Reproductive microbiome and cytokine profiles associated with fertility outcomes of postpartum beef cows.

Shifts from commensal bacteria (for example, Lactobacillus in the phylum Firmicutes) within the reproductive tract have been associated with changes in local reproductive immune responses and decreased fertility in humans. The objective of this study was to characterize the microbiome and cytokine concentrations before artificial insemination (AI) in vaginal and uterine flushes from postpartum beef cows. Twenty Bos indicus-influenced beef cows (approximately 60 d postpartum and free of reproductive, health, or physical issues) were enrolled. The B. indicus prostaglandin (PG) 5-d + controlled intervaginal drug-releasing estrus synchronization protocol was initiated on day -8 of the study with timed AI on d0. Blood samples were collected on days -3, -1, and 28 via coccygeal venipuncture. Vaginal and uterine flushes were collected on days -3 and -1. Based on days 28 pregnancy status determined by transrectal ultrasonography, cows were identified as either Open (n = 13) or Pregnant (n = 7). Bacterial community analyses were conducted targeting the V4 hypervariable region of the 16S rRNA gene. Cytokine analyses were performed using the RayBiotech Quantibody Bovine Cytokine Array Q1 and MyBioSource ELISA kits per the manufacturer's instructions. Statistical analyses for bacteria relative abundance were conducted using PROC NPAR1WAY and for cytokine concentrations using PROC GLM in SAS 9.4. Uterine concentrations of interferon γ, interleukin (IL)1α, and IL21 were greater in Open than in Pregnant cows (P < 0.05). Regardless of pregnancy status, uterine IL13 increased from days -3 to -1 (9.76 vs. 39.48 ± 9.28 pg/mL, respectively; P < 0.05). Uterine relative abundance of the phylum Firmicutes decreased from days -3 to -1 in Open cows (60.4% ± 0.9% vs. 48.5% ± 3.2%; P = 0.004). In Open cows, the genus Blautia decreased in relative abundance within the uterus from days -3 to -1 (2.1% ± 0.2% vs. 0.9% ± 0.1%; P = 0.002). Uterine relative abundance of the phylum Tenericutes increased from days -3 to -1 in Pregnant cows (1.0% ± 0.1% vs. 7.6% ± 4.1%; P = 0.002). In Pregnant cows, the genus Ureaplasma tended to increase within the uterus from days -3 to -1 (0.08% ± 0.06% vs. 7.3% ± 4.1%; P = 0.054). These findings suggest a distinct difference in the reproductive microbiome and cytokine profiles before AI for resulting Open vs. Pregnant cows.

Efficiently producing cattle to feed a growing population can come with many challenges. A few challenges occur soon after a cow has given birth, and subsequent reproductive performance can be impacted. Bacteria within the reproductive tract can trigger an immune response and together play a role in affecting fertility in cows. The objectives of this experiment were to distinguish the commensal vs. harmful bacteria that reside in the reproductive tract and to characterize the immune response in beef cattle via uterine and vaginal flushes. The results demonstrated that bacteria within the reproductive tract of beef cattle changes before breeding. The current study also suggests that changes in immune response before breeding can be associated with fertility outcomes. Additional research may be worthwhile to evaluate management tactics to positively shift bacteria within the reproductive tract and reduce inflammatory immune responses to improve fertility and increase reproductive efficiency. Future research is necessary to identify the causes of bacterial shifts and how it relates to pregnancy establishment.

Shifts in bacterial communities in the rumen, vagina, and uterus of beef heifers receiving different levels of concentrate.

This experiment investigated the effects of diet composition on rumen, vaginal, and uterine microbiota of beef heifers. Fifteen rumen-cannulated, pubertal Angus-influenced heifers were used in a replicated 3 × 3 Latin square design (28-d periods and 21-d washout intervals). Dietary treatments included diets based on (as-fed) 100% grass hay (HF), 60% grass hay + 40% corn-based concentrate (INT), or 25% grass hay + 75% corn-based concentrate (HG). Treatments were offered individually to heifers once daily at 2% body weight. Rumen, vaginal, and uterine samples were collected on days 0 and 28 of each period. Data were analyzed using orthogonal contrasts (linear and quadratic), using results from day 0 as independent covariates and heifer as the experimental unit. Ruminal pH on day 28 decreased linearly (P < 0.01) as concentrate inclusion increased. Uterine and vaginal pH on day 28 were not affected by treatments (P ≥ 0.35). Within the rumen samples, Bacteriodetes was the most abundant phylum and its relative abundance linearly decreased (P ≤ 0.01) with the inclusion of concentrate. Prevotella was the most abundant genus within the rumen but was not affected by treatments (P ≥ 0.44). Genera with relative abundance ≥1% (average across treatments) in the rumen that were impacted by treatments (P ≤ 0.01) included Bacteroides, Pedobacter, Dysgonomonas, Caloramator, and Ruminococcus. Firmicutes was the most abundant phylum in the vagina and uterus, but it was unaffected by treatments (P ≥ 0.16). Prevotella was the most abundant genus in the vagina, and its relative abundance increased (P < 0.01) with the inclusion of concentrate. Other genera with relative abundance ≥1% that were significantly affected (P ≤ 0.05) by treatments were Clostridium, Pedobacter, Roseburia, Oscillospira, Faecalibacterium, Caloramator, Paludibacter, Rhodothermus, and Porphyromonas. In uterine samples, Prevotella was the most abundant genus but was unaffected by treatments (P ≥ 0.29). Genera with relative abundance ≥1% in the uterus that were significantly affected (P < 0.01) by treatments were Caloramator, Paludibacter, and Thalassospira. Collectively, inclusion of concentrate in the diet altered the bacterial composition within the rumen as well as shifting bacterial populations within the vagina and uterus. Research is warranted to further understand the impacts of these diet-induced microbiota changes on reproductive function and performance of beef heifers.

According to the United Nations, worldwide beef production must increase by 120% by 2050 to feed an additional 2.3-billion people. With a growing population and a reduction in available resources, the overall efficiency of beef production needs to advance to meet the increasing demand. Cow­calf operations serve as the foundation of the beef industry and supply all calves for beef production; however, poor reproductive performance limits the productivity in this system. Hence, management strategies to promote reproductive success are warranted for optimal reproductive and overall efficiency in cow­calf operations. Bacterial communities in the reproductive system of cattle have been shown to contribute to fertility and can be affected by several factors such as dietary changes. The objective of this experiment was to evaluate the impact of different diets on the reproductive bacterial communities. Overall, it was concluded that the reproductive tract can shift the abundance of bacteria due to changes in the diet, and more research is needed to better understand the impact of these changes and their consequences to beef production systems.

Short Communication: Influence of estrus activity and reproductive tract size and position scores on fertility in Bos indicus and Bos taurus suckled beef cows.

The primary objective of this study was to determine if estrus activity and reproductive tract size and position score (SPS) are associated with pregnancy outcomes in Bos indicus (Nelore) and Bos taurus (Angus) beef cows. In study 1, multiparous Nelore cows (n = 1,280) were artificially inseminated at a fixed time (FTAI, day 0) using an estradiol and progesterone (P4)-based estrus synchronization protocol. In study 2, multiparous Angus cows (n = 764) were artificially inseminated at a fixed time (FTAI, day 0) using a gonadotropin-releasing hormone and P4-based estrus synchronization protocol. Estrus activity was assessed using Estrotect heat detector patches and scored on day 0 using the following scoring system: 0 (patch was lost, most likely due to repeated mounting), 1 (<25% activation), 2 (≥25%, <50% activation), 3 (≥50%, <75% activation), or 4 (>75% activation) where patch scores of 1 and 2 signified no or limited estrus activity, whereas scores of 0, 3, and 4 had increased estrus activity. Reproductive tract SPS were assigned on day 0 as SPS1: small and compact resting within the pelvic cavity; SPS2: intermediate, resting partially outside the pelvic cavity; and SPS3: larger and resting outside the pelvic cavity. Pregnancy diagnosis was performed by ultrasound on day 30 and 100 after FTAI. Cows were determined as undergoing pregnancy loss if a viable embryo with heartbeat was detected at day 30 but was no longer present at day 100. Pregnancy rate at day 30 was influenced by estrus activity and SPS in both Nelore (P = 0.004) and Angus (P = 0.009) cows. Specifically, cows with smaller reproductive tracts (SPS1) had greater (P < 0.001) pregnancy rate when estrus was expressed before FTAI. There was no effect of estrus activity nor reproductive tract size on pregnancy loss between day 30 and 100 for both breeds. In summary, estrus activity before FTAI may influence reproductive outcomes differently depending on size and position of the reproductive tract at time of breeding.

The livelihood of a cow-calf producer relies on reproductively sound cattle that give birth to live offspring once a year. Two factors, pregnancy rate and pregnancy loss, are informative measures of fertility. Herein, the authors identified that pregnancy rates are interactively influenced by estrus activity and reproductive tract size and position scores. Specifically, cows with smaller reproductive tracts that exhibited estrus activity had greatest pregnancy rates. Neither estrus activity, reproductive tract size and position score, nor their interaction were associated with pregnancy loss.

Positive relationship of rectal temperature at fixed timed artificial insemination on pregnancy outcomes in beef cattle.

The overarching aim was to examine the relationship of rectal temperature at fixed time artificial insemination (FTAI) on pregnancy outcomes in a typical breeding season with expected pregnancy rates approaching 50% using Bos indicus and Bos taurus cattle. This represents a continuum of steps to test the hypothesis that elevated body temperature at or around insemination is functionally important to maximize pregnancy outcomes. Rectal temperature of Bos indicus cattle at FTAI ranged from 37.0 to 40.9 °C; 60.6% were hyperthermic. Positive factors impacting pregnancy outcomes were rectal temperature at FTAI, body condition, and estrus patch scores. Rectal temperature at FTAI was positively associated with pregnancy outcomes (P < 0.0001); per each 1 °C increase pregnancy odds increased 1.9 times (95% CI: 1.4 to 2.6). Highest pregnancy outcomes occurred with rectal temperatures exceeding 40 °C (P = 0.0004). Rectal temperature before FTAI in Bos taurus cattle ranged from 37.8 to 41.8 °C; 43.3% were hyperthermic. Factors impacting pregnancy were rectal temperature at FTAI, estrus activity, parity, and ambient conditions on day of FTAI. Rectal temperature of Bos taurus cattle at FTAI was positively associated with pregnancy (P = 0.0286); odds increased 1.45 times (95% CI: 1.0 to 2.0) per each 1 °C increase. Highest pregnancy outcomes occurred with rectal temperatures at FTAI exceeding 40 °C (P = 0.057). Moreover, positive relationship of rectal temperature at FTAI to pregnancy persisted in estrual females (71.25% of total; P = 0.0408; OR 1.5; 95% CI: 1.0 to 2.2). Mindful that 1) elevated temperatures observed in Bos indicus and Bos taurus cattle directly promote meiotic resumption of the oocyte in vitro and that 2) in vivo hyperthermia alters intrafollicular components which others have shown to potentiate ovulation and promote meiotic resumption, it is biologically plausible that an acute elevation in body temperature at or around time of insemination is functionally important to maximize pregnancy outcomes.

Reproductive efficiency remains a major challenge for beef producers with 35% to 55% of females failing to become pregnant after a single insemination. While basis for failure is multi-factorial, heightened estrus activity matters for pregnancy outcomes, even when synchronizing ovulation for fixed time artificial insemination. Body temperature increases of 1.5 °C+ are common during estrus. We hypothesize that higher estrous-associated temperatures (HEAT) at/near insemination are functionally important to maximize pregnancy outcomes. Elevated temperatures equivalent to what is observed in females exhibiting HEAT have been shown to induce oocyte meiotic resumption. An acute episode of hyperthermia after the LH surge alters intrafollicular components known to potentiate ovulation and affect the oocyte. Effort to examine the relationship of rectal temperature at fixed time artificial insemination with pregnancy outcomes in a breeding season with expected pregnancy rates >50% represents a next step in the continuum of hypothesis testing. The positive relationship of rectal temperature at insemination with pregnancy outcomes that was discovered adds to foundational knowledge. Because the degree of hyperthermia is related to highest pregnancy outcomes, a case is made for HEAT to be biologically and functionally important to maximize pregnancy outcomes in cattle.

Technical Note: Coccygeal vein catheterization for sampling of reproductive tract-derived products from the uterine-ovarian drainage.

Blood sample collection from the caudal vena cava at the site of uterine-ovarian drainage provides a more exact evaluation of the concentration and pattern of secretion of uterine or ovarian secreted products for studies of reproductive processes in cyclic and pregnant cattle compared with samples collected from general circulation. This paper describes a thorough and updated procedure for cannulating the coccygeal vein into the caudal vena cava for the collection of serial blood samples at or near the site of uterine-ovarian drainage. Concentrations of progesterone were quantified in cows of different reproductive tract sizes with an active corpus luteum to assess the distance for proper catheter placement compared with circulating concentrations collected from the jugular vein. This procedure has a low risk for side effects, can be used effectively in pregnant animals with no major consequence to the viability of the pregnancy, and provides means for frequent collections up to 12 d.

Administering an additional prostaglandin F2α injection to Bos indicus beef cows during a treatment regimen for fixed-time artificial insemination.

Two experiments were conducted to evaluate if an additional prostaglandin F2α (PG) injection during an estradiol-based estrous synchronization treatment regimen affects pregnancy rates resulting from fixed-time artificial insemination (FTAI) in Nelore cows. In Experiment 1, 1039 cows were administered estradiol benzoate and an intravaginal progesterone releasing device (CIDR) on d -11, an injection of PG on d -4, with CIDR removal and administration of estradiol cypionate and eCG occurring on d -2, and FTAI on d 0. Cows were administered an additional injection of PG (PG2) or saline (PG1) on d -2. Percentage pregnancy per FTAI on d 30 was greater (P = 0.01) in cows of the PG2 than PG1 group (54.5 % and 46.6 %, respectively). In Experiment 2, there was use of the same treatment regimen for estrous synchronization of 934 cows as that for Experiment 1 followed by FTAI, with there being diagnosis of whether cows had or did not have a corpus luteum on d -4. Response to estrous synchronization, largest follicle diameter at FTAI, and pregnancy per FTAI were greater (P ≤ 0.05) in cows of the PG2 than PG1 group with a body condition score (BCS) < 5.0 at FTAI (81.2 % and 72.6 %, 11.9 and 11.2 mm, 55.5 and 45.6 %; respectively). These treatment responses did not differ (P ≥ 0.18) in cows with BCS ≥ 5.0. Collectively, results indicate that treatment with PG2 increased pregnancy per FTAI in B. indicus cows deficient in body energy reserves, by enhancing follicle development and estrous synchronization response.

The effect of clitoral stimulation post artificial insemination on pregnancy rates of multiparous Bos indicus beef cows submitted to estradiol/progesterone-based estrus synchronization protocol.

Clitoral stimulation after artificial insemination (AI) in beef cattle is a common practice utilized by AI technicians; however, the effect of clitoral stimulation during fixed-time AI (TAI) is still unknown. The objective of the present experiment was to evaluate the effect of clitoral stimulation on temperament responses and pregnancy rates to TAI in Bos indicus beef cows. A total of 1,186 multiparous Nellore cows across three different locations were assigned to an estradiol/progesterone-based estrus synchronization + TAI protocol (day -11 to 0). Cows were randomly assigned to receive either 3 s of clitoral stimulation (n = 602) or no clitoral stimulation (n = 584) immediately after TAI (day 0). Cow body condition score (BCS) was recorded on day -11. Estrus expression was evaluated based on estrus detection visual aid patch activation on day 0 (estrus, ≥50% activated; no estrus, <50% activated). Temperament was assessed by individual chute score based on a 5-point scale before TAI, and individual exit velocity was measured after clitoral stimulation. Pregnancy diagnosis was performed 30 d after TAI via transrectal ultrasonography. Pregnancy rate to TAI was positively affected by BCS (P < 0.01) and estrus expression (P = 0.03). Pregnancy rates of cows receiving clitoral stimulation did not differ (P = 0.39) from cows non-stimulated (47.5 ± 4.6% vs. 44.3 ± 4.6%, respectively). No interaction of clitoral stimulation and estrus expression was observed (P = 0.26). Chute score was positively correlated to exit velocity (P < 0.01; r = 0.29); however, clitoral stimulation did not affect exit velocity (P = 0.86). In summary, pregnancy rates to TAI are influenced by various factors and multiple strategies have the potential to increase the fertility of beef cows submitted to TAI; however, clitoral stimulation of Bos indicus beef cows did not improve TAI pregnancy rate.

Effects of estrous expression and intensity of behavioral estrous symptoms on variables associated with fertility in beef cows treated for fixed-time artificial insemination.

This experiment was conducted to evaluate effects of estrous expression and intensity of behavioral estrous expression, assessed by evaluating physical activity, on variables associated with fertility in beef cows. The following treatment regimen was imposed on lactating, multiparous cows (n = 273): 100-µg injection of GnRH and an intravaginal progesterone (P4) releasing device (CIDR) administered on d -10, 25-mg injection of prostaglandin F2α at CIDR removal on d -3, and another GnRH injection + fixed-time artificial insemination (TAI) 60-66 h following CIDR removal (d 0). Cows were fitted with a pedometer on d -10, and an estrous detection patch on the tail-head on d -3. Cows were classified as not expressing estrus (NOESTR) or expressing estrus with net physical activity greater (HIESTR) or less than the median (LWESTR). Only data from cows responsive to the estrous synchronization treatment regimen were utilized (NOESTR, n = 119; LWESTR, n = 50; HIESTR, n = 50). Diameter of dominant follicles on d 0 and corpus luteum volume on d 7 were greater in HIESTR compared with LWESTR and NOESTR groups (P < 0.01). Plasma P4 concentrations on d 7 and pregnancy rates to TAI were greater in HIESTR and LWESTR than the NOESTR group (P < 0.01). Hence, if there was estrous expression during the period of the GnRH-based TAI treatment regimen, there were greater pregnancy rates to TAI, whereas when there was greater intensity of behavioral estrous symptoms, there were larger dominant follicles and corpora lutea dimensions but not greater pregnancy rates.