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.

Evaluating the use of luteal color Doppler ultrasonography and pregnancy-associated glycoproteins to diagnose pregnancy and predict pregnancy loss in Bos taurus beef replacement heifers.

The objective of this study was to evaluate the use of corpus luteum (CL) color Doppler (CD) ultrasonography and pregnancy-associated glycoproteins (PAG) for early pregnancy diagnosis and examine their ability to predict late embryonic/early fetal mortality (LEM) in Bos taurus beef replacement heifers. Beef heifers (n = 178) were exposed to a 7-d CO-Synch + CIDR protocol followed by fixed-time artificial insemination (day 0). On days 20 and 22, B-mode and CD ultrasonography were performed to evaluate CL morphometries and blood perfusion, respectively. Heifers were considered nonpregnant when CL area was <2 cm2 or estimated luteal blood perfusion was ≤30% of the total luteal area. Blood samples were collected on days 25 and 29 to estimate circulating concentrations of PAG. Conventional ultrasonography on days 29 and 94 was utilized to determine pregnancy status and considered the gold standard method for pregnancy diagnosis. Pregnant heifers had greater (P < 0.01) CL diameter, area, volume, and blood perfusion when compared with nonpregnant heifers on days 20 and 22. Accuracy of CD on days 20 and 22, and PAG on days 25 and 29 were 91%, 94%, 96%, and 98%, respectively. No false-negative results were observed for CD on both days 20 and 22 (negative predicted value = 100%) and false-positive results represented 8% and 6% of the diagnoses. Heifers that experienced LEM between days 29 and 94 of gestation had decreased luteal (P = 0.02) volume on day 20 and tended (P = 0.07) to have decreased concentrations of PAG on day 29 compared with heifers that maintained pregnancy. However, both CD and PAG failed to predict embryonic mortality. In conclusion, CD successfully detected most nonpregnant replacement heifers as early as day 20 of gestation, while resulting in no false negative diagnoses. Both CD and PAG failed to predict LEM in the present study.

Identifying nonpregnant females early after breeding allows cattle producers to rapidly rebreed females that failed to conceive after their first artificial insemination and consequently increases reproductive efficiency. Additionally, embryonic and fetal mortality during early gestation are the main drivers of pregnancy failure and represents a significant economic burden to both the beef and dairy industries. This study evaluated the use of color Doppler (CD) ultrasonography of specific ovarian structures and blood concentrations of pregnancy-associated glycoproteins (PAG) as potential methods to diagnose pregnancy earlier than industry-standard techniques. Moreover, the present study investigated the use of CD and blood PAG as predictor of pregnancy loss. Data generated here indicates that CD and blood PAG can accurately detect pregnancy as early as 20 and 25 d after insemination, respectively. In the present study, heifers that experienced pregnancy loss between days 29 and 94 of gestation tended to have decreased plasma concentrations of PAG during early pregnancy. Heifers experiencing pregnancy loss between days 29 and 94 of gestation were also more likely to have a luteal cavity on day 20 of gestation and had decreased luteal volume on the same day. However, results reported herein indicate that both CD and blood concentrations of PAG failed to predict pregnancy loss in replacement heifers.

Publication bias casts doubt on implicit processing in inattentional blindness.

Two recent meta-analyses on inattentional blindness (Kreitz, Pugnaghi, & Memmert, 2020; Nobre et al., 2020) concluded that objects can be processed implicitly even when attention is directed elsewhere. However, signs of publication bias are evident in both of these meta-analyses. Here, we employed multiple tools to correct for publication bias in the data aggregated in those meta-analyses. Analyses using the Precision-Effect Test (PET) and robust Bayesian meta-analysis (RoBMA) suggest that the estimates in the original meta-analyses were inflated, together with strong evidence of publication bias. Indeed, the data are consistent with no overall implicit effects. We suggest that more evidence, particularly from well-powered pre-registered experiments, is needed before solid conclusions can be drawn regarding implicit processing during inattentional blindness.

Uterine Involution of Mares Supplemented with Dietary Algae-Derived Omega-3 Fatty Acids During the Peripartum Period.

Different approaches have been used to improve conception rates at foal heat. Omega-3 fatty acids family and derivatives have improved reproductive efficiency in ruminants, but literature lacks studies evaluating these components on equines. The objective of this study was to evaluate effects of mare dietary supplementation with microalgae rich in docosahexaenoic acid (DHA) during peripartum on follicular dynamics and uterine involution in early post-partum. Eighteen pregnant mares, no particular breed, 410 ± 39.5 kg body weight (BW), and 7.83 ± 2.01 yr old were used. Mares were randomly assigned to control (CONT) or supplementation with microalgae rich in DHA at 0.06 g/kg BW (ALG). Treatments were supplied from 90 d prior to expected foaling date until 7 d after first ovulation. Reproductive evaluations were performed during early post-partum until 7 d after first ovulation through rectal palpation and ultrasonography of the following parameters: uterine and endometrium diameters, intrauterine fluid (IUF), uterine echogenicity, uterus tone, and follicular dynamics. Endometrial cells samples were collected to assess mRNA expression of CRP, IL-1ß and AKR1C4, using RT-qPCR. Data were analyzed by mixed procedure of SAS. ALG mares had smaller uterine horns diameters and greater uterine echogenicity during post-partum in comparison with CONT. No treatment effects were detected for other characteristics evaluated, but a day effect was observed for uterine and endometrium diameter, IUF, uterine echogenicity, and transcript abundance of endometrial AKR1C4. Supplementation with DHA during peripartum may benefit uterine involution process and odds of early conception, but more studies should be performed regarding fertility.

New approaches to diagnose and target reproductive failure in cattle.

Reproductive failure and pregnancy loss in cattle are some of the largest economic burdens to cattle producers and one of most perplexing factors influencing management decisions. Pregnancy loss may occur at any point during gestation with the largest percentage of loss occurring in the first 30 days and, subsequently, decreasing as the pregnancy progresses. Losses may be attributed to numerous factors, predisposed issues or environmental conditions such as nutritional stressors or disease. From a research perspective, determining the exact causes of pregnancy loss or embryonic mortality in cattle have been difficult, due to limitations of accurately determining early gestation pregnancy status. Until methods that precisely determine embryo success early in gestation are available, our understanding of in vivo pregnancy loss will lack clarity necessary to develop management strategies to decrease such loss. In this review, we will briefly discuss the pivotal periods of pregnancy loss affecting beef and dairy cattle, methods and technologies to determine pregnancy status and embryo viability and potential opportunities to decrease reproductive failure.

Implicit processing during inattentional blindness: A systematic review and meta-analysis.

The occurrence of implicit processing of visual stimuli during inattentional blindness is still a matter of debate. To assess the evidence available in this debate, we conducted a systematic review of articles that explored whether unexpected visual stimuli presented during inattentional blindness are implicitly processed despite not being reported. Additionally, we employed meta-analysis to combine 59 behavioral experiments and investigate the statistical support for such implicit processing across experiments. Results showed that visual stimuli can be processed when unattended and unnoticed. Additionally, we reviewed the measures used to assess participants' awareness of the unexpected stimuli. We also employed meta-analysis to search for differences in awareness of the unexpected stimuli that may result from adopting distinct criteria to categorize participants as aware or unaware. The results showed that the overall effect of awareness changed depending on whether more demanding or less demanding measures of awareness were employed. This suggests that the choice of awareness measure may influence conclusions about whether processing of the US is implicit or explicit. We discuss the implications of these results for the study of implicit processing and the role of attention in visual cognition.

Early pregnancy-induced transcripts in peripheral blood immune cells in Bos indicus heifers.

Immune cells play a central role in early pregnancy establishment in cattle. We aimed to: (1) discover novel early-pregnancy-induced genes in peripheral blood mononuclear cells (PBMC); and (2) characterize the temporal pattern of early-pregnancy-induced transcription of select genes in PBMC and peripheral blood polymorphonuclear cells (PMN). Beef heifers were artificially inseminated on D0 and pregnancies were diagnosed on D28. On D10, 14, 16, 18, and 20, blood was collected for isolation of PBMC and PMN from heifers that were retrospectively classified as pregnant (P) or non-pregnant (NP). PBMC samples from D18 were submitted to RNAseq and 220 genes were differentially expressed between pregnant (P) and non-pregnant (NP) heifers. The temporal abundance of 20 transcripts was compared between P and NP, both in PBMC and PMN. In PBMC, pregnancy stimulated transcription of IFI6, RSAD2, IFI44, IFITM2, CLEC3B, OAS2, TNFSF13B, DMKN and LGALS3BP as early as D18. Expression of IFI44, RSAD2, OAS2, LGALS3BP, IFI6 and C1R in PMN was stimulated in the P group from D18. The novel early-pregnancy induced genes discovered in beef heifers will allow both the understanding of the role of immune cells during the pre-attachment period and the development of technologies to detect early pregnancies in beef cattle.

Applied use of interferon-tau stimulated genes expression in polymorphonuclear cells to detect pregnancy compared to other early predictors in beef cattle.

We aimed to evaluate the accuracy of Interferon-tau stimulated genes (ISG) abundance in peripheral blood polymorphonuclear cells (PMNs) on D20 after fixed-time artificial insemination (FTAI; D0) as a pregnancy diagnosis method against CL evaluation by Doppler ultrasonography and progesterone (P4) concentrations on D20, as well as Pregnancy Associated Glycoproteins (PAG) concentrations on D25. Additionally, we evaluated the potential of ISG abundance in PMNs as pregnancy loss predictors. Nelore heifers (n = 103) and cows (n = 144) underwent estrous synchronization and were artificially inseminated on D0. Pregnancy was diagnosed by B-mode ultrasonography on D30 and D70, and after the final diagnosis, females were classified in four groups: Pregnant; Non-pregnant; Functional CL on D20 but non-pregnant (CL-NP) and Pregnancy loss between D30 and D70 (PL). After determining cutoff values, the Sensitivity (SE), Specificity (SP), Positive Predictive Value (PPV), Negative Predictive Value (NPV) and Accuracy (ACC) were determined for each method. All methods were classified as significant (P < 0.05) predictors of pregnancy. Both ISG expression and PAG concentrations were greater (P < 0.05) in pregnant females than in non-pregnant and CL-NP females but did not differ (P > 0.05) from the PL group. ISG15 expression was greater (P < 0.05) in heifers than in cows, but this difference was not found in OAS1 expression and PAG concentrations. All the methods evaluated were proven to be adequate predictors of pregnancy, but greater accuracies were obtained through PAG concentrations and Doppler-US, due to the decreased number of false positive and false negative results.

Use of color-Doppler ultrasonography for selection of recipients in timed-embryo transfer programs in beef cattle.

We aimed to study the association between CL characteristics assessed by color-Doppler ultrasonography (Doppler-US) at the time of embryo transfer (ET) and pregnancy rate (P/ET) in beef recipients. Estrous cycles of crossbred beef recipients were synchronized for timed-ET. On the day of ET (Day 7), CL area, proportion of luteal blood perfusion (BP), and the relationship between the largest dominant follicle (DF) and CL (ipsilateral or contralateral) were determined. Animals (n = 444) received an in vitro produced embryo from Nelore donors, placed in the uterine horn ipsilateral to the CL. Recipients were split retrospectively in three subgroups according to CL area [small (<3 cm2), medium (3-4 cm2), or large (>4 cm2)] and three subgroups according to luteal signals of BP [low (≤40%), medium (45-50%) or high (≥55%)]. Pregnancy was detected on Days 30-45 by transrectal ultrasonography and P/ET was analyzed considering the effects of cow's category (suckling or non-suckling), CL area, luteal BP and side of DF. P/ET increased along with BP category [low, 45.9%B, (62/135); medium, 54.1%AB (93/172); and high, 58.4%A (80/137)]. When luteal BP was evaluated as a continuous variable, a significant (P < 0.05) linear and positive effect was observed on P/ET. A greater (P < 0.05) CL area and serum progesterone concentrations were observed in the medium and high BP than in the low BP category. Although an effect of luteal size category was not significant on P/ET [small, 49% (76/155); medium, 59.7% (83/139); and large, 50.7% (75/148); P > 0.1], when CL area was evaluated as a continuous variable, a quadratic effect (P < 0.05) indicated a positive relationship between P/ET and CL area until luteal tissue reached 4.07 cm2, followed by a negative relationship. The location of the first-wave DF in relation to the CL did not affect P/ET (P > 0.1). In conclusion, Doppler-US is an innovative tool that has the potential to be used for selection of suitable embryo recipients based on luteal BP. Selection of recipients that have a greater chance of maintaining pregnancy will increase the success of timed-ET programs.

Sex and the housing: Effects on behavior, cortisol levels and weight in zebrafish.

Studies with zebrafish use acclimatizing periods of at least one week immediately before the experiments. During this time, animals can be housed in sexually segregated conditions (only females or males in the tank) or in mixed-sex conditions (both sexes in the tank). The influence of sex and housing conditions regarding the presence of one or two sexes is largely unknown in zebrafish. Our aim was to evaluate the influence that sex and housing regarding the sex of animals had in the open tank task, in the inhibitory avoidance memory test, in cortisol levels and weight in zebrafish. Four groups of animals were used: 1) segregated housed females (only females were kept in the tank); 2) segregated housed males (only males were kept in the tank); 3) mixed-sex housed females (only females were analyzed from a tank containing 50% ratio of each sex); 4) mixed-sex housed males (only males were analyzed from a tank containing 50% ratio of each sex). Males showed higher total distance travelled and mean speed when compared to females. In the inhibitory avoidance memory, sexually segregated animals had higher latencies than their mixed-sex counterparts in the 1day test and sexually segregated females presented a memory that persisted longer and was able to be reinstated. Whole-body cortisol levels were higher in mixed-sex animals while weight was lower in these fish. To the best of our knowledge, this is the first time that effects of sex and housing regarding sex were investigated in behavior and physiology of zebrafish.

Use of Doppler ultrasonography in embryo transfer programs: feasibility and field results.

The intensive use of Doppler ultrasonography in several studies in the last decade allowed the characterization of vascular perfusion and the estimation of function of the reproductive organs and tissues along the estrous cycle and pregnancy in cattle. We aim to discuss the possibility of using Doppler imaging and to explore the potential of its inclusion in reproductive programs in cattle industry. Recent studies in dairy and beef cows indicated a high accuracy and sensitivity when Doppler ultrasonography is used to evaluate corpus luteum function and to diagnosis pregnancy between days 20 and 22. Moreover, resynchronization programs starting 5 to 7 days after timed embryo transfer (FTET) coupled with early pregnancy diagnosis were developed for beef cattle, and have been implemented in commercial embryo transfer programs. These strategies allow a reduction in the interval between two FTET from ≈ 40 to 24 days and may improve the gains in reproductive efficiency when compared to traditional programs than begin resynchronization after the pregnancy diagnosis at 30 days. A second alternative to use Doppler imaging is the evaluation of luteal blood perfusion at the time of embryo transfer for selection of recipients with greater receptivity potential. This may increases fertility in FTET, as embryos would not be transferred to females with non-functional CL, and in cases with recipients surplus, females with higher receptivity would be prioritized.

Assessing the influence of geographic distance in parasite communities of an exotic lizard.

The decay of similarity between biological communities with increasing geographical distance is a well-established pattern in ecology, but there are more complex factors acting on host population connections that influence this association for parasite communities, such as parasites' colonization ability and degree of connectivity between host populations. Here we aim to determine the helminth communities associated with different populations of the host lizard Hemidactylus mabouia, testing if the similarity of parasite communities decreases as the distance between them increases. For this, we collected samples of lizard populations in seven sites from Northeastern coast of Brazil and identified parasite species of helminths and pentastomids in each host, calculated the Sørensen indices of presence/absence and abundance of each pair of communities and related them to the geographical distance. We did not find a relationship of decaying similarity with increasing distance between the parasite communities of the host populations. This can be explained by factors such as the characteristics of the contact between the host populations, and by modes of transmission of most parasite species. Furthermore, it may be related to the exotic nature of the host in Brazil so that parasite communities have not reached equilibrium.

Effects of an H3R antagonist on the animal model of autism induced by prenatal exposure to valproic acid.

Autism spectrum disorders (ASD) are a group of neurodevelopmental disorders primarily characterized by impaired social interaction and communication, and by restricted repetitive behaviors and interests. Ligands of histamine receptor 3 (H3R) are considered potential therapeutic agents for the treatment of different brain disorders and cognitive impairments. Considering this, the aim of the present study is to evaluate the actions of ciproxifan (CPX), an H3R antagonist, on the animal model of autism induced by prenatal exposure to valproic acid (VPA). Swiss mice were prenatally exposed to VPA on embryonic day 11 and assessed for social behavior, nociceptive threshold and repetitive behavior at 50 days of life. The treatment with CPX (3 mg/kg) or saline was administered 30 minutes before each behavioral test. The VPA group presented lower sociability index compared to VPA animals that were treated with CPX. Compared to the Control group, VPA animals presented a significantly higher nociceptive threshold, and treatment with CPX was not able to modify this parameter. In the marble burying test, the number of marbles buried by VPA animals was consistent with markedly repetitive behavior. VPA animals that received CPX buried a reduced amount of marbles. In summary, we report that an acute dose of CPX is able to attenuate sociability deficits and stereotypies present in the VPA model of autism. Our findings have the potential to help the investigations of both the molecular underpinnings of ASD and of possible treatments to ameliorate the ASD symptomatology, although more research is still necessary to corroborate and expand this initial data.

Resveratrol prevents social deficits in animal model of autism induced by valproic acid.

Autism spectrum disorders (ASD) involve a complex interplay of both genetic and environmental risk factors, such as prenatal exposure to valproic acid (VPA). Considering the neuroprotective, antioxidant and anti-inflammatory effects of resveratrol (RSV), we investigated the influence of prenatal RSV treatment on social behaviors of a rodent model of autism induced by prenatal exposure to VPA. In the three-chambered apparatus test, the VPA group showed a reduced place preference conditioned by conspecific and no preference between exploring a wire-cage or a rat enclosed inside a wire cage, revealing sociability impairments. Prenatal administration of RSV prevented the VPA-induced social impairments evaluated in this study. A bioinformatics analysis was used to discard possible molecular interactions between VPA and RSV during administration. The interaction energy between RSV and VPA is weak and highly unstable, suggesting cellular effects instead of a single chemical process. In summary, the present study highlights a promising experimental strategy to evaluate new molecular targets possibly involved in the etiology of autism and developmental alterations implicated in neural and behavioral impairments in ASD.