Fixed-time Artificial Insemination Protocol Research Articles

PSVI-24 Gene expression profiles of granulosa and peripheral white blood cells from fertile and sub-fertile beef heifers

Abstract Beef heifer fertility issues contribute to a major economic loss in the cow-calf production industry. Therefore, identifying beef heifers with superior genetic potential for improved fertility would increase profitability. This study aimed to identify differences in the transcriptome profiles from granulosa and peripheral white blood cells (pWBCs) of beef heifers with varying reproductive potential. For this, Angus-Simmental crossbred heifers were subjected to an estrus synchronization and fixed-time artificial insemination (AI) protocol (7-D CO-Synch + CIDR) followed by exposure for 60-d to a fertile bull. Depending on the presence or absence of conceptus 120 d post-AI, heifers were classified as fertile (pregnant by AI) or sub-fertile (non-pregnant by AI or bull-breeding). Pregnancies were terminated, and all animals in both groups (fertile, n = 8; and sub-fertile, n = 5) were cycling when the blood and ovaries were collected from each heifer. Total RNA was extracted from the pWBCs and granulosa cells and subjected to library preparation and sequencing on the Nova-Seq platform. The read counts were obtained after data quality control using FastQC v0.11.9 and MultiQC v1.12 and alignment to the Ensemble’s ARS UCD1.2 Bos taurus genome reference using STAR aligner v2.7.5. The filtered data were subjected to differential expression analysis using DESeq2. We identified 1,061 and 72 significantly differentially expressed genes (DEGs) with P-values ≤ 0.05 and absolute (log2 fold change) ≥ 0.5 from pWBC and granulosa, respectively. Notably, 12 targets including 9 protein coding genes (PLCL1, DNER, GNAS, CDH3, PER1, ITGA2B, CXCL12, ENSBTAG00000048613, ENSBTAG00000051519), bta-mir-2887-1, 5-8S-rRNA and U5 were found as DEGs in both the tissues. Based on a differential co-expression analysis using PCIT, we identified GNAS and DNER as hub genes in pWBC and the granulosa cells of the sub-fertile heifer group. The 12 shared genes were over-represented for pathways such as NF kappa B and chemokine signaling, regulation of actin cytoskeleton, and platelet activation. Some of the identified genes have been previously associated with fertility, while others are novel. A detailed understanding of the underlying biological mechanisms of the top genes and a follow-up study with a larger sample size at different time points could validate the candidates identified in this study for their role as potential therapeutic targets.

PSV-25 Investigating the effects of Chromium Propionate supplementation on productive and reproductive performance of beef cows

Abstract We investigated the effects of Chromium Propionate supplementation on reproductive performance of beef cows from pre-partum to weaning. Suckled beef cows at nine locations were enrolled in the study. Within location, cows were stratified by parity, dam age, and day postpartum (DPP) and randomly assigned to one of two treatments: 1) Control, supplementation of a mineral product at 113 gּ animal-1ּ d-1 (n = 464 cows; 13 experimental units); or 2) CrP, supplementation of mineral product at 113 gּ animal-1ּ d-1 containing 1.4 g of Chromium Propionate (0.4%KemTRACE Chromium 0.4%, Kemin Industries, Inc., Des Moines, IA; n = 489 cows; 13 experimental units). Treatments were initiated at approximately 37 d pre-calving until weaning for a total of 250 ± 12 d. Cows were enrolled in the 7-d CO-Synch+CIDR fixed-time artificial insemination (TAI) protocol followed by natural service for a 70-d breeding season. Pregnancy diagnosis was performed by ultrasonography at 55 d after TAI protocol and 40 d after the end of the breeding season. Cow body weight (BW) and body condition score (BCS) were recorded at initiation of the TAI protocol (d -10), at pregnancy diagnosis (d 55 and 110), and at weaning (d 285). Within group, mineral disappearance was determined by the difference in weight between offered mineral and refusals. Data were analyzed using the GLIMMIX and MIXED procedures of SAS with pasture/group as the experimental unit with the effect of treatment and included group (treatment × location), cow (group), and location as random variables. Age, DPP, d -10 BCS, and BW were similar (P > 0.05) between treatments (5.4 and 5.3 ± 0.61 yr, 79.2 and 79.9 ± 4.61 d, 5.1 and 5.2 ± 0.05, 515.7 and 515.6 ± 15.11 kg for Control and CrP, respectively). Mineral disappearance was similar (P = 0.999) between treatments (165.5 and 152.9 gּ animal-1ּ d-1, for Control and CrP, respectively). Cow BW was similar (P > 0.05) between treatments on d 55, 110 and 285 (558.5 and 551.4 ± 14.50 kg, 580.8 and 594.6 ± 13.51 kg, and 574.2 and 570.9 ± 14.32 kg for Control and CrP, respectively). Similarly, cow BCS did not differ (P > 0.05) between treatments on d 55, 110 and 285 (5.4 and 5.4 ± 0.09, 5.3 and 5.3 ± 0.10, and 5.1 and 5.2 ± 0.10 for Control and CrP, respectively). Calf birth and weaning BW were similar (P > 0.05) between treatments. However, CrP-supplemented cows tended (P = 0.081) to have greater estrus expression (60.2 and 68.3 ± 3.1 %, for Control and CrP, respectively) and CrP-supplemented cows had greater (P = 0.045) TAI pregnancy rate when compared with Control cows (49.9 and 55.2 ± 2.1 %). Supplementation of Chromium Propionate to beef cows from pre-partum through weaning did not affect cow BW and BCS, tended to increase estrus expression and increased TAI pregnancy rate.

PSIII-10 Flaxseed oil-based supplement alters water consumption and plasma fatty acid profile of beef heifers

Abstract The objective of this study was to determine the effects of a flaxseed oil-based supplement (FlaxLic, New Generations, SD) on reproductive parameters, feeding behavior, and lipid profile in beef heifers. Crossbred Angus heifers [n = 60 body weight (BW) = 397kg ± 5.2 kg) were blocked by BW and antral follicle count and randomly assigned to two supplementation treatments: free-choice commercial lose mineral supplement (CON; n = 30) or free-choice flaxseed oil-based supplement (FLAX; n = 30) for 7 wk. Heifers were individually fed via the Insentec feeding system (Insentec RIC, Hokofarm, Marknesse, Netherlands), where basal diet (TMR) and supplements were provided in separate feeders to measure supplement intake and feeding behavior. Further, water intake and behavior were measured via Insentec water system. Individual intake was compiled over 24 h for each animal. Body weight, transrectal ultrasonography, and blood samples were collected weekly. On wk 5, heifers were enrolled in a fixed-time artificial insemination (FTAI) commercial protocol. Largest follicle diameter (LF) and corpus luteum (CL) presence were recorded weekly and on d of the FTAI protocol (d 0, d 7, and d 0). Pregnancy diagnosis was performed 30 d and 60 d following AI. Blood samples were analyzed for progesterone (P4) by radioimmunoassay (RIA) to determine puberty attainment before wk 5, and fatty acid profile was determined via gas chromatography-flame ionization detection. Heifers with a CL or P4 ≤ 1.0 ng/mL were considered pubertal. Data were analyzed using SAS 9.4. ADG was similar between groups (P = 0.90) but was affected by week (P < 0.01). TMR intake was similar in duration and quantity (P > 0.18) between groups. Supplement intake was greater (P < 0.01) for FLAX (0.73 ± 0.03 kg/d) compared with CON (0.18 ± 0.02 kg/d) and from wk 2 to wk 8 (P = 0.01). Time spent consuming the supplements was greater for FLAX compared with CON heifers (62.65 ± 2.11 vs 0.10 ± 0.11 min/d, respectively) over the entire trial (P < 0.01). Further, FLAX heifers consumed less water than CON heifers (32.2 ± 0.39 vs 33.93 ± 0.35 kg/d, respectively; P = 0.03) and from wk 2 to wk 7 (P = 0.03). LF was not different between groups on any of the days of the protocol (d 0, d 7, d 0; P > 0.20). Additionally, P4 (P ≤ 0.20), puberty attainment by wk 5 (P = 0.24), and pregnancy rates (P > 0.48) were similar between groups. Concentrations of arachidonic acid (3.00 ± 0.24 vs 2.32 ± 0.19 mg/mL; P = 0.04), linoleic (60.11 ± 4.34 vs 40.35 ± 4.04 mg/mL; P = 0.002), αLinolenic (16.73 ± 0.26 vs 15.86 ± 0.23 mg/mL; P = 0.02), and total polyunsaturated fatty acids (PUFA; 130.52 ± 5.97 vs 111.19 ± 5.56 mg/mL; P = 0.02) were greater on wk 8 for FLAX compared with CON heifers. Collectively, these findings did not show evidence that the flaxseed oil-based supplement affects variables that impact reproductive performance. However, it altered the plasma lipid profile, increasing omega-3, arachidonic, and linoleic fatty acids, as well as total PUFA.

185 Effect of synchronized fixed-time AI and non-pregnant heifer delayed feedlot entry on reproductive performance and system economics

Abstract Heifer reproduction management research is being conducted at the NDSU–Dickinson Research Extension Center. Using crossbred heifers, the objective was to evaluate reproductive performance and economic efficiencies comparing a drylot synchronized fixed-time AI protocol with clean-up bulls to two grazing systems using a fixed-time AI protocol without clean-up bulls. Synchronization protocol:14-d CIDR-PG-GnRH (TAI 66 h ± 2 h). Fixed-time AI treatments: 1) Drylot Control (DLOT) with clean-up bulls, 2) Integrated diverse crop-heifer annual forage grazing (IAF, no clean-up bulls), and 3) Native range grazing (NR, no clean-up bulls). Following pregnancy ultrasound on d 85, non-pregnant heifers continued to bale-graze until late November, when they were transferred to the University of Wyoming SAREC feedlot, finished, harvested at Cargill Meat Solutions packing plant, Ft. Morgan, CO, and grid marketed. Data were analyzed using the MIXED procedure of SAS. Year 1 and 2 grazing days were 121, and 149, respectively. Synchronized TAI pregnancy rates yr 1 and 2 for DLOT, IAF, and NR were 59.4, 43.8, and 62.5%; 68.8, 46.9, and 34.4%, respectively. Total DLOT control pregnancy rates yr 1 and 2 were 90.6 and 100%. Grazing heifer feedlot performance Yr 1 did not differ for starting body weight (BW; 452 vs 436 kg, P = 0.29), ending BW (610 vs 600 kg, P = 0.74), gain (157 vs 164 kg, P = 0.69), average daily gain (ADG; 1.42 vs 1.48 kg, P = 0.70), dry matter (DM) feed intake (15.0 vs 14.7 kg, P = 0.73), F:G (4.81 vs 4.56 kg, P = 0.42), daily feed cost ($7.45 vs $9.47, P = 0.73), feed cost/kg BW gain ($3.11 vs $2.95, P = 0.51), and feed and yardage cost ($3.41 vs $3.24, P = 0.55). Year 2 grazing heifer feedlot performance for the IAF and NR were starting BW (501 vs 456 kg, P = 0.007), ending BW (670 vs 604 kg, P = 0.004), feedlot finishing gain (143 vs 123 kg, P = 0.05), ADG (1.08 vs 0.93 kg/day, P = 0.06), daily DM feed intake (20.1 vs 20.1 kg/d, P = 0.98), feed to gain ratio (7.13 vs 8.22 kg/d, P = 0.11), daily feed cost ($9.61 vs $9.54/d, P = 0.94), feed cost/kg BW gain ($4.05 vs $4.67/kg, P = 0.11), and feed and yardage cost/kg gain ($4.45 vs $5.13/kg, P = 0.092). IAF and NR Yr 1 HCW (P = 0.83), dressing % (P = 0.69), marbling score (P = 0.78) did not differ. Year 2, except for hot carcass weight (HCW; 379 vs 341 kg, P = 0.004), dressing pct (P = 0.99), fat depth (P = 0.29), marbling score (P = 0.94) did not differ. Systems 2-yr mean net return for DLOT, IAF, and NR were $592, $296, $379, respectively. Systems net return profitability was very good for all management comparisons, but reduced for IAF and NR due to IAF farming, finishing, and transportation costs, and NR finishing and transportation costs.

10 Awardee Talk: Pregnancy losses in Bos indicus-influenced cowherds

Abstract Pregnancy loss is recognized as a major reproductive challenge in beef cattle operations. Most pregnancy losses are associated with early embryonic mortality, but limited research evaluated pregnancy losses throughout the gestation period. Research is warranted to address this gap in knowledge, particularly fetal losses in Bos indicus-influenced females. This subspecies predominates in tropical regions of the planet, which contain more than 80% of the world’s cattle population and supply a major portion of the global demand for beef products. Our group recently evaluated pregnancy losses from d 30 of gestation to calving in 4,926 Nelore females assigned to an ovulation synchronization + fixed-time artificial insemination (FTAI) protocol. Pregnancy loss was greater (P < 0.01) from d 60 to 150 of gestation (10.2%) compared with d 30 to 60 of gestation (6.0%) and with d 150 of gestation to calving (7.4%) and differed (P = 0.04) between these latter two periods. Pregnancy loss from d 30 to 60 of gestation did not differ (P = 0.26) among cow parities (nulliparous, primiparous, and multiparous), whereas total pregnancy losses (d 30 to calving) were greater (P < 0.01) in precocious (28.4%) and conventional (27.1%) heifers compared with primiparous (16.4%) and multiparous (13.0%) cows. In a second experiment, pregnancy losses from fixed-time embryo transfer (FTET) to calving were evaluated in 4,366 FTET events using Nelore × Angus recipient heifers. Pregnancy losses were greater (P < 0.01) from FTET to d 32 of gestation compared with d 32 to 100 and with d 100 to calving (58.7, 39.5, and 36.7%, respectively), and did not differ (P = 0.56) between these latter periods. Recipients that lost the pregnancy from FTET to d 32 gained less (P < 0.01) body condition score after FTET compared with recipients that maintained the pregnancy. Pregnancy losses from d 32 to calving were greater (P < 0.01) in recipients reared in drylots and moved to pastures on d 32 compared with recipients reared on pasture. Collectively, these results provide novel information about pregnancy losses in Bos indicus females, with a substantial proportion of losses occurring as fetal mortality.

PSI-10 Evaluation of factors influencing conception rate within a fixed-time artificial insemination protocol using sex-sorted semen

PSI-10 Evaluation of factors influencing conception rate within a fixed-time artificial insemination protocol using sex-sorted semen

Association of genomic prediction for twin pregnancies with the incidence of double ovulation in primiparous dairy cows.

Twin pregnancies compromise the health and well-being of dairy cattle. A recent genomic prediction model for twin pregnancies has been developed based on twin calving or abortion. However, the incidence of double ovulation is significantly higher than that of twin births. This study aimed to evaluate whether genomic prediction values for twin pregnancies are associated with the incidence of double ovulation in primiparous dairy cows. Factors influencing the double ovulation rate were analysed using binary logistic regression on 676 cows: 475 (70.3%) inseminated at spontaneous estrus and 201 following one of two different estrus synchronization protocols for fixed-time artificial insemination (FTAI). The odds ratio for double ovulations was 0.92 (p = .002) per unit increase in prediction value and 2 (p = .01) for cows subjected to an FTAI protocol. Our findings suggest that genomic prediction values for twin pregnancies can effectively identify the risk of double ovulation at the herd level.

Effect of three different fixed-time AI protocols on follicular dynamics and pregnancy rates in suckled, dual-purpose cows in the Ecuadorian Amazon

Effect of three different fixed-time AI protocols on follicular dynamics and pregnancy rates in suckled, dual-purpose cows in the Ecuadorian Amazon

Equine chorionic gonadotropin (eCG) treatment in heifers: Double ovulation, twinning rate, and pregnancy losses in twin pregnancies

This study aimed to evaluate the effects of different doses of equine chorionic gonadotropin (eCG; 200 and 300 IU) administered at the end of a fixed-time artificial insemination (FTAI) treatment protocol on ovulation, pregnancy, and twin rates in Bos taurus beef heifers. In addition, pregnancy losses in heifers with singleton and twin pregnancies were determined. A total of 2382 Angus heifers treated with a 6-day estradiol/progesterone-based protocol for FTAI (J-Synch protocol) were randomly allocated to two experimental groups to receive 200 or 300 IU of eCG administered intramuscularly at the time of intravaginal progesterone device removal; FTAI was performed from 60 to 72 h after device removal. The pregnancy rate did not differ (P = 0.89) between the 200 and 300 IU eCG groups. The number of corpus luteum induced by both eCG doses was determined by ultrasonographic examination 14 days after insemination and those treated with 300 IU of eCG had a greater double ovulation rate (P < 0.05). In addition, 300 IU eCG treated heifers had a higher twinning rate on day 30 of gestation (P < 0.05) and parturition (P < 0.05). Pregnancy losses from 30 days of gestation to calving did not differ between heifers treated with 200 and 300 IU of eCG (P = 0.70). However, regardless of the experimental group, heifers bearing twins had greater pregnancy losses than heifers with singletons (P < 0.05). In conclusion, reducing the dose of eCG from 300 to 200 IU under FTAI treatment protocol decreases double ovulation and twinning rates, maintaining a similar pregnancy rate in heifers. Nulliparous cows carrying two fetuses suffer greater pregnancy losses than cows with singletons.

Bovine somatotropin increases the pregnancy rate in fixed-time artificial insemination in beef cattle.

This study evaluated the effect of bovine somatotropin (bST) on pregnancy rate (PR) and size of the dominant follicle (DF) on the day of intravaginal progesterone (P4) removal in protocols for fixed-time artificial insemination (FTAI). Bos indicus (Nellore) females (n = 392) were distributed into three groups. The control group (CG; n = 92) received an intravaginal P4 device + estradiol benzoate on day (d)0; prostaglandin F2α on d7 (first application); removal of P4 + estradiol cypionate (EC) + PGF2α (second application) + ultrasound (US) of the DF on d9; the FTAI was performed on d11; and pregnancy diagnosis (PD) was performed on d45. The bST group (bSTG; n = 142) underwent the same protocol as the CG, except that the animals received 125 mg of bST on d7. The equine chorionic gonadotropin (eCG) group (eCGG; n = 158) underwent the same protocol as the CG, except that the animals received 300 IU of eCG on d9. The PRs of the bSTG, eCGG, and CG were 48%, 48%, and 35%, respectively (p < .05); the bSTG and eCGG showed greater PRs, with follicles 6-7.9 mm (p < .05) and 8-8.9 mm in diameter, respectively. The bSTG exhibited a greater dimension of the DF on d9 of the protocol (p < .05). The eCGG had higher PRs with a body condition score (BCS) of 2.5, and the bSTG had a BCS of 3.0 (p < .05). It was concluded that bST increased PR, bST showed better performance in smaller DF and larger follicular diameter on d9 of the protocol, eCG acted better on animals with lower BCSs, and bST can be used in FTAI.

Use of long-acting injectable progesterone in fixed-time artificial insemination protocols

Knowledge of the physiology of the estrous cycle in bovine females has been extremely important in monitoring and improving strategies in the use of drugs to control their estrous cycle and follicular development, providing better recruitment, selection, growth, and maturation until ovulation, with strategies to further optimize estrus and ovulation synchronization protocols. The objective of this work is to carry out a bibliographical analysis comparing the use of slow-release progesterone devices in relation to the use of long-acting injectable progesterone, taking into account their advantages, disadvantages and characteristics within a fixed-time artificial insemination program.

Combining Embryo Transfer and Artificial Insemination to Achieve Twinning in Beef Cattle, and Effects of Different Twin Calf-Raising Methods on Neonatal Behavior and Growth

As the beef industry moves towards efficient animal production to improve sustainability in agriculture, new production and management approaches are emerging. Among the many facets of the beef industry, cow–calf operations have the most opportunity for efficiency improvement, including improvements in fertility. This project accounts for measures and methods of (1) twinning reproductive technologies and (2) twin calf perinatal care and pre-weaning rearing. The overall objective was to produce twin calves using two reproductive technologies—embryo transfer and artificial insemination. The subobjectives were to determine accuracy of twin pregnancies embryo/fetal losses using ultrasonography, evaluate parturition and dystocia, and determine the effects of different twin-raising methods on neonatal behavior and growth. A fixed-time artificial insemination (FTAI) protocol was applied to 77 multiparous Angus-cross cows from a commercial beef herd in northcentral South Dakota during the summer of 2019. Cows were assigned to two different treatments groups: only artificially inseminated (AI) or received an embryo transfer following artificial insemination (ET + AI). They were estrous-synchronized, artificially inseminated (AI) with black Angus semen at day 0, and received and embryo transfer (ET) at day 7. Ultrasound examination detected 56% pregnancy risk for both groups, with sensitivity, specificity, and accuracy of 75%, 100%, and 90.5%, respectively, for bilateral twin detection. Calves were born during spring 2020. Twin calves (n = 34) and singleton calves (n = 11) were assigned to one of three raising methods: (1) twin born and twin raised (TT; n = 16), (2) twin born and single raised (TS; n = 18), and (3) single born and single raised (S; n = 11). Neonatal nursing behavior and birth weights were recorded, and adjusted day 200 and day 280 were calculated measures of vitality and growth. Blood samples were collected at age 24 h for colostrum intake measures (total serum protein, IgG1, and IgM). Twin calves were born 20% (p &lt; 0.05) lighter in body weight than singletons; however, weights did not differ at day 280 between TT and S calves. TS calves had the shortest average latency to stand, but immunoglobulin concentrations did not differ among treatments. At weaning, cows that had birthed and raised twins produced more kilograms of live weight per pregnancy than cows birthing and raising singletons. Using ET + AI proved to increase twinning rate, and growth was maintained when raising both twins with their dam.

130 Effect of an appeasing substance application on growth, stress, handling quality and reproduction of Bos indicus beef heifers synchronized to fixed-time AI

Abstract This experiment evaluated the effects of an analogue of bovine appeasing substance (BAS) on growth, stress, handling quality, and reproduction of Nellore heifers. Twenty-nine heifers [body condition score (BCS) = 4.84 ± 0.13; body weight (BW) = 371 ± 0.77 kg; 23 ± 2 months of age)] were assigned to 1 of 2 treatments: 1) Saline solution (0.9 NaCl; n = 14) or BAS (Secure Cattle, IRSEA Group, Quartier Salignan, France; n = 15). Both solutions were applied topically in the nuchal skin area (5 mL/heifers) at the beginning (d -9) of the fixed-time artificial insemination protocol (FTAI). Heifers were allocated in two different pastures according to the treatment (one pasture per treatment) for 15 d (time of action of BAS) and then combined in a single group (both treatments in the same pasture). Heifers were inseminated on d 0, and BW and BCS were collected on d -9 and 40. The temperament in the squeeze chute (entry and exit scores; scale 1 – 3; chute score; scale 1 – 5; greater values mean more reactive heifers) and blood samples were collected on d -9, 0, 7, 14 and 30. The percentual of aggression, accident, and dirtiness in the perineal region were evaluated on d 0 along with time to inseminate, estrus expression scores (scale 1 – 3; 1 = no expression, 2 = low expression, 3 = greater expression), and dominant follicle size. The corpus luteum size was measured on d 7 and 14, and the pregnancy and embryo size on d 40. The BAS vs. Saline-treated heifers tended to have greater BW change (23.6 vs. 20.8 ± 1.03 kg; P = 0.09) and had greater BCS change (0.05 vs. -0.21 ± 0.05; P &amp;lt; 0.01). The BAS vs. Saline-treated heifers tended to have lesser entry scores (1.42 vs. 1.67 ± 0.09; P = 0.09) and percentual of dirtiness in the perineal region (48.3 vs. 71.1 ± 8.67 %; P = 0.06), and no effects of treatment were detected (P ≥ 0.39) for chute score, exit score, aggression, accident and time to inseminate. The BAS vs. Saline-treated heifers had greater estrus scores (2.00 vs. 1.27 ± 0.21; P = 0.03), and no effects were detected (P ≥ 0.11) for ovarian variables, pregnancy rate, and conceptus size. Furthermore, no treatment effects were detected (P ≥ 0.25) for serum concentration of cortisol and plasma concentration of haptoglobin, ceruloplasmin, and progesterone. Therefore, BAS application at the beginning of the FTAI protocol could decrease the temperament and dirtiness in the perineal region and increase the growth and estrus expression of beef heifers.

Human Chorionic Gonadotrophin (hCG) induces changes in IFN-pathway and Interferon-Stimulated Genes (ISGs) on the bovine endometrium at Day 18 of pregnancy.

We hypothesized that the hCG modulates the expression of IFNT-pathway and ISGs in bovine endometrium during early pregnancy. The aim of the current study is to evaluate the effect of hCG on IFNT-pathway signals and ISGs expression in endometrial cells. For this, 29 non-lactating cross-bread cows were used in the study and submitted to a 9-day fixed-time artificial insemination (FTAI) protocol. The day of the AI was considered Day 0 (D0), and five days (D5) after the FTAI, the cows were allocated into two groups: Control and hCG group, when a hCG group received a single dose of 2.500UI of hCG. On day 18 after FTAI (D18) cows were slaughtered and endometrial tissue samples were collected. There was no difference between the embryo recovery rate of the cows in C compared to the hCG. The hCG group increased the accessory corpus luteum formation rate. The hCG resulted in greater serum progesterone concentration in the hCG group compared to the C on Day 14. Only the expression of IFNAR2 and STAT1 were upregulated on pregnant cows of the hCG group compared to the C group. The pathway genes (JAK1, STAT2, and IRF9) were not regulated. The mRNA abundance of ISG15, MX1, MX2, and OAS1 was upregulated in pregnant cows for hCG group, compared to C group. The results show that the administration of hCG, 5 days after AI, in addition to increasing the serum progesterone, modulates the expression of IFNT-pathway and ISGs on bovine endometrium on Day 18 of pregnancy.

Characterizing pregnancy losses in Bos indicus beef females receiving a fixed-timed artificial insemination protocol

This experiment evaluated pregnancy losses from day 30 of gestation to calving in Bos indicus females, and evaluated if serum haptoglobin concentrations during early gestation impacts subsequent pregnancy losses. A total of 4926 Nelore females were used, being 1802 nulliparous heifers inseminated as yearlings (precocious heifers), 1356 nulliparous heifers inseminated at 24 months of age (conventional heifers), 887 suckling primiparous cows, and 881 suckling multiparous cows. Cows were assigned to an ovulation synchronization + fixed-time artificial insemination (FTAI) protocol from day −11 to 0 of the experiment. Pregnancy status was verified using transrectal ultrasonography on days 30 and 60 after FTAI, via transrectal palpation on day 150 after FTAI, and according to calf birth. Blood samples were collected from all animals diagnosed pregnant on day 30 after FTAI, and analyzed for serum concentrations of haptoglobin and pregnancy associated glycoproteins (PAG). Pregnancy loss was greater (P < 0.01) from day 60–150 of gestation (10.2 %) compared with day 30–60 of gestation (6.0 %) and with day 150 of gestation to calving (7.4 %), and differed (P = 0.04) between these latter two periods. Pregnancy loss from day 30–60 of gestation did not differ (P = 0.26) among parities, whereas total pregnancy losses (day 30 to calving) were greater (P < 0.01) in precocious (28.4 %) and conventional (27.1 %) heifers compared with primiparous (16.4 %) and multiparous (13.0 %) cows. Serum PAG concentrations on day 30 after FTAI were less (P ≤ 0.03) in cows that lost the pregnancy (5.63 ng/mL) from day 30–60 of gestation, as well as those that lost the pregnancy from day 30 to calving (8.59 ng/mL) compared with cohorts that maintained the pregnancy (9.39 and 9.32 ng/mL, respectively). No differences in serum PAG concentrations on day 30 after FTAI were noted (P ≥ 0.23) according to pregnancy losses from day 60 to calving. Serum haptoglobin concentration on day 30 after FTAI also did not differ (P ≥ 0.48) between cows that maintained or lost the pregnancy. This experiment provides novel information about pregnancy losses after day 30 of gestation in B. indicus cattle, with most losses occurring as fetal mortality and not affected by systemic inflammation during early gestation. Pregnancy losses were nearly doubled in precocious and conventional heifers compared with parous cows, demonstrating the relevance of this reproductive failure to B. indicus replacement heifers.

Uterine microbial profiles in healthy postpartum dairy cows do not vary with sampling techniques or phases of estrous cycle

In this study, we aimed to compare uterine microbial profiles in postpartum dairy cows, determined by bacteriological culture and next-generation sequencing, using three uterine sampling techniques (swab, cytobrush, and lavage) and induced phases of the estrous cycle (estrus and diestrus). Fifteen healthy postpartum dairy cows at 53 ± 5 days postpartum were enrolled in the study. Uterine samples were collected during a fixed-time artificial insemination protocol. Viable bacteria were aerobically cultured from part of each sample, and bacterial isolates were identified through Sanger sequencing of the 16S rRNA gene. Total genomic DNA was extracted from the remainder of undiluted samples to quantify bacterial load using 16S rRNA qPCR and characterize the microbiome by metagenomic sequencing of the V1–V3 region of the 16S rRNA gene. Microbial profiles and composition were analyzed using the Shannon-Weaver diversity index and principal component analysis, respectively. Out of 87 samples, 88 % (77/87) were culture positive. The proportion of culture-positive uterine samples did not differ between sampling techniques (P = 0.39) or estrous cycle phases (P = 0.99). However, swab, cytobrush, and lavage techniques yielded 1.5, 9 and 9 times greater bacterial loads (P < 0.01), respectively, during diestrus than estrus phase. Moreover, during diestrus phase, the cytobrush method yielded 3 and 6 times more bacteria (P < 0.01) than both the lavage and swab methods. The most abundant bacterial genera identified from both bacteriological culture and metagenomic sequencing were Bacillus and Enterococcus, regardless of sampling technique or phases of the estrous cycle. Bacterial genera in moderate to low abundance through metagenomic sequencing included Streptococcus, Oscillospiraceae, and Lachnospiraceae. Notably, the uterine microbial profiles and composition, determined by metagenomic sequencing, did not differ by sampling techniques (P = 0.55 and P = 0.60, respectively) or estrous cycle phases (P = 0.34 and P = 0.17, respectively). In conclusion, our results suggest that any of the sampling techniques can be reliably used to study the uterine microbiome of healthy cows at random phases of the estrous cycle. However, it is important to consider potential differences in bacterial yield as a confounding factor.

171 Supplementing Ca Salts of Soybean Oil Via Low-Moisture Molasses-Based Blocks to Improve Reproductive Performance and Overall Productivity of Beef Cows

Abstract This experiment evaluated reproductive and productive responses of beef cows receiving self-fed low-moisture blocks (LMB) enriched or not with Ca salts of soybean oil (CSSO) throughout the breeding season. Non-pregnant, suckling multiparous Angus-influenced cows (n = 554) were assigned to a fixed-time artificial insemination (AI) protocol (day -10 to 0) followed by natural service (day 15 to 70). Cows were managed in 12 groups (46 ± 4 cows/group) maintained in individual pastures, and groups received LMB enriched with 25% (as-fed basis) of CSSO or ground corn (CON) from day -10 to 100. Both treatments were designed to yield a daily LMB intake of 0.454 kg/cow (as-fed basis). Groups were enrolled in an estrus-synchronization + fixed-time AI protocol from day -10 to 0 (AI occurred on day 0) of the experiment and beginning 15 days after AI cows were exposed to mature Angus bulls for 55 d. Pregnancy status was verified on day 55 and 100 of the experiment, and cows that gave birth during the initial two weeks were considered pregnant to fixed-time AI. Calf birthdate and weight was recorded during the subsequent breeding season. All calves were weaned at the same time, and a calf BW and age were recorded. Blood samples were also collected from a subsample of cows on days -10, 0 (fixed-time AI), and 55 (pregnancy diagnosis). All data were analyzed using group as the experimental unit and Satterthwaite approximation to determine the denominator df for the tests of fixed effects. Quantitative data were analyzed with the MIXED procedure of SAS, and binary data analyzed with the GLIMMIX procedure of SAS with a binomial distribution and logit link function. Data were analyzed with group (treatment × location), cow(group), and location as random variables. Significance was set at P ≤ 0.05 and tendencies were determined if P &amp;gt; 0.05 and ≤ 0.10. Cows receiving CSSO had greater (P &amp;lt; 0.01) mean concentrations of ω-6 fatty acids in plasma samples collected on days 0 and 55. Cows receiving CSSO had greater (P = 0.05) pregnancy rate to fixed-time AI (67.2 vs. 59.3%), whereas final pregnancy rate did not differ (P = 0.92) between treatments. Pregnancy loss was less (P = 0.03) in CSSO cows (4.50 vs. 9.04%), which also calved earlier during the calving season (treatment × week; P = 0.04). Weaning rate tended to be greater (P = 0.09) in CSSO (84.8 vs. 79.4%), although calf weaning age and weight did not differ (P ≥ 0.72) between treatments. Kilos of calf weaned/cow exposed was greater (P = 0.04) in CSSO cows (234 vs. 215 kg). Therefore, supplementing CSSO to beef cows via LMB during the breeding season improved their reproductive and overall productivity during a cow-calf cycle.

Yeast estrogen screen assay applied in the assessment of estrogenic activity removal from dairy cattle wastewater treated by anaerobic digestion

The presence of estrogen-like endocrine disrupting chemicals (EEDC) in surface waters, soils, and groundwater has been frequently reported in the literature. Since these compounds can be excreted by humans and animals, the release of domestic sewage and animal wastewaters may be an important source of EEDC in the environment. In this context, there is a growing concern regarding the presence of these substances in the environment and their potential adverse effects on human health. However, most studies have been focused on the presence of EEDC in domestic wastewaters and the available information regarding EEDC in livestock wastewater is still limited. Therefore, this work aimed to quantify the estrogenic activity (EA) in the liquid phase of dairy cattle wastewaters and evaluate the removal of EA during anaerobic digestion, applying an adapted methodology based on the yeast estrogen screen (YES) assay, which has been widely used to analyze EA in domestic sewage. Influent and effluent dairy cattle wastewater samples from a full-scale anaerobic digester placed in an experimental farm were collected during seven months. EA concentrations in the liquid phase of raw wastewater ranged from 228 to 2182 ng.L−1 E2-eq. Anaerobic digestion was not efficient in EA removal, achieving effluent concentrations ranging from 109 to 946 ng.L−1 E2-eq. Results also indicate that the application of an estradiol-based fixed time artificial insemination protocol did not statistically contribute to the increase of EA concentrations in dairy cattle wastewater which reuse in cleaning of animal feeding operation facilities and fertigation are beneficial and economically advantageous to dairy production systems, reducing water demand in parallel to promoting organic matter and nutrient recycling. However, the potential environmental and health risks associated with the presence of EEDC in dairy cattle wastewaters should be better elucidated and evaluated.

PSVI-21 Effects of Calcium Salts of Soybean Oil Supplementation to Nellore Heifers During Second and Third Trimesters of Gestation and Offspring Sex on Milk Production and Performance of the Offspring

Abstract The study aimed to evaluate the effects of supplementing calcium salts of soybean oil to Nellore heifers during the second and third trimesters of gestation and offspring sex on cow productivity and their male and female offspring growth. Forty-eight pregnant Nellore heifers [body weight (BW) = 387 ± 38.4 kg] were assigned to this experiment at day 123 ± 13 of gestation. All heifers were conceived during the same fixed-time artificial insemination protocol at 14 and 24 months of age. Heifers were randomly used in a completely randomized block design with a 2 × 2 factorial treatment arrangement, consisting of supplementing or not (CON) calcium salts of soybean oil (CSSO; 3 g/kg of BW; Table 1) and the sex of the offspring (male or female), totaling four treatments and twelve reps per treatment. Throughout the experimental period, heifers were maintained in a pasture composed of Brachiaria brizantha cv.Marandu. After birth, all animals remained under the same pasture conditions and received the same supplementation until weaning. Data were analyzed using the MIXED procedure of SAS and heifer as the experimental unit. Treatment × offspring sex interaction was not detected (P &amp;gt; 0.36) for any of the variables evaluated. Calves born from heifers supplemented with CCSO were 2 kg heavier (P = 0.03) at birth and 5 kg heavier (P = 0.03) at 120 days of age compared with calves born from CON heifers. At weaning (210 days), no dietary treatment effects were observed (P = 0.17) in offspring BW. Male offspring were 3.5 kg, 9 kg, and 12 kg heavier (P ≤ 0.01) at birth, day 120, and at weaning, respectively, compared with female offspring (Table 2). Throughout the experiment, male offspring gained 0.040 kg more daily (P = 0.01) than female offspring. The female offspring had 0.1 thicker subcutaneous fat per 100 kg of BW (P = 0.02) than the male offspring. Heifer milk production was not impacted (P &amp;gt; 0.10) by the dietary treatments or sex of the offspring in any of the periods evaluated (days 30 and 210; Table 3). Hence, supplementing CSSO to Nellore heifers during the second and third trimester of gestation altered offspring BW up to 120 days after birth, suggesting programming effects on postnatal offspring growth resultant from the CSSO treatment. Male offspring were heavier from birth to weaning compared with female offspring. These outcomes indicate that supplementing CSSO to Nellore heifers during pregnancy might be a feasible alternative to optimize offspring productivity, and carcass merit in grazing systems.

PSIX-20 Differential Gene Expression in Peripheral White Blood Cells of Fertile and Sub-Fertile Beef Heifers

Abstract Infertility in beef heifers remains a major economic loss to the cow-calf production industry. Identifying beef heifers with superior genetic potential for improved fertility would increase profitability. Therefore, the study aimed to identify differences in transcriptome profile from peripheral white blood cells (pWBCs) of beef heifers with varying reproductive potential. To accomplish this, Angus-Simmental crossbred heifers were subjected to an estrus synchronization and fixed-time artificial insemination (AI) protocol (7-Day CO-Synch + CIDR). Fourteen days after AI, all heifers were exposed to fertile bulls for a 60-day natural breeding season. Depending on the presence or absence of conceptus 120 days post-AI, heifers were classified as fertile (pregnant heifers) or sub-fertile (non-pregnant by AI or bull-breeding). Pregnancies were terminated, and all animals in both groups were cycling when pWBCs were isolated. Total RNA was extracted from the PWBCs and subjected to the library preparation and sequencing on the Nova-Seq platform. The read counts were obtained after data filtering and quality control using FastQC v0.11.9 and MultiQC v1.12 and alignment to the Ensemble’s ARS UCD1.2 Bos taurus genome reference using STAR aligner v2.7.5 for fertile (n = 8) and sub-fertile (n = 5) beef heifers. The filtered data were subjected to differential expression analysis using DESeq2. We identified 300 significantly differentially expressed genes (DEGs) with P-value ≤ 0.05 and absolute (log2 fold change) ≥ 0.5. Among them, 34 genes including FKBP5, ADCY3, REPS1, LAMP1, CTSH, ADGRE5 were identified with padj &amp;lt; 0.05. DEGs were over-represented for pathways such as response to thyroid stimulus, immune response, response to lipid, neutrophil degranulation, and calcium ion binding. Some of these genes were reported as fertility-related, while the other genes are novel and have the potential to predict heifers with varying reproductive potential. A detailed understanding of the underlying biological mechanisms of the top genes and a follow-up study with a higher sample size at different time points could validate the potential candidates identified in this study.