7-day CO-Synch Research Articles

Reproductive outcomes in Holstein heifers synchronized with timed-AI protocols that provide for a lengthened proestrus.

This study compared reproductive outcomes among two protocols for synchronization of ovulation that provide for a lengthened proestrus with the conventional oestradiol-based protocol currently used for timed-AI (TAI). Holstein heifers (13-15 months) at one location were assigned randomly to one of three TAI protocols. Heifers (n = 150) in the 7-day oestradiol benzoate (EB) group received a progesterone device (Cue-Mate) and 2 mg EB on Day 0; 500 μg of cloprostenol (PGF) and Cue-Mate removal on Day 7; 1 mg of EB on Day 8 and TAI on Day 9 (54 h after Cue-Mate removal). Heifers (n = 150) in the 5-day CO-Synch (CO) group received a Cue-Mate and 100 μg of gonadotropin-releasing hormone (GnRH) on Day 2; Cue-Mate removal and PGF (twice, 12 h apart) on Day 7; and GnRH along with TAI on Day 10 (72 h after Cue-Mate removal). Heifers (n = 150) in the J-Synch (JS) group received a Cue-Mate and 2 mg of EB on Day 1; PGF and Cue-Mate removal on Day 7; GnRH and TAI on Day 10 (72 h after Cue-Mate removal). Heifers were inseminated by one technician with frozen-thawed conventional semen from one of four commercially available sires. Plasma progesterone (P4) concentrations (ng/mL) were determined at Cue-Mate removal and TAI. Ovarian ultrasonography was done in a subset of 217 heifers at the initiation of protocols, at Cue-Mate removal; TAI; and 7 days after TAI. Approximately, 28 and 50 days after TAI pregnancy status was determined by ultrasonography. Mean (±SEM) plasma P4 concentration at Cue-Mate removal was greater (p < .01) in CO (6.02 ± 0.2) and JS (6.51 ± 0.2) compared to EB heifers (4.53 ± 0.2). Mean (±SEM) plasma P4 concentration at TAI was lowest in the JS (0.28 ± 0.05), intermediate in CO (0.46 ± 0.02), and greatest in EB heifers (0.66 ± 0.05, p < .01). The diameter of the ovulatory follicle (mean ± SEM) was the smallest in the JS group compared to that in the CO and EB groups (15.8 ± 0.5; 13.9 ± 0.5; and 12.7 ± 0.5 mm for EB, CO and JS, respectively). More (p < .01) heifers in the JS group had their oestrous cycle synchronized (50.0, 78.8 and 82.4% for EB, CO and JS groups), and were pregnant at 28 (40.3, 51.3 and 63.3% for EB, CO and JS groups) and 50 days after TAI (32.6, 46.0 and 60.0% for EB, CO and JS groups). In summary, heifers subjected to the J-Synch TAI protocol had lower P4 at TAI, and better overall response to hormonal treatments, which resulted in increased P/AI at 28 and 50 days after TAI compared to those heifers subjected to either a 7-day EB protocol or a 5-day CO-synch protocol.

On-farm study: cytokine profiles and vaginal microbiome of Bos indicus cattle before artificial insemination

Prior studies in beef cattle have shown shifts in the reproductive microbiome prior to artificial insemination (AI), yet few have characterized both the reproductive microbiome and immune responses prior to AI, particularly in purebred Bos indicus. Therefore, the aim of this study was to characterize the circulating cytokines and the vaginal microbiome of Bos indicus females prior to fixed-time artificial insemination (FTAI). Bos indicus females (n = 79) on four separate ranch operations within a 20-mile radius in East Texas were subjected to the 7-day CO-Synch + controlled intervaginal drug-releasing (CIDR) protocol beginning on day (d)-9 with FTAI on d0. Blood samples were collected on d-9 (CIDR IN), d-2 (CIDR OUT), and d0 (AI DAY) for cytokine concentration analyses using the RayBiotech Quantibody Bovine Cytokine Array Q1 kit per the manufacturer’s instructions. Sterile vaginal swabs were inserted past the vulva, rotated, and stored at -80°C for microbiome analysis. Bacterial community analyses targeted the V4 hypervariable region of the 16S rRNA gene. Pregnancy status was determined by transrectal ultrasonography approximately 60 days after FTAI for resulting open females (n = 45) and pregnant females (n = 34). Regardless of pregnancy status, the vaginal relative abundance of Firmicutes differed between CIDR IN, CIDR OUT, and AI DAY (63.74% vs. 28.31% vs. 60.86% ± 3.66%, respectively; P &amp;lt; 0.01). Genera with phylum Firmicutes including Ruminococcus, Clostridium, Blautia, Butyrvibrio, and Mogibacterium followed a similar trend (P &amp;lt; 0.05). Butyrvibrio tended to have greater relative abundance in the vaginal samples of Cows than Heifers (4.17% ± 0.75% vs. 3.26% ± 0.77%; P = 0.07). Concentrations of the interferon (IFN)γ (2005.98 ± 471.94 pg/mL vs. 1185.40 ± 482.65 pg/mL; P &amp;lt; 0.01), interleukin (IL)1F5 (153.89 ± 141.07 pg/mL vs. 627.30 ± 149.28 pg/mL; P &amp;lt; 0.01), and interferon gamma-induced protein (IP)10 (9363.26 ± 2929.83 pg/mL vs. 5905.53 ± 2983.60 pg/mL; P = 0.05) were greater in Cows than Heifers. There was a parity-by-status interaction for IP10, with Open Cows having the greatest concentration compared all other groups (P &amp;lt; 0.05). These results indicate differences in circulating cytokines and shifts in the vaginal microbiome for in Bos indicus cattle prior to FTAI.

PSXII-8 Effects of Pre-Synchronization with Short-Term Melengesterol Acetate in Nulliparous Beef Heifers

Abstract Conception rates in nulliparous beef heifers can be greatly influenced by their ability to reach puberty at or prior to the initiation of a breeding season. Previous research has analyzed the efficacy of utilizing both long and short-term exposure to a source of progesterone via a controlled internal drug release insert (CIDR) prior to estrous synchronization with timed artificial insemination (TAI) and found improved conception rates. The purpose of this study was to evaluate the effects of feeding melengesterol acetate (MGA) immediately and 10 days before estrous synchronization. Ninety-three crossbred heifers (395.67 ± 5.37 kg) were blocked by BW, genetics, and reproductive tract scores and allotted to 3 treatments. The 3 treatment groups were: 7-day Co-synch + CIDR without presynchronization (CON, n = 31); 2cc PGF2α (Lutalyse) followed by melengesterol acetate feeding for 7 d before the start of the 7-day Co-synch + CIDR program (PRE, n = 31); and 2 mL Lutalyse followed by MGA feeding for 7 d followed 10 d later by the start of the 7-day Co-synch + CIDR program (PRE+10, n = 31). The 7-day Co-synch + CIDR protocol was initiated on d 0 by administering a 2 mL IM injection of GnRH and placing a CIDR into the vagina. The CIDRs were removed 7 days later and accompanied by 2 ml IM injection of PGF2α. An injection of GnRH occurred 60 to 66 hours following PGF2α at TAI with frozen semen from a single bull. Ten days after TAI, heifers were exposed to a bull. Estrotect patches were applied throughout the study to assess estrous behavior. Ovaries were visualized 24 hours post TAI to determine whether ovulation occurred. Pregnancy was determined on days 40, 64, and 109 post TAI via transrectal ultrasonography. Blood was collected via jugular venipuncture (d -19, MGA initiation and termination, d 0, d 7, and d 9) and serum progesterone determined. Performance data were analyzed using the MIXED procedure and conception data were analyzed using the GLIMMIX procedure of SAS. Heifers in the PRE+10 treatment group had higher levels of progesterone (P = 0.04) at d 0 than PRE heifers. At d7 there was a tendency (P = 0.07) for PRE+10 heifers to have greater concentrations of progesterone than PRE, but did not differ by d 9 (TAI, P = 0.36). TAI conception rates in heifers in the PRE treatment group (63%) tended (P = 0.09) to be greater compared with the controls (35%) and not different from PRE+10 (43%), with no differences in season-long pregnancy rates (P &amp;gt; 0.15). Presynchronization with MGA immediately before TAI synchronization can increase conception rates early in the breeding season in beef heifers.

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.

180 Differences in Gene Expression in Granulosa Cells Harvested from Fertile and Sub-Fertile Beef Heifers

Abstract Infertility in beef replacement heifers is one of the leading contributors to economic losses in the cow-calf sector of the beef production industry. Successful reproduction is complex, involving multiple tissues and biological systems. This complexity makes identifying the leading causes of subfertility difficult. One potential source of fertility issues is poor oocyte health. As developing oocytes are dependent on granulosa cells (GCs) and physically coupled with GCs, we hypothesized that the gene expression profile would be different between the GCs from fertile heifers and those from sub-fertile heifers. The objective of this study was to identify differences in gene expression in GCs collected from beef heifers of differing fertility status, either fertile or sub-fertile. To gather this information, Angus-Simmental crossbred heifers underwent an estrus synchronization and fixed-time artificial insemination (AI) protocol (7-Day CO-synch + CIDR). At 41 days post-AI, heifers were pregnancy checked through ultrasound and palpation. Heifers pregnant to AI were classified as fertile (n = 8). Any heifers that remained non-pregnant following AI were exposed to bulls of proven fertility for 61 days. Bull-exposed heifers were evaluated for pregnancy 34 days after the bulls were removed. The non-pregnant heifers following both AI and natural service were classified as sub-fertile (n = 5). Pregnancies were terminated in fertile heifers, and heifers from both groups were allowed to resume normal cyclicity before GCs being harvested. RNA was extracted from GCs and quality was checked for each sample. Total RNA from GCs of 13 heifers was subjected to the library preparation and sequencing on the Nova-Seq platform. Through the application of DESeq2, 79 genes were identified as significantly differentially expressed genes (DEGs) with P-value &amp;lt; 0.05 and absolute (log2 fold change) &amp;gt; 0.5 including DNER, HOXD10, CD52, CXCL12, and HOXD11. DEGs, along with co-expressed genes, were over-represented in pathways associated with the regulation of MAP kinase activity, regulation of serine/threonine kinase activity, MHC protein binding, and plasma membrane signaling receptor complex. Through the pursuit of knowledge regarding the function of top DEGs, increased sample size, and more diverse sample collection time points, we may improve our understanding of the causation of heifer fertility issues. Improving our knowledge of heifer infertility has the potential to develop therapeutic options, and in turn, increase the efficiency and sustainability of cow-calf production systems.

PSIV-19 The Identification of Possible Microrna Biomarkers of Successful Pregnancy in Timed Embryo Transfer Recipients

Abstract The objective of the current study was to look for potential microRNA (miRNA) biomarkers indicative of a successful pregnancy in timed embryo transfer (ET) recipients. Commercial beef cows were synchronized using a 7-day CIDR co-synch protocol and GnRH injection given 66 hours following CIDR removal. At 6.5 days post GnRH administration, cows (n = 78) had a frozen thawed embryo delivered into the uterine horn ipsilateral to the corpus luteum detected by rectal palpation. A blood sample was collected by coccygeal venipuncture and serum isolated and frozen at -80°C 24 days post embryo transfer. At 38 days post embryo transfer pregnancy was determined by rectal ultrasound by a trained individual and confirmed by Biopryn assay. A total of 42 recipients were determined to be pregnant and 36 were determined to be non-pregnant. Of the 42 pregnant recipients, 10 did not calve and 32 delivered a calf at full term. Day 24 post ET serum samples from pregnant cows that calved normally (n = 4), pregnant cows that did not calve (n = 4), and pregnant cows that had an abnormal pregnancy at time of ultrasound and did not calve (n = 4) were selected for further analysis. These samples had RNA extracted with the microRNA fraction enriched and were then subjected to RNA sequencing (LC Sciences). Differential miRNA abundance was determined using ANOVA adjusted for false discovery rate using the Benjamini-Hochberg method with significance set at P &amp;lt; 0.05. A total of 747 miRNAs were detected and annotated to the bovine genome. A total of 36 miRNA showed differential abundance (P &amp;lt; 0.05) between groups of recipients. Of them, bta-mir-185-p3_1ss21CT, bta-miR-224_R-1, bta-mir-125b-1-p3, bta-miR-363_L+1, and bta-miR-494_R+1 had increased (P &amp;lt; 0.01) abundance in animals that calved to ET compared with cattle that did not calve to ET. MicroRNA PC-5p-120366_31 and PC-5p-89234_47 had increased abundance (P &amp;lt; 0.01) in recipients that had an abnormal appearing pregnancy compared with the other two recipient groups. MicroRNA bta-mir-1306-p3 had increased abundance (P &amp;lt; 0.01) in the two groups of recipients that had normal appearing pregnancies compared with the groups whose pregnancies were abnormal at time of ultrasound. These microRNAs could be used as biomarkers for an insufficient pregnancy that will not go to full term and therefore could be used to increase the efficiency of large scale ET.

PSIII-18 Effect of Industrial Hemp Supplementation on the Salivary Melatonin Concentrations of Angus Cattle at Different Phase of Reproductive Cycle

Abstract Melatonin is an indoleamine, a neurotransmitter and neurohormone mainly produced in pineal gland but also in the ovary. It has an important role in reproduction, antioxidant, immune system and circadian behavior. Industrial hemp is increasingly being included in animal feeds and it is therefore important to evaluate the effect of industrial hemp supplementation on the profiles of melatonin following different phases of reproductive cycle. Twenty apparently healthy Angus heifers, aged 19 months with average body weight of 363 kg and body condition score of 6.5 were randomly divided into two groups of ten heifers each in a completely randomized design. Group 1 (n = 10) were supplemented with grounded industrial hemp at 20/200mg of the concentrate, and group 2 (n = 10) no industrial hemp supplementation. Hay and water were provided ad libitum. The animals were acclimatized for 1 week after which they were synchronized using the 7-day CO-Synch protocols. Salivary samples were collected at preestrus, during estrus and at post estrus. Melatonin in the saliva was assayed with a competitive ELISA kit. The salivary melatonin concentrations were greater (P &amp;gt; 0.05 in the treated groups at estrus (25.29 ± 1.48 pg/mL), pro-estrus (23.98 ± 2.3 pg/mL) and post-estrus (20.31 ± 3.0 pg/mL) compared with the untreated groups at pre-estrus (18.9 ± 1.5 pg/mL), estrus (20.4 ± 2.22 pg/mL) and post estrus (17.22 ± 2.6 pg/mL).The profiles of the salivary melatonin were greater (P &amp;gt; 0.05) at estrus phase in the hemp treated groups compared with the pre-estrus and post estrus phase of the treated groups; however, numerical but non-significant increases were observed in the untreated group at estrus phase compared with the pre-estrus and post estrus phases. It was concluded that industrial hemp supplementation could improve melatonin concentrations and hence improve reproductive, antioxidant and immune function of the cattle.

PSII-9 Circulating Cytokine Profiles of Bos Indicus Cattle Prior to Artificial Insemination in Relation to Pregnancy Status

Abstract Cytokines and chemokines (cytokines with chemotactic activity) influence inflammatory responses and can upregulate pro- or anti-inflammatory immune responses and influence fertility. The aim of this study was to characterize the circulating cytokines of Bos indicus females prior to fixed-timed artificial insemination (FTAI). Bos indicus females on operations within a 20-mile radius were subjected to the 7-day CO-Synch + CIDR protocol on day (d) -9 and FTAI on d 0. Blood samples were collected on d -9 (CIDR IN), d -2 (CIDR OUT), and d 0 (AI). Cattle were body condition scored (BCS) on d 0. Pregnancy status was determined by transrectal ultrasonography for resulting non-pregnant females (n = 44) and resulting pregnant females (n = 34). Cytokine analyses on blood plasma samples were conducted using the RayBiotech Quantibody Bovine Cytokine Array Q1 kit per manufacturer's instructions. Cytokines and chemokines measured included interferon alpha (IFNa), interferon-gamma (IFNg), interleukin (IL)1a, IL1F5, IL13, IL21, chemokine-ligand (CXCL) 9, CXCL10, macrophage inflammatory protein (MIP)1b, and tumor necrosis factor alpha (TNFa). Data were analyzed using PROC GLM in SAS 9.4 with fixed effects for pregnancy status, ranch, day, and parity. Body condition scores did not significantly differ by pregnancy status (P &amp;gt; 0.05). There were no differences in cytokine concentrations by day of the synchronization protocol or between cows and heifers (P &amp;gt; 0.05). There were differences by location (P &amp;lt; 0.05) for most cytokines except MIP1b and TNFa. Circulating concentrations of the pro-inflammatory cytokines IFNg, IL1a, and TNFa were greater (P &amp;lt; 0.05) in resulting non-pregnant females compared with pregnant females (2,323.5 ± 260.5 vs. 1,502.8 ± 246.8 pg/mL, 894.6 ± 96.8 vs. 598.3 ± 91.7 pg/mL, and 6,052.6 ± 627.4 vs. 4,156.6 ± 594.3 pg/mL, respectively). Circulating concentrations of the chemokines CXCL9, CXCL10, and MIP1b were also greater (P &amp;lt; 0.05) in resulting non-pregnant females compared with pregnant females (4,887.0 ± 538.3 vs. 2,905.3 ± 509.8 pg/mL, 12,167.3 ± 1,496.2 vs. 6,295.4 ± 1,417.2 pg/mL, 586.4 ± 56.8 vs 324.4 ± 53.8 pg/mL, respectively). Interestingly, circulating concentrations of the anti-inflammatory cytokines IL13, IL1F5, and IL21 were greater (P &amp;lt; 0.05) in resulting non-pregnant females compared with pregnant females (11,147.7 ± 1,009.9 vs. 6,752.1 ± 956.6 pg/mL, 1,189.3 ± 111.7 vs. 744.5 ± 105.8 pg/mL, and 1,1153.6 ± 898.0 vs. 8,409.7 ± 850.6 pg/mL, respectively). No differences by pregnancy status were found for concentrations of IFNa (P = 0.13). These results suggest that an increase in circulating cytokines and chemokines prior to FTAI negatively impacts fertility in Bos indicus cattle. Future research is necessary to determine the threshold of circulating cytokine concentrations that subsequently impact pregnancy establishment and maintenance.

PSI-18 Vaginal Microbiome of Bos Indicus Cattle in Relation to Fertility

Abstract Shifts in commensal bacteria (e.g., Firmicutes) in the human vaginal microbiome are associated with compromised fertility. The objective of this study was to characterize the reproductive microbiome in the vagina of Bos indicus females prior to fixed-timed artificial insemination (FTAI). Bos indicus females on operations within a 20-mile radius (RANCH 1, RANCH 2, RANCH 3, RANCH 4) were subjected to the 7-day CO-Synch + CIDR protocol on day (d)-9 and FTAI on d0. Sterile vaginal swabs were collected on d-9 (CIDR IN), d-2 (CIDR OUT), and d0 (AI). Cattle were body condition scored (BCS) on d 0. Pregnancy status was determined by transrectal ultrasonography (Non-pregnant, n = 44 and Pregnant, n = 34). Bacterial community analyses were conducted targeting the V4 hypervariable region of the 16S rRNA gene. Data were analyzed using PROC GLM in SAS 9.4. Body condition scores did not differ by pregnancy status or ranch. Additionally, there was no difference in bacterial relative abundance between pregnant or non-pregnant (P &amp;gt; 0.10). The vaginal relative abundance of Firmicutes differed between CIDR IN, CIDR OUT, and AI days (63.54% vs. 28.11% vs. 60.66% ± 1.96%; P &amp;lt; 0.0001). Pregnancy rate did differ by ranch (RANCH 1 63.6% ± 14.7%, RANCH 2 60.0% ± 10.9%, RANCH 3 28.0% ± 9.7%, RANCH 4 36.4% ± 10.4%; P = 0.074). There was a difference observed between ranches with RANCH 4 having the greatest relative abundance of Firmicutes and RANCH 1 having the least (58.84% ± 2.12% vs. 45.12% ± 3.17%; P = 0.0001). A tendency for increased relative abundance of Firmicutes was noted in cows compared with heifers (53.03% ± 1.71% vs. 48.51% ± 1.89%; P = 0.099). Under the phylum of Firmicutes, the genera of Ruminococcus, Clostridium, Blautia, Butyrivibrio, and Mogibacterium followed a similar trend with a shift in relative abundance between CIDR IN, CIDR OUT, and AI days (Table 1). RANCH 1 had the lowest relative abundance of Ruminococcus and Clostridium and the greatest relative abundance of Blautia compared with other ranches (P &amp;lt; 0.05). A shift in relative abundance between CIDR IN, CIDR OUT, and AI days was observed for the phylum of Proteobacteria (7.84% vs. 26.44% vs. 9.69% ± 1.82%; P &amp;lt; 0.0001) and Bacteroidetes (12.19% ± vs. 15.62% vs. 12.67% ± 1.07%; P = 0.046). A tendency for differences in the relative abundance of Bacteroidetes between ranch were observed with RANCH 2 having the greatest relative abundance and RANCH 3 having the least (15.36% ± 1.29% vs. 11.73% ± 1.21%, respectively; P = 0.066). These results suggest a distinct difference in the vaginal microbiome before FTAI. In addition, microbial relative abundances within the vagina also differ between ranches of similar genetics and region.

PSII-10 Effect of Equine Chorionic Gonadotrophin-Like Glycoprotein Treatment on Fertility in Angus Cows Subjected to a 7-Day Co-Synch Protocol

Abstract This study evaluated the effect of administering a glycoprotein with equine chorionic gonadotropin like activity produced by recombinant DNA technology (eCG-like) associated with a 7-d Co-Synch protocol on estrus rate (ER) and pregnancy per timed AI (P/TAI). A total of 1,344 suckled Angus cows (primiparous, n = 381; multiparous, n = 963) from 11 herds were scanned at initiation of synchronization protocol by transrectal ultrasonography and the presence of corpus luteum (CL) and uterine tone was used to classify them by cycling. On day -10, cows received 100 μg GnRH analogue and a progesterone intravaginal device (IVD; 1.0g progesterone) for 7 days. At IVD removal, cows were administered 500 μg of cloprostenol and randomly assigned to receive 400 IU of eCG-like glycoprotein (Syntex S.A., Argentina, PCT/EP2019/073277; eCG-like, n = 665) or to remain as untreated (Control, n = 679). Also on day -3, paint was applied on the sacrum to the tail head of each cow. Finally, 100 μg GnRH analog were injected concurrently with TAI on Day 0 (66h after IVD removal) and the ER was evaluated by the degree of paint loss. Pregnancy to TAI was determined on day 30 by transrectal ultrasonography when an embryo and fluid were detected in the uterus. Body condition score (BCS; scale = 1-9) was evaluated on days -10 and 30. Optimal BCS was considered ≥ 5 and BCS balance between days -10 and 30 was classified as negative (lost BCS) or positive (maintained or gained BCS). The associations among treatment, parity, cyclicity at initiation of protocol, optimal BCS on day -10, and their interactions and ER and P/TAI were analyzed by GLIMMIX (SAS 9.3). Estrus expression and BCS balance were also included as effects in the model of pregnancy per TAI. The effect of herd was included as a random effect. The model specifications included an option to retrieve the mean, SEM, and an adjustment for multiple comparisons (Tukey-Kramer). The percentage of acyclic cows was 31.3%. The ER in cyclic cows was 64.9% and the eCG-like treatment increased ER only in anestrus cows (55.8 vs. 44.3%; Treatment x cyclicity interaction; P = 0.03). The percentage of cows with negative BCS balance was 27.2%. The P/TAI in cows with positive BCS balance was 48.4% and the eCG-like treatment increased P/TAI only in cows with negative BCS balance (44.7% vs. 30.5%; Treatment x BCS balance interaction; P = 0.01). In conclusion, the addition of a recombinant eCG-like glycoprotein to a 7-day Co-Synch protocol increased estrus expression in anestrus Angus cows and P/TAI in those with negative body condition score balance. However, this addition did not influence the estrus expression in cycling cows or on P/TAI in those with positive BCS balance.

74 Ovulatory Follicle and Corpus Luteum Development ofBos Taurus Cows Under Nutrient Restriction

Abstract Establishment and maintenance of pregnancy is a central concern to the cattle industry, as it strongly impacts efficiency and profitability of beef cow-calf operations. The objective of this study was to determine if nutrient restriction impacts ovulatory follicle size and corpus luteum (CL) volume of Angus cows enrolled in estrous synchronization. A total of 26 Angus cows were housed at the Virginia Tech – Shenandoah Valley Agricultural Research and Education Center (SVAREC) equipped with a Calan gate system for individual animal intake, stratified by body weight (BW) and randomly assigned to one of two treatments: 1) a maintenance diet that provided 100% of nutrient requirements (MTN; n = 13) or 2) a diet providing 70% of nutrient requirements (REST; n = 13). Nutrient requirements were calculated and adjusted weekly based on BW using the 2016 NRC version of Beef Cattle Nutrient Requirements Model Software. Cattle underwent an acclimation period of4 days and were exposed to nutritional treatments for 30 days before estrous synchronization. Body weight was measured daily using an automated scale, and a conventional livestock scale at the beginning and end of the experiment. Cows were synchronized using a 7-day CO-synch + CIDR protocol beginning on day -10. An estrus alert patch was applied on day -3 and read on day 0 to assess expression of estrus. Ultrasonography of the ovaries was performed at each event of the estrous synchronization protocol. Data were analyzed using the MIXED procedure of SAS. Initial BW was similar between treatments (P = 0.07; 600 ± 32 kg), but MTN compared with REST cows had greater final BW (P &amp;lt; 0.01; 687 ± 24 and 556 ± 27 kg, respectively) and greater average daily gain (1.35 ± 0.18 and -0.72 ± 0.21 kg/d, respectively). Estrus expression was numerically greater (P = 0.185) MTN (84.62%) than REST (61.54%) cows. Diameter of the largest follicle was similar (P = 0.851) between treatments at the CIDR insertion (12.6 ± 0.6 mm) and CIDR removal (12.9 ± 0.4 mm), but were greater for MTN than REST cows at 60 hours after CIDR removal (14.01 ± 0.6 and 12.37 ± 0.5 mm, respectively). Volume of CL was similar (P &amp;gt; 0.01) at 5 (3211 ±13 mm3) and 7 (5280.3 ± 212 mm3) days after ovulation. In conclusion, nutrient restriction during estrous synchronization negatively affects diameter of the ovulatory follicle, but it did not affect subsequent CL volume in mature Angus cows.

55 Investigating the Potential Benefits of an Immunostimulant (Mycobacterium Cell Wall Fraction) Treatment on Increased Pregnancy Success of Embryo Transfer Recipients

Abstract Modulating the vaginal and uterine microbiome through the use of immunostimulants has the potential to improve fertility in cattle. The object of this study was to evaluate the potential benefits of an immunostimulant, mycobacterium cell wall fraction (MCWF), to increase pregnancy success of embryo transfer (ET) recipients. The three main objectives of this study were: determining if injecting ET recipient cows with MCWF increased proportion of cows with a corpus luteum (CL) on ET day, identifying differences in CL volume and progesterone concentration at the time of ET, and determining if ET recipients that received MCWF have greater pregnancy rate. A total of 926 beef cows at eight different locations were enrolled in this study. Primiparous and multiparous cattle were enrolled in a 7-day CO-Synch + CIDR estrous synchronization protocol. During this protocol, cows received an injection of GnRH (100-μg) at CIDR (controlled internal drug release) insertion [d -9], a prostaglandin F2α injection (25 mg), and CIDR removal [d-2] followed by another injection of GnRH (100 μg; d 0) within 60-66 hours after CIDR removal. At each location at (d 0), cows were sorted based on BCS and days post-partum and randomly assigned to one of two treatments: 1) MCWF- a single intramuscular injection (5 mL) of MCWF at (d 0; Amplimune, NovaVive Inc.; n = 452) or 2) Control- a single intramuscular injection (5 mL) of saline (n = 474). Seven days after (d 7), cows were examined by ultrasonography to determine if a CL was present. Recipient cows that had a CL received ET with a frozen or fresh in vivo or in vitro produced embryo in the ipsilateral uterine horn to the CL, with embryo type and grade balanced between treatments. Blood samples were collected in one location (n = 98) for analysis of plasma concentration of progesterone on (d 7) ET day. Pregnancy diagnosis was then performed by ultrasonography between 30 and 90 days of gestation. Data were analyzed using the GLIMMIX and MIXED procedures of SAS. Cow BCS and days postpartum were similar (P &amp;gt; 0.10) between treatments (5.5 ± 0.6 and 103 ± 14 days, respectively). Recipient cow utilization rate was similar between treatments (P = 0.49; 81.2 ± 0.42%). At the time of ET MCWF had larger CL than Control cows (5,768.6 ± 133.7 and 5,408.0 ± 128.5 mm3, respectively, P = 0.03). and increased progesterone concentration (2.68 ± 0.17 and 2.18 ± 0.14 ng/mL, respectively, P = 0.01). However, pregnancy rate to ET was similar (P = 0.922) between treatments (60.9 and 61.3 ± 0.05% for Control and MCWF, respectively). In conclusion, treatment of ET recipient cows with MCWF increased CL volume and concentration of progesterone at the time of ET but failed to generate greater pregnancy rates.

Effects of two protocols of ovulation synchronization on corpus luteum size and blood flow, progesterone concentration, and pregnancy rate in beef heifers

The objective of this study was to evaluate the effect of 7-day estradiol-progesterone-based [Treat(C)] and 5-day Co-Synch plus progesterone [Treat(Co-Sy)] protocols on ovulation time, pre-ovulatory follicle diameter, corpus luteum (CL) size and blood flow, progesterone (P4) concentration and pregnancy rate (PR) in beef heifers. In Experiment 1, a crossover design was applied (n = 9). For Treat(C), a progesterone intravaginal (PI) device was inserted, plus 2 mg of estradiol benzoate (day 0). On day 7, 500 µg of cloprostenol plus 0.5 mg of estradiol cypionate were administered, and PI was removed. For Treat(Co-Sy), on day 0, a PI was inserted plus 100 µg gonadotropin-releasing hormone (GnRH). On day 5, PI was removed, plus 500 µg of cloprostenol and 100 µg of GnRH were administered at 69–70 h later. From day one to ovulation day, dominant follicle was evaluated by ultrasonography. On days 4 and 8 post-ovulation, CL was evaluated by color Doppler, and P4 concentration was determined by chemiluminescence. In Experiment 2, a split-plot experimental design was used. Protocols followed were the same as in Experiment 1 [Treat(C); n = 310 and Treat(Co-Sy); n = 314]. Heifers were fixed-time artificially inseminated. Pregnancy was determined on day 41. In Experiment 1, the interval between PI removal and ovulation time was different between protocols (P < 0.01). In addition, P4 concentration was related to the CL size (P < 0.001), CL blood flow (P < 0.01) and protocols (P < 0.03). In Experiment 2, PR did not differ between protocols.

GnRH dose at initiation of a 5-day CO-Synch + P4 for fixed time artificial insemination in suckled beef cows

The objective of the present study was to evaluate the effect of GnRH dose administered at initiation (GnRH-1) of a 5-day CO-Synch + P4 protocol on ovulatory response, expression of estrus, and fertility in suckled beef cows. Suckled beef cows (n = 1101) at four locations were randomized to receive either 100 or 200 µg of gonadorelin acetate at initiation (D-8) of a 5-day CO-Synch + P4 protocol concurrently with insertion of an intravaginal progesterone (P4) device. On D-3 the P4 device was removed, two doses of prostaglandin F2α were administered concurrently and a patch was applied to evaluate expression of estrus. Artificial insemination was performed 72 h after P4 device removal (D0) simultaneously with the administration of 100 µg of gonadorelin acetate (GnRH-2). Increasing GnRH dose at initiation of a 5-day CO-Synch + P4 did not enhance ovulatory response (P = 0.57) to GnRH-1, expression of estrus (P = 0.79), nor pregnancies per AI (P/AI; P = 0.91). Both follicle size (quadratic) and circulating P4 (linear) affected (P < 0.01) ovulatory response to GnRH-1 independent of dose. Cows that had ovulation to GnRH-1 had smaller (P < 0.001) follicle size on D-3 and reduced (P = 0.05) expression of estrus compared to cows that did not have ovulation to GnRH-1, however, P/AI did not differ (P = 0.75). In conclusion, increasing the dose of GnRH-1 in the 5-day CO-Synch + P4 protocol did not enhance ovulatory response, expression of estrus, or P/AI in suckled beef cows.

Comparison of the 7-day CO-Synch and 8-day estradiol-based protocols for estrus synchronization and timed artificial insemination in suckled Bos taurus cows

Two experiments were conducted to compare, follicle diameter (FD) on Day −1, corpus luteum (CL) area on Day 7, progesterone (P4) concentration on Day 7 and 18, pregnancy per timed artificial insemination (TAI) on Day 30, and pregnancy loss (PL) between Days 30 and 60 after TAI (TAI, Day 0) using two different synchronization protocols. In Experiment 1, Angus cows (n = 1148) were randomly assigned to either 7-d progesterone CO-Synch (7-d CO-Synch) or 8-d progesterone + estradiol (8-d P + ES) synchronization protocols for TAI. On Day −10, cows in the 7-d CO-Synch treatment group (n = 574) received a progesterone-releasing intravaginal device (PIVD; 0.5 g P4) and GnRH (0.105 mg), on Day −3 the PIVD was removed and cows received cloprostenol (0.150 mg), then, on Day 0 (64 h after PIVD removal), cows received GnRH (0.105 mg) and were TAI. On Day −10, cows in the 8-d P + ES treatment group (n = 574) received a PIVD (0.5 g P4) and estradiol benzoate (2.0 mg), on Day −2 the PIVD was removed, and cows received cloprostenol (0.150 mg) and estradiol cypionate (0.5 mg), then, on Day 0 (48 h after PIVD removal), cows were TAI. Pregnancy per TAI was determined on Days 30 and 60. In a subset of cows (7-d CO-Synch, n = 41; 8-d P + ES, n = 40), serum P4 concentration was evaluated on Day 18. In Experiment 2, anestrus (n = 34) and cyclic (n = 34) suckled beef cows were selected and submitted at random on Day −10, to either 7-d CO-Synch or 8-d P + ES treatment groups. Follicle diameter on Day −1, CL area, and serum P4 concentration on Day 7 were determined. In Experiment 1, pregnancy per TAI on Day 30 did not differ (7-d CO-Synch = 48.9%; 8-d P + ES = 45.6%) between treatments but it was greater for cows with BCS ≥5 (P < 0.01). Pregnancy loss between Days 30 and 60 did not differ between treatment groups but tended to be greater in cows with BCS <5.0 (P < 0.1). In a subset of cows, serum P4 concentration on Day 18 did not differ between treatment groups but tended to be lower (P < 0.1) in cows that had PL between Days 30 and 60 compared to cows that had no PL. In Experiment 2, FD tended to be greater (P < 0.1) and CL area was greater (P = 0.05) in anestrus cows from 7-d CO-Synch treatment. In cyclic cows, the treatment did not affect the FD or CL area. In conclusion, there was no difference in pregnancy per TAI on Day 30 and PL between Days 30 and 60 between cows using 7-d CO-Synch + PIVD or 8-d estradiol-based + PIVD protocols for estrus synchronization and TAI.

Resposta do adesivo marcador do estro, inseminação artificial em tempo fixo e protocolo de GnRH em bovinos de corte na Inseminação artificial dividida

Abstract An estrous-detection patch was used to determine the optimum timing for STAI and the necessity of GnRH at STAI on a 7-day CO-Synch+CIDR protocol. Crossbred beef cows (n=216) were stratified into the following treatment groups: CTRL=TAI (n=67) at 72 h post CIDR removal, or TRT=STAI (n=149) at 72 or 84 h post CIDR removal. All females received GnRH (100 mcg) plus a CIDR on d0, PGF2α, CIDR removal, and an Estrotect estrous-detector patch on d7. At 72 h post-CIDR removal, a patch score was assigned (PS1&lt;50% removed; PS2&gt;50% removed) to all females. Cows in the CTRL group were administered a second GnRH (100 mcg) at 72 h TAI. Cows in the TRT group with PS2 were not administered GnRH. At 84 h, the remaining TRT group cows were given a second PS; cows with PS1 received a GnRH (100 mcg), and cows with PS2 were not administered. Results: The TAI pregnancy rates were similar (P=0.81) between the CTRL (45.6%) and TRT (44.8%) groups. Pregnancy rates tended to be higher (P=0.07) for cows with PS2 (50.3%) than for those with PS1 (29.4%). However, by extending TAI to 84 h in unresponsive cows, 82.0% of TRT cows did not receive a second injection of GnRH at TAI. It was concluded that the estrous detector patches reduced the percentage of cows that required GnRH at TAI without compromising pregnancy rates. The estrous detector patches significantly reduced the number of cows that received a second GnRH injection at TAI.

Effects of temperament on reproductive performance of Bos taurus heifers enrolled in the 7-day CO-Synch + controlled internal drug release protocol.

Cattle temperament significantly impacts production traits such as reproduction. The objective of this study was to assess the effects of temperament on pregnancy rates to fixed-timed artificial insemination (TAI) in Bos taurus beef heifers. A total of 297 Angus influenced heifers from 3 different locations were evaluated for temperament based on chute score and exit velocity on the first day of the estrus synchronization protocol (d-9) and classified by temperament type based on temperament score (calm ≤ 3 < excitable). Pregnancy status was determined by transrectal ultrasonography approximately 40 d after TAI. Hair from the tail switch was collected at d-9 and at d0 (TAI) for cumulative cortisol evaluation. A subset of 43 heifers from location 3 had blood samples collected at all handling events and evaluated for cortisol concentrations. Overall, 71% of heifers were classified as calm whereas 29% as excitable. Pregnancy rates to TAI were reduced (P = 0.042) in excitable heifers compared to calm heifers (36% vs. 55%, respectively). Mean concentration of cortisol in the hair was reduced over time (P < 0.001) from d-9 (3.5 ± 0.3 pg/mg) to d0 (1.74 ± 0.3 pg/mg) in all heifers, regardless of temperament. In addition, excitable heifers had increased circulating concentrations of cortisol when compared to calm heifers in all handling events (P = 0.015). More interestingly, despite of temperament, concentrations of circulating cortisol were reduced as the protocol events progressed, with greater cortisol concentrations on d-9, intermediate on d-3, and lesser on d0 (P = 0.031). Therefore, the present study demonstrates that heifer temperament has negative effects in pregnancy rates to TAI programs. However, according to blood cortisol concentrations, no chronic stress response was detected due to cattle handling for the TAI protocol. In fact, blood cortisol concentration was reduced between the initiation and completion of the protocol, indicating that heifers were acclimated to handing. These results support the adoption of acclimation protocols and proper cattle handling as a strategy to increase fertility of heifers that are exposed to TAI.

PSI-5 Comparison of 7&amp;7 Synch and 7-day Co-Synch + Cidr Protocols in a Fixed-Time ai System on Beef Cows Using Y-Sorted sex-Selected Semen

Abstract The 7&amp;7 Synch and the 7-day CO-Synch+CIDR fixed-time estrous synchronization protocols were compared among mature and two-year-old lactating postpartum Angus and SimAngus cows (n=201). Mature (n=63) and two-year-old (n=36) cows treated with the 7&amp;7 Synch received an injection of prostaglandin F2α (PGF; 25 mg dinoprost tromethamine) at the same time as insertion of an intravaginal progesterone-releasing insert (CIDR; 1.38g progesterone) on Day -17, gonadotrophin-releasing hormone (GnRH; 100 μg gonadorelin) on Day -10, and PGF at time of CIDR removal on Day -3. Mature (n=65) and two-year-old (n=37) cows in the 7-day CO-Synch+CIDR groups received an injection of GnRH on Day -10 that coincided with CIDR insertion and received PGF at the time of CIDR removal on Day -3. Fixed-timed AI (FTAI) by a single technician using Y-chromosome-bearing sex-sorted semen (4x106 sperm/unit of SexedULTRA 4M) from one of two Charolais bulls occurred on Day 0, 70 hours after PGF injection. Seven days later bulls were pastured with cows for the remainder of the breeding season. Transrectal ultrasound was performed 77-83 days after AI and fetal size was used to differentiate AI and natural service pregnancies. Within the mature cows and two-year-old age groups, the GLIMMIX procedure in SAS was used to compare FTAI and breeding season pregnancy rates with estrous synchronization treatment and sire as main effects. No differences (P=0.41) were observed amongst mature cows in FTAI pregnancy rates between the 7&amp;7 Synch (52.4%) and the 7-day CO-Synch+CIDR (44.9%) treatments. A greater (P=0.05) proportion of two-year-old cows were pregnant to FTAI following treatment with the 7-day CO-Synch+CIDR (56.7%) as compared to the 7&amp;7 Synch (33.4%). A greater (P=0.02) proportion of two-year-old cows in the 7-day CO-Synch+CIDR treatment (91.9%) were pregnant at the conclusion of the breeding season as compared with the 7&amp;7 Synch (69.7%).

PSV-A-1 Estrus Response and Pregnancy Rates of Beef Cows Enrolled in two Fixed-Time Artificial Insemination Protocols, with or without pre-Synchronization

Abstract Pre-synchronization strategies with prolonged exposure to exogenous progesterone have the potential to alter estrus expression and improve fertility in beef cows. The objective of this study was to evaluate the estrus response and pregnancy rate of suckled beef cows enrolled in two fixed-time artificial insemination (TAI) protocols. A total of 1,216 cows (angus-crossed) at 10 locations across VA were enrolled in the study. Within location, cows were ranked by body weight, and days postpartum and randomly assigned to one of two treatments: 1) 7&amp;7 (n = 610), cows were administered prostaglandin F2α (PGF; 25 mg) and a controlled internal drug releasing (CIDR) insert [Day -14], an injection of GnRH (100 μg [Day -7]), PGF injection and CIDR removal [Day 0], and a second injection of GnRH concurrently with TAI 64 ± 2 h later [Day 2]; 2) 7-day CO-Synch+CIDR (7-day; n = 606), cows were administered GnRH (100 μg) and a CIDR insert [Day -7], PGF (25 mg) injection and CIDR removal [Day 0], and a second injection of GnRH concurrently with TAI 64 ± 2 h later [Day 2]. All cows received an estrus alert patch (ESTROTECT) on Day 0 and were evaluated for patch activation at TAI on Day 2. Pregnancy was diagnosed by transrectal ultrasonography between 45 and 65 days after TAI. The percentage of cows exhibiting estrus between Day 0 and 2 was greater (P &amp;lt; 0.001) for 7&amp;7 compared with 7-day cows (72.8% and 55.9%, respectively). However, pregnancy rate to TAI was similar (P = 0.328) between treatments (63.8% and 61.1%, for 7&amp;7 and 7-day, respectively). We conclude that suckled beef cows enrolled in the 7&amp;7 TAI protocol had increased estrus expression, but similar pregnancy rate when compared with cows enrolled in the 7-day CO-Synch+CIDR TAI protocol.

63 Impact of Supplementing Rumen-Protected Methionine During the Periconceptional Period in Fetal and Postnatal Growth in Beef Cattle

Abstract Previous evidence has suggested that fertilization and preimplantation embryonic development are sensitive periods that can be influenced by changes in maternal nutrition. We hypothesized that feeding rumen-protected methionine (RP-Met) during the periconceptional period will program bovine gestation in a manner that enhances fetal and postnatal growth. A total of 114 cows were fed a roughage-based diet and randomized to receive corn gluten supplemented with 15 g of RP-Met (RP-Met; Smartamine, Adisseo) or not (Con) from d -7 to +7 relative to artificial insemination (AI) with sex-sorted semen. Estrus synchronization was conducted using the 7-day CoSynch + CIDR protocol. Pregnancy diagnosis, embryo, and fetal morphology were conducted using transrectal ultrasonography 30, and 60 days after AI. After calving, 40 calves (Cont = 19; RP-Met = 21) were considered for birth weight (BW) analysis. Female calves (n=34) were weighed at 2 months of age. Pregnancy per AI at day 30 (Con = 50%, RP-Met = 55%) and day 60 (Con = 50%; RP-Met = 53.4%) were not different between groups (P ≥ 0.05). At 30 days of pregnancy, embryonic vesicle diameter (Con=11.0 ± 0.7 mm; RP-Met =11.6 ± 0.7 mm; P &amp;lt; 0.05) and circumference (Con=0.88 ± 0.09 cm2; RP-Met =0.99 ± 0.09 cm2 P &amp;lt; 0.05) were larger in the RP-Met group but embryo length and abdominal cavity were not different between groups. No difference was observed in fetal morphology at d 60. A treatment by sex interaction was observed (P =0.04). Male calves (n=6) from cows fed RP-Met showed greater BW than those from Con group (Con=31.9 ± 2.3 kg; RP-Met =41.4 ± 2.3 kg) but no difference was observed in females (n=34). There was no difference in BW at 2 months of age between groups. In conclusion, using RP-Met increased embryonic vesicle diameter and circumference, and BW in males.