Optimizing ReBreed21 II: Fertility and reproductive efficiency in different parities during a shortened breeding season in beef cattle.

This study evaluated the efficiency of a rapid reinsemination program allowing timed AI (TAI) every 21d (ReBreed21) in a commercial beef cow-calf operation. Nelore females from different parities (n = 2085) were synchronized for first TAI (D0 = TAI) using an estradiol/progesterone (E2/P4) protocol and assigned to one of three reinsemination programs: Resynch33 (n = 753), traditional resynch program with second TAI at D42 after first TAI; ReBreed21 (n = 687); or ReBreed21+EC (n = 670). The ReBreed females (n = 1357) received intravaginal P4 insert on D12, on D19 P4 was removed, and a dose of equine chorionic gonadotropin (eCG) was administered, then, ReBreed21 females received 0.6 mg of EC (ReBreed21+EC) or nothing (ReBreed21) and on D21, nonpregnancy (NP) was determined using Doppler ultrasound to detect corpus luteum (CL) blood flow (BF) (NP: <25 % BF pixels of total CL area) and NP cows received immediate TAI and GnRH to induce ovulation. Pregnancy diagnosis was performed at D33 after TAI following all TAIs. Cows considered pregnant at D21, based on CL BF, but NP on D33 were designated False-Positives (FP) and false negatives (FN) were number of nonpregnant cows/heifers on d21 based on the CL BF found to subsequently be pregnant on D33 divided by the total number pregnant. Pregnancy/AI (P/AI) did not differ for the first TAI (55.1 %) among the treatments. Heifers had similar P/AI at the second AI in all groups and similar to the first AI. Primiparous had greater P/AI in ReBreed21+EC and Resynch33 at s TAI compared to ReBreed21, 51.7 %, 55.8 %, 34.2 %, respectively. Multiparous had greater P/AI at second TAI in Resynch33 (60.9 %) than ReBreed21 programs (34.7 %). The percentage FP and FN among ReBreed21 programs did not differ, 13.8 and 0.2 %, respectively. Overall accumulative pregnancies on D21 of the breeding season were greater for ReBreed21 and ReBreed21+EC than Resynch33 (69.7 %, 71.6 %, and 55.5 %, respectively). However, on D42 of the breeding season, only heifers had greater pregnancies in ReBreed21 programs than Resynch33 (73.3 %, 74.3.6 %, and 63.2 %, respectively). Average days to pregnancy were less (P = 0.01) for ReBreed21 and ReBreed21+EC than Resynch33. Thus, the ReBreed21 strategy can improve the efficiency of TAI programs in beef cattle. Of interest, ReBreed21 was particularly effective in nulliparous, somewhat effective in primiparous when EC was added to the program, but relatively ineffective in multiparous beef cattle.

Optimizing ReBreed21 I: Evaluation of endocrine and ovarian dynamics in non-bred Bos indicus heifers.

The present study evaluated follicular and endocrine dynamics during ReBreed21, a reproductive strategy that allows resynchronization of ovulation every 21 days in Bos indicus (Nelore) heifers. A synchronized estrous cycle was induced using a standard timed ovulation protocol (d -10: P4 implant inserted + 2 mg estradiol benzoate; d -2: P4 removed+ 0.5 mg cloprostenol + 0.6 mg estradiol cypionate + 200 IU equine chorionic gonadotropin (eCG); d0: 8.4 µg buserelin) without AI to ensure nonpregnancy in heifers. Day of GnRH was designated d0 of estrous cycle. On d12, heifers (n = 80) were randomized into three experimental groups: (1) ReBreed21 (n = 28) d12 P4 device inserted, d19 P4 device withdrawal plus 200 IU eCG, and d21 8.4 µg buserelin (GnRH); (2) ReBreed21+G (n = 26) same as ReBreed21 plus GnRH (16.8 µg) treatment on d12; and (3) Control (n = 26) no treatment. ReBreed21+G increased two-fold (62.9%; 18/26) percentage of heifers with synchronized follicular wave emergence compared to Control (34.6%; 9/26) whereas ReBreed21 (53.6%; 15/28) was intermediate. The ReBreeed21 groups (eCG on d19) increased (P < 0.01) follicular growth between d19 and d21 in ReBreed21 (2.3 ± 0.2 mm) and ReBreed21+G (3.4 ± 0.2 mm) compared with Control (1.2 ± 0.3 mm), resulting in greater (P < 0.01) follicle diameter on d21 for ReBreed21 (10.7 ± 0.4 mm) and ReBreed21+G (10.8 ± 0.4 mm) compared with Control (9.1 ± 0.5 mm). Structural luteolysis was similar among groups (P = 0.51), although the average day when P4 was <1 ng/mL was later (P < 0.01) for ReBreed21 (20.5 ± 0.2) and ReBreed21+G (20.7 ± 0.2) compared to Control (19.2 ± 0.4). Overall ovulation at the end of the estrous cycle was increased (P = 0.03) for ReBreed21 groups (83.3%; 45/54) compared with Control (57.7%; 15/26). Synchronized ovulation on day 22-23 was greater (P < 0.01) for ReBreed21 (78.6%; 22/28) and ReBreed21+G (76.9%; 20/26) compared with Control (30.8%; 8/26). Thus, the ReBreed21 resynchronization program produced acceptable endocrine and follicular dynamics, including synchronized ovulation at the end of the protocol in nonpregnant heifers providing good rationale for testing the fertility and practical implementation of this protocol under field conditions.

Profiles of interferon-stimulated genes in multiple tissues and circulating pregnancy-associated glycoproteins and their association with pregnancy loss in dairy cows†.

Pregnancy loss (PL) in lactating dairy cows disrupts reproductive and productive efficiency. We evaluated the expression of interferon-stimulated genes (ISG) in blood leukocytes, vaginal and cervical epithelial cells, luteolysis-related genes, progesterone, and pregnancy-associated glycoprotein (PAG) profiles in lactating dairy cows (n = 86) to gain insight about PL. Expression of ISG on d17, d19, and d21 was greater in cows that maintained the pregnancy (P33) compared to nonpregnant with no PL (NP). Greater ISG differences between groups were observed in the cervix (96.7-fold) than vagina (31.0-fold), and least in blood leukocytes (5.6-fold). Based on individual profiles of ISG and PAG, PL was determined to occur either before (~13%) or after (~25%) d22. For cows with PL before d22, ISG expression was similar on d17 but by d21 was lower and OXTR was greater than P33 cows and similar to NP; timing of luteolysis was similar compared to NP cows suggesting embryonic failure to promote luteal maintenance and to attach to the endometrium (no increase in PAG). For cows with PL after d22, ISG expression was similar to P33 cows on d17, d19, and d21 and luteolysis, when it occurred, was later than NP cows; delayed increase in PAG suggested later or inadequate embryonic attachment. In conclusion, PL before d22 occurred due to embryonic demise/failure to signal for luteal maintenance, as reflected in reduced ISG expression by d21. Alternatively, embryos with PL between d22 and 33 adequately signaled for luteal maintenance (ISG) but had delayed/inadequate embryonic attachment and/or inappropriate luteolysis causing PL.

Research on timed AI in beef cattle: Past, present and future, a 27-year perspective.

This review aimed to (1) summarize the results from fixed-timed artificial insemination (TAI) fertility studies performed during the last 27 years; (2) compile and evaluate, as examples from the literature base, the direct comparisons made of specific manipulations to synchronization protocols; (3) evaluate the impact of the TAI programs on the reproductive performance during the breeding season, and (4) provide perspective on the future of TAI programs in beef cattle. A search of the literature published from 1995 to 2021 was conducted to identify experiments in which synchronization of ovulation and TAI in beef cattle was performed. The primary outcome of interest was fertility expressed as pregnancies per TAI. The literature included two search engines, the SIS Web of Science and the US National Library of Medicine Institutes of Health through PubMed. After the initial search and screening, a total of 228 manuscripts were selected containing a total of 272,668 TAI. A dramatic increase in the number of publications and TAIs occurred throughout the years. Most of them were from Brazil and United States, followed by Canada, Argentina, Uruguay, and Australia. Two main types of TAI programs were identified: GnRH-based and E2/P4-based protocols. In terms of GnRH-based programs, two variations were evaluated in the present manuscript. First, we evaluated the effect of the progesterone implant during the protocol. The progesterone implant increased pregnancy/TAI (P/TAI) from 44.3 to 54.3%. Second, the use of a second prostaglandin F2α treatment in 5-d CO-synch program increased the P/TAI from 53.2 to 60.9%. In E2/P4-based programs, use of GnRH at TAI increased P/TAI from 54.7 to 59.2% in cows. However, no increase was detected in heifers. Other research showed that use of TAI can increase the overall proportion of the cows pregnant at end of the breeding season and produce earlier calvings compared with bulls. In conclusion, there have been a large number of excellent research studies that have been performed during the last 27 years on TAI in beef cattle. This technology is being utilized successfully in the beef cattle industry. This success is largely because of the valid research that underlies the application of the technology and the economic value of the technology.

Review: Maintenance of the ruminant corpus luteum during pregnancy: interferon-tau and beyond.

This manuscript reviews the mechanisms that maintain the corpus luteum (CL) of pregnancy in ruminants. In mammals, ovulation and luteinization of the remaining cells in the CL are due to a surge in Luteinizing Hormone (LH). In cattle, continued secretion of pulses of LH is essential for full development and function of the CL during the estrous cycle (LH pulses), however, the few studies on the CL after d20 of pregnancy do not indicate that LH is essential for maintaining the CL of pregnancy. The first essential step in maintaining the CL of pregnancy in ruminants is overcoming the mechanisms that cause regression of the CL in non-pregnant ruminants (d18-25 in cattle; d13-21 in sheep). These mechanisms have a uterine component involving oxytocin-induced prostaglandin F2α (PGF2A) pulses and a luteal component involving decreased progesterone production and luteal cell death. There is a critical role for embryonic interferon-tau (IFNT) in suppressing the uterine secretion of PGF2A during early pregnancy (d13-21 in sheep; d16-25 in cattle) and preventing luteolysis. There are also effects of IFNT on the expression of interferon-stimulated genes in other tissues including the CL but the physiologic role of these interferon-stimulated genes is not yet clear. After the IFNT period, there is another mechanism that maintains the CL of pregnancy in ruminants since embryonic IFNT is inhibited as attachment occurs and trophoblastic binucleate/giant cells begin secretion of pregnancy-associated glycoproteins. The second mechanism for luteal maintenance has not yet been defined but acts in a local manner (ipsilateral to pregnancy), and remains functional from d25 until just before parturition. The most likely mechanisms mediating later maintenance of the CL of pregnancy are increased uterine blood flow or decreased prostaglandin transporter expression in the utero-ovarian vasculature, preventing PGF2A reaching the CL. Finally, implications of these ideas on pregnancy loss in cattle are explored, highlighting the importance of inappropriate regression of the CL of pregnancy as a mechanism for pregnancy loss in cattle.

Transcriptomic analysis reveals gene expression changes in peripheral white blood cells of cows after embryo transfer: Implications for pregnancy tolerance.

Most embryo losses occur in the first trimester of pregnancy in cows and include losses following embryo transfer. There is a resulting negative economic impact on cattle production systems when this occurs. Cellular and molecular mechanisms behind the maternal immune response to the growing embryo have not been fully characterized. The objective of this study was to examine the gene expression profiles of peripheral white blood cells (PWBCs) from pregnant cows 21 days after an embryo was transferred, and cows that were treated equally but lost the embryo. Specifically, we obtained and compared the transcriptome of PWBC from heifers that became pregnant at day 21 (N = 5) or failed to become pregnant after the embryo transfer (N = 5). Sequencing data can be accessed by Gene Expression Omnibus (GEO) with the accession number GSE210665. A total of 13,167 genes were evaluated for differential expression between groups. A total of 682 genes showed differential expression (p-value <.01), 302 genes were up-regulated while 380 were down-regulated due to pregnancy. The most significant genes were COL1A2, H2AC18, HTRA1, MMP14, CD5L, ADAMDEC1, MYO1A and RPL39, among others. Most of the significant genes are related to the up-regulation of inflammatory chemokine activity and immune defence response. Our findings extend the current knowledge that pregnancy alters the PWBC by promoting immune tolerance, cell chemotaxis, blood coagulation, angiogenesis, inflammatory response, cell adhesion and cytokine secretion. Our data suggest that pregnancy and ectoparasites could trigger poorly described genes in PWBC of cows, and a few previously escribed genes, such as IFI44. These results could shed light on the genes and mechanisms that promote tolerance to pregnancy and allow survival of the developing embryo.

Is pregnancy loss initiated by embryonic death or luteal regression? Profiles of pregnancy-associated glycoproteins during elevated progesterone and pregnancy loss.

Because progesterone (P4) is essential for pregnancy establishment and maintenance, we investigated the effect of increased concentrations of P4 on embryonic attachment and concentrations of pregnancy-associated glycoproteins (PAG). Additionally, we investigated the relationships among luteal regression, pregnancy loss, and PAG concentrations in cows undergoing pregnancy loss by d 33 of pregnancy. Lactating dairy cows were allocated into control (n = 40) and human chorionic gonadotropin (hCG; 3,300 IU on d 7 and 13 to promote greater circulating P4; GnRH = d 0; n = 46) groups. Progesterone was measured daily from d 7 to 33, and PAG was measured daily from d 17 to 33; both hormones were also measured on d 47 and 61. An increase in PAG >10% compared with d 17 was considered a marker for pregnancy. The gold standard for pregnancy diagnosis was ultrasound evaluation of embryonic heartbeat on d 33. Statistical analyses were done with PROC MIXED from SAS Institute Inc. Concentrations of P4 were greater from d 8 onward in the hCG group. Concentrations of PAG did not differ between groups from d 17 to 33, suggesting no effect of increased P4 on hastening embryonic attachment and placental development. Nevertheless, PAG was greater in the hCG group on d 47 and 61, suggesting greater placental area or PAG secretory capacity. Pregnancy loss between d 20 and 33 occurred in 24.6% of cows. About 50% of pregnancy loss was due to luteal regression and about 50% was due to conceptus failure; that is, a decrease in PAG in the absence of luteal regression. In conclusion, increased P4 does not hasten embryonic attachment or early placental development but it leads to increased PAG in the second half of the second month of gestation. Additionally, pregnancy loss seems to be initiated by either corpus luteum regression or conceptus failure.

Short communication: profiling the expression of Let-7d-5p microRNA in circulating blood of pregnant and nonpregnant cows.

The objective of this work was to determine if specific circulating microRNA (miRNA) differed due to pregnancy status in heifers. Blood samples were collected from heifers 21 d after receiving an in vitro-produced embryo. Pregnancy status was diagnosed 21 d after embryo transfer, equivalent to day 28 of gestation, with rectal ultrasonography. Blood samples from 10 pregnant and 10 nonpregnant heifers were then evaluated for miRNA expression. There were five different miRNAs quantified using delta-delta Ct and qPCR methodology. These miRNAs had previously been associated with early pregnancy in cattle. The miRNA Let-7d-5p was decreased in nonpregnant as compared to pregnant females (P < 0.05). There were no changes in 16-5p, 16-1-3p, 16-2-3p, and 26a-5p associated with pregnancy (P > 0.05). Results demonstrate an opportunity to identify and study the differential expression of miRNAs from the blood of pregnant cows. The Let-7d-5p miRNA is a potential early pregnancy marker and is critical to better understand the early relationships of the cellular and molecular interactions of the cow and embryo.

MicroRNAs are critical molecules in cell homeostasis and can bind their target in the cellular cytoplasm by miRNA recognition of the 3ʹUTR sequence in the messenger RNA (mRNA) or other RNAs and inhibiting the mRNA translation. This molecular mechanism is called gene silencing. These miRNAs are stable in blood and can be useful as biomarkers of early pregnancy in the bovine. We demonstrated a decrease in the miRNAs, Let-7d-5p, at day 21 post embryo transfer for nonpregnant samples contrasted with pregnant blood samples (P < 0.05). This research opens new methodologies to study and understand miRNAs circulating in blood of pregnant vs. nonpregnant cows at early stages.

Effect of increased serum progesterone on luteal morphology and function, pregnancy, and gestational loss in Nelore cows.

The aim of the present study was to determine the impact of different strategies for increasing the level of serum progesterone (P4) on luteal morphology and function in bovine females. The effects of increasing P4 on pregnancy rate and gestational loss (GL) in Nelore cows subjected to timed artificial insemination (TAI) were also evaluated. A total of 939 cows were divided into three groups: P4LA (n = 305), 150 mg of long-acting injectable P4 7 days after TAI; GnRH (n = 306), 10 µg of buserelin acetate 7 days after TAI; and control (n = 328), no hormone treatment after TAI. Doppler ultrasound assessments and P4 measurements were performed on days 7 and 16 after TAI. The pregnancy rate and GL as a function of treatment were compared using the SAS GLIMMIX procedure. Corpus luteum (CL) vascular perfusion, volume, and plasma P4 concentration were analysed using the SAS PROC MIXED procedure. No significant difference was found among the treatments in terms of volume, number of pixels, and CL intensity or in the serum P4 concentration at 7 days after ovulation. The CL blood flow at 16 days after ovulation was lower in the P4LA and GnRH groups than that in the control group (p < .01). Serum concentrations of P4 at 16 days after ovulation were higher in the P4LA and GnRH groups than those in the control group (p = .04). A difference in the pregnancy rate (p = .003) and a trend in GL (p = .07) as a function of treatment were found. Overall, long-acting injectable P4 supplementation on day 7 after TAI or GnRH administration affected CL vascularization and increased the serum concentrations of P4 16 days after ovulation, promoting better pregnancy rates than the control.

Mitigation of low pregnancy rate in excitable Nellore cows by administration of GnRH or P4.

This research aimed to determine the effect of temperament on reproductive parameters including cortisol and progesterone (P4) in Nellore cows. Additionally, two methods for increasing plasma progesterone (P4) levels in excitable animals to enhance pregnancy rate (P/AI) and reduce pregnancy loss were investigated. In total, 939 cows were subjected to timed artificial insemination (TAI) and divided into three groups: (P4LA; n = 305) 150 mg of injectable long-acting progestogen 7 days after TAI; (GnRH; n = 306), 10 µg of buserelin acetate on day 7 after TAI; control group (CG; n = 328) without hormonal treatment. In 213 cows, randomly chosen from each group, ultrasound evaluations of the preovulatory follicle (Mode B) were performed on the day of insemination and of the corpus luteum (Color Doppler) 7 and 16 days after TAI. Blood samples were obtained from 20% of the 939 animals, randomly chosen from each group, on the day of insemination and after 7 and 16 days to measure cortisol and progesterone, respectively. At the time of insemination, subjective temperament evaluations were performed with the animals being classified as excitable (EXC) or adequate (ADQ). The SAS GLIMMIX procedure was used to compare the pregnancy rate (P/AI) and gestational loss within each temperament for the three experimental groups. Continuous variables were analyzed utilizing SAS PROC MIXED procedure. Cortisol concentration was higher and POF (preovulatory follicle) and CL (corpus luteum) volumes at the time of insemination and 7 days after AI, respectively, were lower in EXC animals than in ADQ. No significant difference was observed between the number of pixels, CL intensity, and plasma concentration of P4, 7 days after TAI. However, 16 days post-insemination, among the animals classified as EXC, higher concentrations of P4 were observed in the GnRH and P4LA groups than in the control. Regarding P4 concentrations, there was a tendency to be lower in animals classified as EXC than in ADQ within the control group (P = 0.06), while rate of blood flow from the CL was lower in EXC animals than in ADQ animals (P = 0.04). Among the ADQ animals, the GnRH and P4LA groups showed a lower CL flow rate than that observed in the control (P = 0.04), 16 days after the TAI. Among EXC animals, a higher pregnancy rate was observed in the GnRH and P4LA groups than in the control group (P = 0.01). In the control group, the pregnancy rate (P/AI) of the ADQ animals was higher than that of the EXC animals (P = 0.05). No statistically significant differences were observed between gestational losses when the treatments or temperaments were compared. In conclusion, the use of GnRH or P4LA, 7 days after insemination, improves pregnancy rates in excitable animals and is a viable alternative to minimize the negative impact of stress and improve reproductive efficiency in beef cattle.

Accessory corpus luteum induced by human chorionic gonadotropin on day 7 or days 7 and 13 of the estrous cycle affected follicular and luteal dynamics and luteolysis in lactating Holstein cows.

Our objective was to determine the effect of inducing an accessory corpus luteum (CL) with human chorionic gonadotropin (hCG; 3,300 IU) on d 7 (hCG7) or 2 accessory CL with hCG on d 7 and 13 (hCG7+13) of the estrous cycle in noninseminated lactating Holstein cows. Cows (n = 86) between 39 and 64 DIM were pretreated with an Ovsynch + CIDR protocol, and only synchronized cows were used (n = 64). The day of the last GnRH of Ovsynch was considered d 0 of the estrous cycle. Follicular and luteal dynamics of cows were evaluated daily during an entire estrous cycle by ovarian ultrasonography. Blood samples were collected daily to measure serum concentration of progesterone (P4). Cows were randomly assigned to CON (n = 22, no treatment), hCG7 (n = 20), or hCG7+13 (n = 22) treatments. Two cows from hCG7+13 failed to ovulate after hCG and were removed from the analyses post-hCG treatment. The first day of luteolysis was considered the day that P4 declined to more than 2 SD of the mean for the 4 consecutive P4 concentrations with the greatest mean in late diestrus for each individual cow. The P4 cut-off for complete luteolysis was <1.0 ng/mL. Mean P4 on d 7 (3.23 ± 0.16 ng/mL) did not differ among treatments. Cows treated with hCG had greater total luteal and original CL volume and serum P4 during diestrus than CON. Cows treated with hCG7+13 had greater serum P4 after d 13 of the cycle than hCG7. Cycles were classified as having atypical cycles if the dominant follicle or future dominant follicle at the time of luteolysis did not ovulate (delayed ovulation; CON, n = 2; hCG7, n = 4; hCG7+13, n = 3), had a short cycle (CON, n = 1), delayed (CON, n = 2) or incomplete luteolysis (CON, n = 1; hCG7, n = 4; hCG7+13, n = 5). The remainder of cycles with normal complete luteolysis followed by ovulation were considered to be typical. Based on blood perfusion, the CON cow with incomplete luteolysis had 2 original CL remaining functional after first onset of luteolysis. The rest of the cows with incomplete luteolysis (9/10) had one or more CL regressing and at least one remaining functional after first onset of luteolysis. No specific pattern for CL side (ipsilateral vs. contralateral to a CL with complete regression) was observed for nonregressed CL. Cows with incomplete luteolysis had a second onset of luteolysis to undergo complete functional luteolysis. The proportion of cows with typical cycle was 73% (16/22) for CON, 60% (12/20) for hCG7, and 55% (11/20) for hCG7+13. Cows with typical cycles treated with hCG (hCG7 and hCG7+13) had a later onset of luteolysis, prolonged time to undergo complete luteolysis, and greater proportion of cows with 3 follicular waves than CON, resulting in a longer interovulatory interval for hCG7 and hCG7+13 than CON. In summary, accessory CL induced by hCG during diestrus not only altered follicular and luteal dynamics but also deferred and prolonged the luteolytic process.