Fixed-time Artificial Insemination Research Articles

Influence of Ovarian Follicle Sizes and Estrous Signs on Pregnancy Following Progesterone-Based Fixed Time Artificial Insemination in Water Buffaloes

The objectives of the present study were to elucidate the importance of follicle sizes and estrous signs during Controlled Internal Drug Release-Synch-human Chorionic Gonadotropin (CIDR-Synch-hCG) protocol for Fixed Time Artificial Insemination (FTAI) and to evaluate their association with pregnancy in water buffaloes. Data from riverine buffaloes (n = 207) under the CIDR-Synch-hCG protocol were analyzed. Buffaloes were administered with Gonadotropin-Releasing Hormone (GnRH) with insertion of CIDR on Day 0. Prostaglandin (PGF2α) was given on Day 7 with the removal of CIDR. hCG was given on Day 9, and AI was performed on Day 10. Follicle measurements by ultrasonography were done on Days 0, 7, and 10, and follicle sizes on those days were categorized into I, II, and III. Estrus signs were taken on the day of AI. The pregnancy diagnosis was done on Day 30-35 post-AI. The average size of follicles in Category III is significantly higher than those of Categories I and II, regardless of the Days of the protocol. Pregnancy is significantly (P<0.001) associated with Pre-Ovulatory Follicle (POF) size and uterine tonicity on the Day of AI but not with follicle sizes on Days 0 and 7, nor with mucus discharge discharge (P>0.05). The overall pregnancy rate is 44.44% while performing AI with POF size ≥12.0 mm increased the probability of pregnancy rate to 56.25%. In conclusion, the present study demonstrated a follicle size-based CIDR-Synch-hCG protocol providing new fertility indicators to improve FTAI efficiency in buffaloes with huge application in other livestock species.

Effects of cortisol levels on reproductive success in cattle of different temperaments during fixed-time artificial insemination (FTAI)

The present study was designed to assess the effect of stress on the reproductive performance of cattle. For this purpose, a total of 137 cows were divided into two groups: 65 belonging to the Azores Lydia breed and 72 crossing Aberdeen-Angus with Limousine, free of IBR, Chlamydia and BVD diseases and with a body condition score ranging from 2.75 to 4.0. Eight days before starting the experimental procedures, animals passed through the containment sleeve every other day for routine and human presence. For the synchronization protocol, a progesterone-impregnated controlled internal drug release (CIDR®) insert was placed intravaginally, and an injection of GnRH was given. After 7 days, PGF2α was administered, and the CIDR® was removed. Sixty hours after removal, cows were inseminated once after another injection of GnRH to induce ovulation, and pregnancy diagnosis was performed by ultrasonography 30 days after artificial insemination. Peripheral blood was collected in 5 ml tubes with a Z Serum Sep Clot Activator, and cortisol was measured using the IMMULITE 2000 Immunoassay System ®. Our results indicated significant differences (p≤0.05) in the cortisol levels among both groups (beef crossbred cows vs Azores Lydia) of 4.3±0.3 ng/dl and 5.8±0.4 ng/dl, respectively. The pregnancy results were also significantly different: 63.7% vs 45.6%, respectively, for beef crossbred and "Azores Lydia" cows. The present study clearly demonstrated a negative correlation between cortisol levels and pregnancy rates after fixed-time artificial insemination. The low levels of cortisol observed in the animals and particularly in the Azores Lydia breed, when compared to other studies carried out on these animals, must be due to the passage of the animals in the sleeve, allowing them to habituate to a routine, as well as to human presence.

178 Optimization of the time of insemination and presynchronization treatment in beef heifers submitted to fixed-time artificial insemination

178 Optimization of the time of insemination and presynchronization treatment in beef heifers submitted to fixed-time artificial insemination

181 Expression of estrus and fertility of different presynchronization strategies for fixed-time artificial insemination in suckled beef cows

181 Expression of estrus and fertility of different presynchronization strategies for fixed-time artificial insemination in suckled beef cows

Comparative evaluation of natural tupping with fixed-time laparoscopic and cervical insemination techniques using chilled semen in estrous synchronized sheep

The present study was conducted in crossbred ewes to compare the efficiency of different artificial insemination techniques with natural tupping following fixed time artificial insemination using fresh semen. Crossbred ewes (n=29) were randomly divided into three groups with 10 animals in natural tupping (NT) and laparoscopic artificial insemination (LAI) groups and nine animals in cervical artificial insemination (CAI) group. Ewes in all the treatment groups were subjected to the same estrus induction protocol: insertion of intravaginal progesterone sponges for 10 days followed by an intramuscular injection of 500 IU eCG at the time of sponge withdrawal. Immediately after removal of sponges, animals of NT group were kept with proven breeding rams up to 72 h. Fixed time cervical or laparoscopic insemination was done at 48 h after sponge removal. The insemination was repeated 12 h later in CAI group. The pregnancy and lambing rates were significantly higher in NT group (90%, 90%), than CAI group (55.5%, 55.5%) and LAI group (20%, 10%). The prolificacy rate was significantly higher in CAI group (180%) than NT group (111.1%) and LAI group (100%). The serum progesterone concentration was higher in pregnant ewes on day 10, day 17 and day 35 than non-pregnant ewes. The fixed time cervical insemination following intravaginal progesterone sponges for 10 day+eCG protocol resulted in better pregnancy rate and prolificacy rate in crossbred ewes during breeding season.

Effects of mineral and vitamin supplementation on pregnancy rates in Nelore cattle submitted to FTAI programs with different reactivity scores

A high-reactivity herd temperament reduces productivity and profitability in cattle farming. In Bos taurus indicus (Nellore) cattle, we hypothesized that a higher reactivity score is associated with a lower pregnancy rate, and that injectable supplementation with minerals (Cu, Zn, Mn, Se) and vitamins (A, E) would increase the pregnancy rate and reduce embryonic mortality in very reactive cows. Injectable supplement or control treatments were administered on days 25 and 10 before fixed-time artificial insemination (FTAI; Day 0 : day of FTAI). We selected 1722 cows 45 ± 7 days postpartum, with different behavioral reactivity levels, and a series of measures related to reproduction were measured. The cows were randomly grouped and received sc 1 mL/50 kg live weight of the supplement (n = 872) or placebo (n = 850), 25 and 10 days before the FTAI protocol (Day–25 and Day–10 respectively). On Day −10, devices containing 1 g progesterone were inserted into the vagina, and 2 mg estradiol benzoate was applied intramuscularly. On Day–2, the devices were removed and 530 µg of sodium d-cloprostenol, 300 IU of equine chorionic gonadotropin, and 1 mg of estradiol cypionate were applied. At 48 h after final injection, the cows were subjected to FTAI (Day 0). On Day - 25 (25 days before IATF), temperament was graded as 3 to 12 using composite reactivity score (RS), which evaluates movement and breathing while the animal is in the cattle chute, and their escape velocity, and was used to classify cows as calm (RS ≤ 4; n = 590), reactive (RS 5 to 8; n = 573), and very reactive (RS ≥ 9; n = 559). On Day 0, calm cows had a higher largest follicle diameter on the FTAI compared to reactive and very reactive (14.4 + 0.2 vs. 13.6 + 0.4 and 13.2 + 0.2 mm, respectively; P = 0.03). Interactions were observed, where supplemented cows compared to those no supplemented had higher pregnancy on Day 30 (P = 0.04; 60.00 vs. 54.10 % calm and 58.00 vs. 52.50 % very reactive) and Day 60 (P = 0.03; 58.70 vs. 47.90 % calm, 52.20 vs. 49.10 % reactive and 48.40 vs. 41.30 % very reactive) and reduced embryonic loss at 30 to 60 d (P = 0.04; 2.00 vs. 6.10 % calm, 4.20 vs. 8.40 % reactive and 9.60 vs. 11.20 % very reactive). Supplementation on Days–25 and–10 improved pregnancy rates and reduced embryonic in calm and very reactive cows.

Effects of a single supplemental dose of gonadotropin-releasing hormone in 2 different fixed-time artificial insemination synchronization protocols among beef cows and heifers

Effects of a single supplemental dose of gonadotropin-releasing hormone in 2 different fixed-time artificial insemination synchronization protocols among beef cows and heifers

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.

Evaluation of later timepoints for fixed-time artificial insemination of beef heifers and cows when using sex-sorted semen

Two experiments were designed to evaluate later timepoints for fixed-time artificial insemination (FTAI) of beef heifers and cows, with the hypothesis that use of a later timepoint would allow a greater proportion of animals to express estrus prior to FTAI and result in greater conception rates among estrous females inseminated with sex-sorted semen. In Experiment 1, estrus was synchronized for 1640 heifers using the 14 d CIDR-PG protocol: insertion of an intravaginal progesterone-releasing insert (CIDR; 1.38 g progesterone) on Day −33 and removal on Day −19, and administration of prostaglandin F2α (PG; 500 μg cloprostenol sodium) on Day −3. Heifers were inseminated at one of three FTAI timepoints: 66 h, 70 h, or 74 h after PG administration. In Experiment 2, estrus was synchronized for 414 beef cows using the 7 & 7 Synch protocol: administration of PG coincident with CIDR insertion on Day −17, gonadotropin-releasing hormone (GnRH; 100 μg gonadorelin) on Day −10, and PG coincident with CIDR removal on Day −3. Cows were inseminated at one of two FTAI timepoints: 66 h or 72 h after PG administration. In both experiments, only animals that expressed estrus prior to FTAI were inseminated with sex-sorted semen. In Experiment 1, the proportion of heifers that expressed estrus prior to FTAI (66 h: 62 %; 70 h: 67 %; 74 h: 71 %) was greater when FTAI was performed at 74 h versus 66 h (P = 0.0097); however, conception rate of heifers that expressed estrus and were serviced with sex-sorted semen did not differ among treatments (P = 0.67; 66 h: 56 %; 70 h: 53 %; 74 h: 53 %). In Experiment 2, the proportion of cows expressing estrus prior to FTAI did not differ between treatments (P = 0.30; 66 h: 71 %; 72 h: 76 %). Additionally, conception rate of estrous cows inseminated with sex-sorted semen did not differ between treatments (P = 0.24; 66 h: 45 %; 72 h: 40 %). These results indicate that performing FTAI later following the 14 d CIDR-PG protocol increases the proportion of heifers that express estrus and are serviced with sex-sorted semen but does not improve conception rates. Later timing of FTAI following the 7 & 7 Synch protocol was not observed to increase the proportion of cows expressing estrus prior to FTAI or improve conception rates among estrous cows inseminated with sex-sorted semen. Together, these results provide further insight into optimal timing of FTAI when using sex-sorted semen.

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.

PSIII-7 Effect of Time of Estrus on Conception Rate for a Sexed Semen Artificial Insemination Protocol in Multiparous Beef Cows

Abstract Sexed semen artificial insemination is a breeding technique that allows producers to shorten breeding seasons, optimize genetics through use of superior semen, create calves based on sex preference, and increase profitability. Sexed semen is a technology that sorts semen by differences in DNA content between X (female) and Y (male) bearing sperm. However, sexed semen practices have yet to be widely used in beef cows due to reduced sperm numbers, low conception rates, and increased costs. The objective of this study was to determine the effect of time of estrus upon fixed-time artificial insemination (FTAI) of sex-sorted semen on conception rate using two different estrous synchronization protocols. Multiparous commercial beef cows (n = 73) were randomized by age, body condition score (BCS), and days post-partum at the University of Arkansas Livestock and Forestry Research Station in Batesville. Both synchronization treatments (TRT) used a controlled internal drug releasing device (Eazi-breed CIDR; 1.38g progesterone) inserted intravaginally on d -14 of the protocol simultaneous to administration of prostaglandin F2α (PGF; 500µg cloprostenol sodium; Estrumate). In the first treatment (TRT 1; 7&amp;7 Synch), gonadotropin-releasing hormone (GnRH; 86µg gonadorelin; Fertagyl) was administered on d -7. In the second treatment (TRT 2; 8&amp;6 Synch), GnRH was administered on d -6. Both treatments were then administered PGF simultaneous to CIDR removal on d 0, 66 ± 2h before FTAI and administration of GnRH. Standing heat (estrus) was visually detected every 3 h for 30 min intervals between CIDR removal and FTAI. Heat detection patches were used in conjunction with visual detection of estrus. Time of estrus was signified as time of standing immobile when mounted in combination with presenting a patch score of 4 (&amp;gt;75% color removed). Time of estrus (TD) was deviated from time of FTAI and categorized into three equal time groups (Grp 1 = 0 to 11.0 h; Grp 2 = 11.01 to 22.0 h; and Grp 3 = 22.01 to 33.0 h). Conception rate (CR) was determined via ultrasound 37 ± 2 d after FTAI. Data were analyzed using the GLIMMIX procedure of SAS 9.4. CR did not differ by age of dam (P = 0.58), BCS (P = 0.48), or TRT (P = 0.83). However, TD had an effect on CR (P = 0.002); and resulted in CR of 45% (Grp 1), 87% (Grp 2), and 42% (Grp 3). In conclusion, these results indicate artificially inseminating cows with sexed semen between 11.01 and 22.0 h after time of standing estrus will increase pregnancy rates for multiparous commercial beef cattle.

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.

292 Tandem Administration of Prostaglandin F2α and Gonadotropin-Releasing Hormone in Beef Heifers and Cows as a Convergent Presynchronization Method in the 7 &amp; 7 Synch Protocol

Abstract An experiment was designed to evaluate modifications to the 7 &amp; 7 synch protocol with the hypothesis that tandem administration of prostaglandin F2α (PG) and gonadotropin-releasing hormone (GnRH) during the presynchronization portion of the protocol would generate a convergent ovarian response among anestrous and estrous cycling females. Nulliparous (n = 153), primiparous (n = 92), and multiparous (n = 413) Angus-cross females across four locations were blocked based on parity and pre-treatment cyclicity status (presence or absence of a corpus luteum) as assessed via transrectal ovarian ultrasonography. Within block, females were randomly assigned to one of three treatments. Females assigned to the 7 &amp; 7 Synch treatment received an intravaginal progesterone-releasing insert (CIDR) and administration of PG on day 0, administration of GnRH on day 7, and administration of PG coincident with CIDR removal on day 14. Females assigned to 7 &amp; 7 Synch + G received the same treatment schedule with the modification that GnRH was administered in tandem with the PG on day 0. Females assigned to 7 &amp; 7 Synch + G + P received GnRH in tandem with PG on both day 0 and day 7 of the treatment schedule. Fixed-time artificial insemination (FTAI) was performed at 54 h after CIDR removal for nulliparous females and at 66 h for primiparous and multiparous females. Transrectal ovarian ultrasonography was performed on day 7 and day 14 to characterize the largest follicle diameter (LFD) as well as the presence/absence and number of corpora lutea (CL). Expression of estrus before FTAI was characterized in three locations using estrus detection patches (Estrotect. Approximately 75 to 90 days after FTAI, pregnancy status was determined via transrectal ultrasonography. Day 7 CL status was affected by treatment (P = 0.002), with a greater proportion of females presenting with CL on Day 7 when treated with the modified treatments (64%) as compared with the 7 &amp; 7 Synch treatment (31%). Day 14 LFD was affected by treatment (P = 0.002), with a larger average LFD among females treated with 7 &amp; 7 Synch + G + P (12.0 mm) as compared with 7 &amp; 7 Synch (11.4 mm) or 7 &amp; 7 + G (10.7 mm). Pregnancy rate to FTAI was affected by pre-treatment cyclicity status (P &amp;lt; 0.0001) and semen type (P = 0.0003) and tended to be affected by treatment (P = 0.09) with 7 &amp; 7 Synch + G + P tending to result in a greater proportion pregnant (58.86%) as compared with 7 &amp; 7 Synch (49.17%) and 7 &amp; 7 Synch + G (48.79%). Tandem administration of GnRH and PG on days 0 and 7 of the 7 &amp; 7 Synch protocol effectively generated a convergent ovarian response among anestrous and estrous cycling beef females and tended to result in increased pregnancy rates to FTAI.

291 Impact of Early Resynchronization in Combination with Color Doppler Ultrasonography on Cumulative Pregnancy Rates in Postpartum Beef Cows

Abstract The objective of this study is to evaluate a GnRH and progesterone-based early resynchronization strategy combined with color Doppler ultrasonography for early pregnancy diagnosis. Postpartum Bos taurus beef cows (n = 375) from four locations were enrolled in a completely randomized design, where cows were randomly assigned to 1 of the 2 treatments: 1) Natural Service (NS), cows were exposed to a 7-d CO-Synch + CIDR protocol followed by fixed-time artificial insemination (day 0). On day 15, cows were exposed to natural service for the remainder of the study; or 2) Early Resynchronization (ER), cows were exposed to a 7-d CO-Synch + CIDR protocol followed by fixed-time artificial insemination (day 0). On day 15, a controlled internal drug release (CIDR) device was inserted. On day 20, CIDR was removed, and pregnancy was estimated based on luteal blood perfusion using color Doppler ultrasonography. Non-pregnant cows received a 25-mg injection of prostaglandin F2α at CIDR removal and were fixed-timed-AI concurrently with a 100-μg injection of GnRH 60 to 66 h later. Pregnancy diagnosis and fetal aging were performed on day 53 using conventional ultrasonography. Pregnancy rate to the first AI was greater (P = 0.03) for NS compared with ER (70.25% vs 59.29%, respectively). Pregnancy rates for the second service were also greater (P = 0.01) for NS compared with ER (65.67 vs 42.93%, respectively). Cumulative pregnancy rates to AI did not differ between treatments (P = 0.38); however, cumulative pregnancy rates (first and second service) were greater (P &amp;lt; 0.01) for NS compared with ER (90 vs 77.73%, respectively). In conclusion, the proposed early resynchronization strategy using color Doppler ultrasonography resulted in fewer pregnancies compared with natural service as the second service strategy. Moreover, the proposed ER decreased pregnancy rates to the first insemination, indicating a detrimental effect of ER on pregnancy establishment.

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.

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.

PSXIV-13 Evaluation of On-Animal Sensors for Monitoring Diurnal Physical Activity of Beef Bulls During the Breeding Season

Abstract Applications of on-animal sensors (OAS), commonly used to monitor behavioral patterns in livestock, remain largely undeveloped for extensively managed beef bulls. Thus, the objective of this study was to characterize and compare two OAS types for monitoring bull activity during the breeding season. Angus and Angus crossbred beef bulls (n = 50) aged 1 to 8 years, from six cow-calf operations in Ohio were placed into multi-sire breeding groups (n = 19) with a bull-to-female ratio of 1:25 (range 1:16 to 1:31). Bulls were placed with a female group from day 10 (range day 5 to 12) until day 60 after fixed-time artificial insemination. Before turnout, each bull was subjected to a breeding soundness examination and fitted with a collar-mounted GPS and an ear tag-mounted triaxial accelerometer (AX3). Raw GPS and AX3 activity measurements, namely speed (m/s) and motion intensity (g), were converted to standardized Z-scores and summarized to an hourly resolution by computing the median hourly activity over a 24-hour cycle at early (day 13 to 28) and late (day 34 to 49) stages of the breeding season. Linear mixed models were fitted to standardized activity measurements (expressed in log scale) to assess the dynamic effect of OAS type throughout the day at early and late stages of the breeding season. Size of the breeding group (males and females), bull age, scrotal circumference, and body condition score were evaluated as potential explanatory covariates using stepwise selection. Diurnal activity curves followed a pattern of relative inactivity overnight from hours 21 to 5 and relative activity during daylight from hours 6 to 20. For both OAS, mean activity early in the breeding season was ~47% greater (P &amp;lt; 0.01) than late. At both breeding season stages, mean activity of bulls differed between OAS types (P &amp;lt; 0.01) at hours 2, 3, 4, 5, and 6 by at least 38%, with greater activity from GPS than AX3 (P &amp;lt; 0.05 in all cases). Conversely, at hours 13, 14, and 16, activity derived from AX3 was greater than from GPS by at least 2.5-fold (14: P &amp;lt; 0.01; 13 and 16: P &amp;lt; 0.1). There was no evidence (P &amp;gt; 0.1) for differences between bull activity derived from GPS or AX3 during the remaining overnight (21-1) and daytime (7 to 12, 15, and 17 to 20) hours. In conclusion, this study provides evidence that simultaneous assessment of bull activity using independent OAS, differed between technologies throughout a 24-h day. Additionally, over the course of the breeding season, both OAS indicated a decline in activity. Bull breeding activities are expected to be less frequent as the breeding season progresses; thus, these may explain the observed differences between stages of the breeding season. Further exploration of benchmarks for bull activity may yield useful tools for the identification of health and reproductive-related behaviors.

179 Cost and Profit Per Pregnancy Associated with Estrous Synchronization Utilizing Estrus Detection And/Or Fixed-Time Artificial Insemination

Abstract This experiment aimed to compare synchronization cost associated with two protocols: one utilizing estrus detection (6d; PG 6-d CIDR+TAI) and one utilizing strictly fixed-time artificial insemination (FTAI; 7d; 7-d CO-Synch+CIDR). Cows and heifers (n = 657) from 10 herds in three states were grouped by days postpartum and age, and randomly assigned to one of two protocols. Cows and heifers assigned to the 6 d protocol were administered prostaglandin F2α (PGF) on d-9, CIDR insertion and administration of gonadotropin-releasing hormone (GnRH1) on d -6, and CIDR removal and PGF administration on d 0. Cows and heifers that were assigned to the 6-d protocol were subsequently grouped into three treatments based on presence and/or timing of estrus and insemination (PG1: Insemination before CIDR insertion, n = 136; PG2: Insemination after CIDR removal, n = 26; T6: Insemination at TAI, n = 166). Cows and heifers assigned to the 7-d protocol (T7: Insemination at FTAI, n = 329) were administered GnRH1 and CIDR insertion on d -7, followed by CIDR removal and PGF administration on d 0. For all animals, FTAI and GnRH2 administration occurred at 54 h (heifers) or 66 h (cows) after CIDR removal. Pregnancy was determined from d 30 to d 40 after FTAI by transrectal ultrasonography. Total cost and profit per pregnancy was calculated for herds of 30, 100, 300, and 500 females with varying estrus response and pregnancy rates. Expenditures were calculated assuming synchronization costs (PG1 = $46.15, PG2 = $75.05, T6 = $77.91, T7 = $73.72), general labor cost considering herd size and handling days, as well as estrus detection labor cost assuming $10.00 per h, a 95% overall herd pregnancy rate, a 1:40 bull:cow ratio, and average bull purchase price of $3,000.00. Revenue was estimated assuming a maximum 205-d weaning age, 1.09 kg/d gain, $200.00/45.4 kg, and 85% weaning rate. Differences in estrus response and pregnancy rate were determined using the GLIMMIX procedure of SAS (v9.4), including herd as a random variable. Estrus response was greater (P &amp;lt; 0.0001) in females assigned to the 6d (73 ± 4%) compared with the 7d (53 ± 4%) protocol; however, pregnancy success was similar among treatment groups (P = 0.32; PG1 = 45 ± 4%, PG2 = 58 ± 10%, T6 = 46±4%, T7 = 52±3%). At the observed estrus response and pregnancy rate for each treatment group, the 6d protocol resulted in a decreased cost per pregnancy compared with the 7d protocol [Table 1; herds of: 30 = ($3.53), 100 = ($5.16), 300 = ($6.58), 500 = ($7.00) females]. Additionally, using the same variables, the 6d protocol resulted in an increased profit per pregnancy compared with the 7d protocol (herds of: 30 = $3.50, 100 = $6.41, 300 = $17.27, 500 = $12.06 females). These results suggest that labor associated with estrus detection may be offset by the proportion of calves subsequently born earlier within the calving season, allowing for improvements to profitability, especially in larger herds.

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.