigned to one of three treatments (0, 5, or 10 µg of a GnRH analog at CIDR removal). Animals were inseminated following detection in estrus. Interval to estrus was calculated for each animal that exhibited estrus (INTERVAL 1). Animals that did not exhibit estrus were administered 100 µg of GnRH at the time of AI and their interval to estrus was designated at 120 h (INTERVAL 2). There was a treatment by age interaction (P = 0.05) on INTERVAL 1. Heifers treated with 5 µg of GnRH tended to have a shorter interval to estrus (P = 0.07; 47 ±â€¯1.4 h) compared to 0 µg (50 ±â€¯1.5 h) and did have a shorter interval compared to 10 µg (P < 0.01; 52 ±â€¯1.5 h). There were no differences between treatments in interval to estrus among cows (P > 0.34). When animals that did not exhibit estrus by 120 h were included in the analysis there was no treatment by age interaction (P = 0.49). This is likely due to the fact that treatment (P < 0.01), but not age (P = 0.96) or treatment by age (P = 0.74) influenced expression of estrus. In addition, there tended to be a treatment by estrus interaction (P = 0.08) on pregnancy success. There was no difference in pregnancy success between treatments among animals that exhibited estrus (P > 0.30). In summary, 5 µg of GnRH at CIDR removal tended to decrease the interval to estrus and increased expression of estrus among heifers but not cows, and 10 µg of GnRH at CIDR removal did not improve estrus expression and lengthened the interval to estrus in comparison to the control.

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.