Indicus Females Research Articles

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.

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 < 0.01). Genera with phylum Firmicutes including Ruminococcus, Clostridium, Blautia, Butyrvibrio, and Mogibacterium followed a similar trend (P < 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 < 0.01), interleukin (IL)1F5 (153.89 ± 141.07 pg/mL vs. 627.30 ± 149.28 pg/mL; P < 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 < 0.05). These results indicate differences in circulating cytokines and shifts in the vaginal microbiome for in Bos indicus cattle prior to FTAI.

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.

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.

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.

Influence of GnRH analog and dose on LH release and ovulatory response in Bos indicus heifers and cows on day seven of the estrous cycle

This study evaluated the influence of GnRH analogs (gonadorelin vs. buserelin) and doses (single vs. double) on LH release and ovulatory response in Bos indicus (Nelore) females on Day 7 of the estrous cycle. Cycling heifers and non-lactating cows were pre-synchronized: Day −10: progesterone (P4) implant insertion plus 2 mg of estradiol benzoate; Day −2: implant removal and 0.53 mg of cloprostenol sodium (PGF); Day 0: 25 μg of lecirelin (GnRH). Over four replicates, heifers (n = 57) and cows (n = 53) that ovulated to the GnRH treatment on Day 0, having a visible corpus luteum (CL) and a dominant follicle (DF) ≥ 8.5 mm, were allocated to receive the following GnRH treatments on Day 7: G-Single (100 μg of gonadorelin); G-Double (200 μg of gonadorelin); B-Single (10 μg of buserelin); and B-Double (20 μg of buserelin). At GnRH treatment, a P4 implant was inserted in heifers (0.5 g) and cows (1 g). Ultrasound examinations were done on Days −10, −2, 0, 2, 7, 9, 12, and 14 to evaluate DF diameter, ovulation and presence of CL. Blood samples were collected on Day 7 at 0, 2, and 4 h from GnRH treatment, to evaluate circulating P4 and LH concentrations. On Day 12, the P4 implant was removed, females received two PGF treatments (24 h apart), and 2 d later, 25 μg of GnRH was given to start the next replicate. In both heifers and cows, P4 concentrations were elevated on Day 7, and similar among groups (3.9 and 4.2 ng/mL, respectively). In heifers, buserelin induced greater LH peak (9.5 vs. 2.6 ng/mL; P < 0.01) and greater ovulation (88.9 [24/27] vs. 16.7% [5/30]; P < 0.01) than gonadorelin treatments, regardless of the dose. Similarly, in cows, buserelin induced greater LH peak than gonadorelin (9.9 vs. 4.9 ng/mL; P < 0.01). However, ovulation was only increased in cows from the B-Double group (90.9% [10/11]), whereas in the other groups the ovulatory response was similar (35.7% [15/42]). Regardless of treatment, heifers had similar P4 concentrations (P = 0.22), but smaller DF (P < 0.01) than cows on Day 7. Only in G-Double group the LH peak was lower (P = 0.05) in heifers than in cows, with no difference within other groups. In heifers, but not in cows, the single dose of buserelin resulted in high ovulatory response, equivalent to that produced by the double dose. In conclusion, in Bos indicus heifers and cows on Day 7 of the cycle, with elevated P4 concentrations, buserelin induced greater LH release and ovulatory response than gonadorelin treatments. Double doses increased the LH release, however, only resulted in greater ovulation in females treated with buserelin. Finally, although circulating P4 concentrations did not differ between parities, heifers were more likely to ovulate in response to a GnRH-induced LH peak than cows.

Oxidative activity of corpus luteum and ovarian parenchyma in Bos taurus indicus heifers.

The aim of this study was to evaluate the oxidative stress in ovaries and corpus luteum (CL) of Bos taurus indicus females and the oxidant effect of CL in ovarian tissues in regions near, intermediate, or distant from it. Ovaries (n=12) of Nelore heifers (n=6) were collected from a slaughterhouse and fragmented. Experiment 1, each ovary was obtained from three fragments, resulting in 18 fragments of ovaries with CL (OV+CL) and another 18 fragments of ovaries without CL (OV-CL). Three fragments were generated from CL, totaling 18 CL fragments. In experiment 2, the ovarian fragments were removed from specific regions near, intermediate, or distant from the CL. All the fragments were placed in Eppendorf-type microtubes (1 mL), kept in a thermal container at 4 ºC, and then stored in a -80 ºC freezer for analysis of oxidative stress (TBARS and NBT) and antioxidant potential (FRAP and ABTS). In the antioxidant activity analysis, luteal tissues showed more antioxidant activity than ovarian tissue (FRAP = P < 0.0001; ABTS = P < 0.02). In the oxidative stress analysis, CL had lower levels of reactive oxygen species (ROS; TBARS = P < 0.03; NBT = P < 0.0001) than ovarian tissues. There was no difference in antioxidant activity and oxidative stress between the fragments obtained from different regions (OV+CL versus OV-CL; P > 0.05). The presence of CL in the ovaries of Bos taurus indicus females did not influence the oxidative stress or antioxidant potential of the gonad. Thus, the removal of ovarian fragments with or without the presence of CL indicates that biotechnologies such as in vitro follicle cultivation is possible.

Follicular development, morphological integrity, and oxidative stress in bovine preantral follicles cultured in vitro with ascorbic acid.

The aim of this study was to evaluate the follicular development, morphological integrity, and oxidative stress of preantral ovarian follicles from Bos taurus indicus females grown in vitro with ascorbic acid. Ovaries (n = 20) from Bos taurus indicus females were collected, fragmented, and were cultured in vitro for 6 or 12 days in minimum essential medium (MEM), or MEM supplemented with 50 or 100 ng/ml ascorbic acid, with an extracellular matrix of agarose gel, in an incubator at 38.5°C; every 2 days, 100% of the culture medium was replaced. The data were analyzed using the chi-squared test and/or Fisher's exact test. In the event of a significant effect, the proportions were compared using a 2 × 2 proportion test. The oxidative stress analysis data were submitted to analysis of variance followed by the Bonferroni test. Values were considered significant when P ≤ 0.05. The addition of 100 ng/ml of ascorbic acid to the in vitro culture medium of preantral ovarian follicles from bovine females promoted follicular development, was efficient in maintaining morphological integrity, as well as the stability of reactive oxygen species, after 6 days of in vitro culture.

Culture of preantral ovarian follicles of Bos taurus indicus with alpha-lipoic acid.

The aim of this study was to evaluate follicular development, morphological integrity, and antioxidant potential of preantral ovarian follicles from Bos taurus indicus females grown in vitro with alpha-lipoic acid. Ovaries (n = 24) of Bos taurus indicus (n = 12) females were collected during slaughter and fragmented. A randomly obtained fragment from each pair of ovaries was fixed in Bouin (non-cultivated control; D0). These fragments were intended for classical histology (morphology and evaluation of follicular growth), and a fragment from each pair of ovaries was frozen at -80°C (non-cultivated control; D0), and assigned for analysis of oxidative stress [thiobarbituric acid reactive substances (TBARS), nitroblue tetrazolium (NBT), and ferric reducing antioxidant power (FRAP)]. The remaining fragments were cultured in vitro for 6 (D6) or 12 (D12) days, containing only minimum essential medium (MEM) or MEM supplemented with alpha-lipoic acid (50, 100, or 250 ng/ml), on an extracellular matrix of agarose gel, in an oven at 38.5ºC. Every 2 days, 100% of the culture medium was replaced. Supplementation with 100 ng/ml was effective for maintaining follicular integrity after 6 days of culture (primordial: 51.28%; development: 36.88%; P < 0.0001). There was no difference (P > 0.05) between treatments compared with the non-cultivated control treatment (D0), using the NBT and TBARS assays. Therefore, supplementation of the in vitro culture medium of bovine preantral ovarian follicles with a concentration of 100 ng/ml of alpha-lipoic acid at 6 days of culture was effective for maintaining follicular integrity and, after 6 days, maintaining stable levels of reactive oxygen species.

Lipid profile of in vitro embryos produced from Bos indicus cows with low and high antral follicle counts

The quantity of lipids available for the initial development processes of in vitro-produced (IVP) embryos can reflect directly on embryonic quality and pregnancy rates. Therefore, the objective of this study was to investigate the lipid profile of IVP embryos from Bos indicus females with different antral follicle counts (AFCs). For this purpose, ovaries from 149 Nelore females were collected at a local slaughterhouse and transported to the laboratory. AFC was determined by visual follicle counting on the surface of both ovaries of each animal. The animals were classified as low AFC (≤ 31 follicles; mean less SD) and high AFC (≥ 92 follicles; mean plus SD). Oocytes were matured, fertilized and cultured in vitro. Cleavage and blastocyst rates were evaluated on days 3 and 7, respectively. Blastocysts collected on day 7 (D7; n = 18 per group) were subjected to lipid profile analysis by desorption electrospray ionization-mass spectrometry (DESI-MS). In vitro production rates were evaluated by logistic regression using the Car R statistical package (R Development Core Team – 2008), and the differences were considered significant at P < 0.05. For lipid profiling, principal component analysis (PCA) followed by Fisher's test was used. There were no differences between groups regarding the cleavage (78.9% in high AFC vs. 79.5% in low AFC) or blastocyst rates (41.7% in high vs. 40.3% in low AFC). The embryos derived from high-AFC cows presented a higher concentration of lipids in the category of TAG than the embryos from low-AFC females. In contrast, embryos from low-AFC donors had the highest concentrations of cholesterol and its derivatives and DAG. In conclusion, the lipid profile of embryos generated from ovaries with a low AFC is different from that of embryos generated from ovaries with a high AFC despite similar IVP rates. Our results may support studies related to the influence of lipid content on embryonic development and viability.

Relationship among serum metabolic hormones with pregnancy rates to fixed-time artificial insemination in Bos indicus beef females

Two experiments compared body parameters and serum concentrations of GH, IGF-I, and leptin in Bos indicus females based on their pregnancy status to fixed-time artificial insemination (FTAI). In Exp. 1, 226 nulliparous heifers were assigned to a puberty induction protocol (d -35 to d -23), followed by an estrus synchronization and FTAI protocol (d -11 to 0). Heifers received a controlled internal progesterone releasing insert (CIDR) previously used three times on d -35, followed by 0.6 mg of estradiol cypionate and CIDR removal on d -23. Heifers received 2 mg of estradiol benzoate and a CIDR previously used twice on d -11, 12.5 mg of dinoprost tromethamine in addition to CIDR removal, 0.6 mg of estradiol cypionate and a 200 IU of eCG on d -2, followed by FTAI on d 0. Heifer BW and BCS were recorded on d -35, -11, and 30, whereas blood samples were collected on d -35, -23, -11, 0, and 30. In Exp. 2, 821 multiparous lactating Nelore beef cows were assigned to the estrus synchronization and FTAI protocol described in Exp. 1, but received 300 IU of eCG on d -2. Cow BW and BCS were recorded on d -11 and 30, and blood samples were collected on d -11, 0, and 30. In both experiments, pregnancy status was verified via transrectal ultrasonography on d 30. In Exp. 1, heifers that became pregnant had greater (P = 0.03) IGF-I concentrations on d 0 and less (P = 0.05) IGF-I concentrations on d 30 compared to heifers that did not become pregnant (pregnancy status × day; P < 0.01). No differences in BW, BCS, GH and leptin were detected (P ≥ 0.23) according to heifer pregnancy status. In Exp. 2, cows that became pregnant had less (P = 0.03) leptin concentrations on d -11, and less (P = 0.01) IGF-I concentrations on d 30 compared to cows that did not become pregnant (pregnancy status × day; P ≤ 0.05). No differences in BW, BCS, and GH were detected (P ≥ 0.15) according to cow pregnancy status. In summary, serum leptin at the beginning of estrus synchronization was negatively associated with pregnancy rates to FTAI in multiparous cows. A negative relationship was also observed between serum IGF-I during early gestation with pregnancy rates to FTAI across parities. These results provide novel insights on the relationships among metabolic hormones with reproductive performance of B. indicus females.

Effect of the Estrus Expression on Follicular and Luteal Morphofunctional Characteristics and Fertility in Bos indicus females Synchronized for FTAI

Background: Recent studies have been conducted with the aim of improving the fertility rates in the FTAI programs in beef females. The observation of the estrus expression constitutes an important indicator of fertility in zebu females. Therefore, this work has as an objective to evaluate the impact of the estrus expression on the follicular, luteal and fertility morphofunctional characteristics of Nelore females synchronized for FTAI.Materials, Methods &amp; Results: Sixty five lactating female Nelore (Bos taurus indicus) were used. On a random day, denominated day 0 (D0), the 65 Nelore females received a progesterone-releasing device associated to the application of 2.0 mg of estradiol benzoate intramuscularly (IM). On D9, the progesterone-releasing intravaginal devices were removed and was administered 500 μg of cloprostenol sodium IM; 0.6 mg of estradiol cypionate IM and 300 UI of Equine Chorionic Gonadotropin IM. At this point, the animals were marked with a marking stick for the determination of the estrus expression. On D11 of the synchronization protocol, the animals were characterized in two groups: without estrus expression (WO/ ESTRUS) and with estrus expression (W/ ESTRUS). The evaluation of the follicle diameter (FOLD), of the follicle wall area (FOLA), of the follicle wall vascularization (FOLV) and the percentage of vascularization in the area of the preovulatory follicle wall (%FOLV) were conducted on D11 using B-mode ultrasonography and color Doppler and then the artificial inseminations were performed. The evaluation of the corpus luteum diameter (CLD), the total area of the corpus luteum (CLA), of the area of vascularization of the corpus luteum (CLV), of the percentage of vascularization of the in the area of the corpus luteum (% CLV) and the collection of blood for the evaluation of the serum levels of progesterone were carried out on D24. The analysis of the serum progesterone concentration were performed by the chemiluminescence method using the Access immunoassay systems Progesterone, with a sensitivity of 0.1 ng/mL. The diagnosis of gestation was carried out by transrectal ultrasonography on D45. The animals of the WITH ESTRUS group presented superior FOLD, FOLA, FOV (P &lt; 0.05) averages to those presented by the cows of the WITHOUT ESTRUS group. In relation to the luteal characteristics, in the WITH ESTRUS group the cows exhibited averages for CLD, CLA, CLV and P4 significantly higher than those of the WITHOUT ESTRUS group. However, the bovines of the WITH ESTRUS group presented a % CLV similar to the presented by the females of the WITHOUT ESTRUS group. As for the fertility rates, there was difference between the experimental groups WITH ESTRUS and WITHOUT ESTRUS.Discussion: The present work demonstrated the impact of the estrus expression on the follicular morphological parameters, therefore in accordance with other studies. These authors show a relation between the estrus expression and levels of estradiol, which is directly related to the sperm transport in the female reproductive tract, oocyte maturation and fertilization directly affecting the fertility of females submitted to the FTAI program. Similarly, there was an interrelation between the estrus expression and the luteal morphological parameters, corroborating with the findings of some researches, where were verified higher luteal morphological parameters associated to higher conception rates in cows that demonstrated estrus behavior in the FTAI. Therefore, the estrus detection can be used as a tool to direct matings in synchronization protocols, promoting the identification of the animals with a higher probability of conception, owing to better follicular and luteal hemodynamic conditions.

385 Awardee Talk: Impacts of temperament on productive and reproductive responses of Bos taurus and B. indicus females

Abstract Temperament is defined as the fear-related behavioral responses of cattle when exposed to human handling. Our group evaluates cattle temperament using: 1) chute score: 1 to 5 scale that increases according to excitable behavior during chute restraining, 2) exit velocity: speed of an animal exiting the squeeze chute, 3) exit score: dividing cattle according to exit velocity into quintiles using a 1 to 5 scale (1 = slowest quintile; 5 = fastest quintile), and 4) temperament score: average of chute and exit scores. Subsequently, cattle are assigned a temperament type; adequate temperament (ADQ; temperament score ≤ 3) or excitable temperament (EXC; temperament score &amp;gt; 3). Our group associated these evaluation criteria with reproductive responses of Bos taurus and B. indicus-influenced females. Cattle classified as EXC had greater (P ≤ 0.04) plasma cortisol vs. ADQ during handling, independent of breed type or parity. Both B. taurus and B. indicus cows classified as EXC had reduced (P ≤ 0.09) annual pregnancy rates vs. ADQ cohorts, resulting in decreased calving rate, weaning rate, and kg of calf weaned/cow exposed to breeding. Bos indicus heifers classified as EXC as yearlings had less (P &amp;lt; 0.01) growth rate and delayed puberty attainment by 15 months of age compared with ADQ heifers. Bos taurus heifers that reached puberty by 12 months of age had less (P ≤ 0.05) temperament score and plasma cortisol concentrations at weaning compared with heifers that did not reach puberty. Acclimating B. indicus-influenced and B. taurus heifers to human handling after weaning improved (P ≤ 0.02) temperament and hastened puberty attainment. Hence, temperament impacts productive and reproductive responses of beef females independent of breed type and parity. Strategies to improve herd temperament are imperative for optimal production efficiency of cow-calf operations based on B. taurus and B. indicus-influenced cattle.

Ovarian follicular dynamics, progesterone concentrations, pregnancy rates and transcriptional patterns in Bos indicus females with a high or low antral follicle count

We evaluated the effect of the antral follicle count (AFC) on ovarian follicular dynamics, pregnancy rates, progesterone concentrations, and transcriptional patterns of genes in Nelore cattle (Bos taurus indicus) after a timed artificial insemination (TAI) programme. Cows were separated based on the AFC, and those with a high AFC showed a larger (P < 0.0001) ovarian diameter and area than those with a very low AFC. Females with a very low AFC exhibited a larger (P < 0.01) diameter of the dominant follicle at TAI (13.6 ± 0.3 vs. 12.2 ± 0.4 mm) and a tendency (P = 0.06) to have different serum progesterone concentrations (2.9 ± 0.3 vs. 2.1 ± 0.3 ng/mL; on day 18, considering day 0 as the beginning of the synchronization protocol) than those with a high AFC. The pregnancy rate was higher (P ≤ 0.05) in animals with a very low (57.9%) and low (53.1%) AFC than in those with a high AFC (45.2%). The expression of genes related to intercellular communication, meiotic control, epigenetic modulation, cell division, follicular growth, cell maintenance, steroidogenesis and cellular stress response was assessed on day 5. In females with a low AFC, 8 and 21 genes in oocytes and cumulus cells, respectively, were upregulated (P < 0.05), while 3 and 6 genes in oocytes and cumulus cells, respectively, were downregulated. The results described here will help elucidate the differences in ovarian physiology and the reproductive success of Bos indicus females with a low or high AFC.

Cattle adapted to tropical and subtropical environments: genetic and reproductive considerations

Efforts to understand biological functions and develop management schemes specific to Bos indicus-influenced cattle raised in tropical and subtropical environments are critical to meet the increasing global demand for protein. In the United States, B. indicus breeds are mostly used to generate B. indicus × B. taurus crosses with increased thermal and parasite tolerance, while retaining some productive characteristics of B. taurus cattle. Although crossbreeding represents a proven strategy to improve cattle adaptation almost immediately, research has also attempted to identify B. taurus genetics that can withstand subtropical and tropical climates. Reduced milk production and delayed reproductive maturation appear to be related with tropical adaptation of B. taurus breeds, as a means to conserve energy under stressful conditions and limited nutrition. Moreover, longevity may be the ultimate adaptation response to unfavorable environments, and retention of bulls and heifers from proven cows is the recommended strategy to improve longevity in B. indicus-influenced herds. Besides selection for longevity, other aspects should be considered when planning reproductive management in tropical and subtropical regions. Bos indicus and B. taurus breeds have multiple differences pertaining to reproductive function, including age at puberty, ovarian dynamics, and pregnancy development. Nutritional strategies such as the stair-step regimen, and use of exogenous progesterone (P4) inserts are options to hasten puberty attainment of late-maturing B. indicus-influenced heifers. Yet, limited pharmacological alternatives are available for reproductive management of B. indicus-influenced females in the United States, which rely on GnRH-based protocols not specifically designed to the reproductive function of B. indicus breeds. In contrast, hormonal protocols based on exogenous P4, estradiol esters, and equine chorionic gonadotropin are available for use in B. indicus females in South America. These include protocols tailored to prepubertal heifers, anestrous cows, and cycling nulliparous or parous females, which often yield pregnancy rates of 50% to fixed-time artificial insemination. The global dairy industry also faces similar challenges in increasing demand and production as the beef industry. Selection of cows capable of sustaining optimal milk yield, reproductive success, and health status in hot and humid conditions is essential for optimal dairy production in subtropical and tropical regions.

41 Reproductive management of beef females in South America

Abstract The majority of beef cow herds in South America are constituted by Bos indicus females, which have particular reproductive features that contribute to reduced reproductive efficiency compared with that of B. taurus cohorts. B. indicus heifers reach puberty at an older age compared with B. taurus heifers. Nutritional challenges are critical in cows maintained on low-quality tropical pastures, given that nutrient intake during the postpartum period is not sufficient to meet their requirements for growth and lactation. Several alternatives to enhance reproductive efficiency of B. indicus heifers and cows have been developed to address their inherent reproductive shortcomings. These research-based technologies include: a) hormonal protocols to induce puberty in nulliparous heifers, b) hormonal protocols to synchronize estrus and/or ovulation in B. indicus females to exploit their reproductive responses to artificial insemination, and c) genetic and environmental factors that influence reproductive success in beef herds, including reproductive diseases and excitable temperament of B. indicus females, which have been investigated to support/promote the development of appropriate mitigation technologies. The objective of this presentation is to discuss pharmacological and management technologies to improve reproductive performance of cow-calf systems from tropical areas in South America. Currently, there are hormonal treatments allowing South American B. indicus producers to reach the benchmark of 50% pregnancy rate to TAI, as well as enhance the percentage of cows becoming pregnant to AI at the beginning of the breeding season. This outcome is expected to optimize use of labor, and increase calf weaning age and weight via genetic improvement and concentration of births at the beginning of the calving season.

34 Reproductive management of beef females in tropical and sub-tropical environments

Abstract Bos indicus and Bos taurus cattle diverged from an evolutionary standpoint more than 110,000 years ago. Since then, Bos indicus cattle have undergone genetic adaptations beyond the commonly discussed increased thermo-tolerance and parasite resistance. Several physiological differences exist between Bos indicus and Bos taurus cattle, and it is important to consider these differences when establishing reproductive management strategies. It has been well described that Bos indicus cattle have a delayed onset of puberty and longer periods of postpartum anestrus, yet through the utilization of estrus synchronization protocols these challenges can be attenuated. However, when Bos indicus females are exposed to these protocols, they are known to have smaller dominant follicles, lower expression of estrus, and decreased pregnancy rates to artificial insemination (AI) when compared to Bos taurus females. These factors can be overcome through the utilization of estradiol and progesterone based synchronization protocols, which improve follicular dynamics and yield acceptable pregnancy rates to assisted reproductive technologies in cattle adapted to tropical or subtropical conditions. However, the use of estrogens for synchronization purposes is not permitted in the United States, and cattle producers need to rely on GnRH-based protocols. Another key difference between subspecies, is that Bos indicus females have greater antral follicle counts than Bos taurus females, which proves beneficial for in vitro embryo production. Therefore, an opportunity exists to explore the greater productivity of donors in embryo production in order to improve genetics in herds that utilize these breeds.

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

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

Absence of a corpus luteum and relatively lesser concentrations of progesterone during the period of pre-ovulatory follicle emergence results in lesser pregnancy rates in Bos indicus cattle

Concentrations of progesterone before AI have had variable effects on fertility in both Bos indicus and Bos taurus cattle. The aim of this study was to determine if fertility and concentrations of progesterone after AI were affected in Bos indicus females when pre-ovulatory follicles develop in the absence or presence of a corpus luteum (CL). Between 6.5–7.5 days after a synchronised oestrus, all follicles ≥4 mm in diameter were aspirated (Day 0) and cloprostenol was administered on Days 0 and 1 (LP4, n = 36) or on Days 4 and 5 (HP4, n = 40). Animals were inseminated on detection of oestrus until Day 9. Breeding continued using natural mating between Days 9 and 19, AI on detection of oestrus between Day 19 and 29 and natural mating between Days 29–90. Mean concentrations of progesterone were less on Days 2–4 in the LP4 compared to the HP4-treated animals but similar on Days 14 and 20. In the LP4- compared to the HP4-treated animals, the odds of being detected in oestrus and ovulating close to the first AI were similar, but odds of pregnancy to first AI (OR = 0.19, 95% CI 0.07 – 0.52) and after 1, 4 and 13 weeks of breeding were less (P ≤ 0.051). Absence of a CL and relatively lesser concentrations of progesterone during emergence of pre-ovulatory follicles resulted in lesser pregnancy rates to AI in Bos indicus cattle but did not affect concentrations of P4 after ovulation.