With demand for the global food supply continuing to increase with a growing world population, reproductive efficiency of cattle on cow/calf operations will need to adapt. When a female fails to establish a pregnancy, yet remains in the breeding herd, the rancher experiences an economic loss (Cooke et al., 2020). While infertility in cattle can be attributed to many factors, such as nutrition, genetics, or disease; the immune system plays an active role in reproduction and may influence reproductive efficiency. Additionally, with Bos indicus-type or Brahman cattle consisting of around 30% of the beef population in the United States, it is important to understand how these factors impact reproductive efficiency in this subspecies. Their adaptivity and hardiness in sub-tropical and tropical climates make them ideal for beef production on the Gulf Coast (NASS, 2017; Cooke et al., 2020).

The innate immune system in an animal is the initial defense in the reproductive tract against invasive pathogens. It plays an active role in pregnancy, especially in preventing the onset of infection (Singh et al., 2008). Upon identification of an infection or pathogenic bacteria in the reproductive tract, the innate immune system will release small communication proteins called cytokines. Cytokines can initiate an inflammatory response in an environment by communicating with immune cells (Wira et al., 2005; Azawi, 2008; Murphy et al., 2022). There are two major classifications of cytokines: pro- inflammatory or anti-inflammatory. Pro-inflammatory cytokines may cause a more sustained and severe immune reaction and can lead to detrimental effects on tissues. In contrast, anti-inflammatory cytokines may aid in healing and returning the immune system back to homeostasis after an immune response (Dinarello, 2000; Cavaillon, 2001). Particularly during the rebreeding period in postpartum cattle, the innate immune system plays an active role in preventing an infection from occurring (Singh et al., 2008). Previous work has demonstrated certain bacterial genera are associated with cytokine concentrations prior to breeding in beef cattle (Smith et al., 2023). Additionally, the immune system’s first reaction for pregnancy establishment appears at insemination (Fair, 2015). During insemination, a variety of inflammatory cells are introduced at the site of semen deposition, potentially affecting the overall inflammatory response and subsequent fertility (Robertson, 2005).

Sheldon et al. (2002) and Williams et al. (2004) postulated that bacterial contamination in the uterus could disrupt ovulation. Other studies have indicated that changes in hormone concentrations lead to shifts in bacterial relative abundances (Sandrini et al., 2015; Org et al., 2016; Poole et al., 2023; Smith et al., 2023). Ault et al. (2019a) observed greater relative abundances of various commensal and pathogenic bacterial phyla in cattle in response to increased or reduced concentrations of progesterone (P4). These shifts in bacterial communities of the reproductive microbiome could impact fertility (Swartz et al., 2014; Laguardia-Nascimento et al., 2015; Luecke et al., 2022; Smith et al., 2023). Evaluating certain factors, such as the reproductive microbiome, provides an opportunity to develop methods that may increase efficiency in animal agricultural practices (Sheldon et al., 2002).

Characterization of the vaginal microbiome and circulating cytokine concentrations on a purebred Bos indicus beef cattle operation remains to be determined. As such, a unique opportunity became available to collect samples from several purebred Bos indicus beef cattle operations subjected to an estrus synchronization protocol. Therefore, the first objective of the current study was to determine the concentrations of specific pro- and anti-inflammatory cytokines in purebred Bos indicus beef cattle in association with a fixed-time artificial insemination (FTAI) protocol on-farm. The second objective was to evaluate the vaginal bacterial relative abundances in the vagina of Bos indicus beef cattle on-farm subjected to a FTAI protocol. The hypothesis was that concentrations of pro- and anti-inflammatory cytokines as well as the taxonomic composition of bacteria within the vagina would shift throughout during the estrus synchronization protocol.

It has been previously established that the immune system plays a role in beef cattle fertility; in addition to the relationship between different bacterial communities and the immune system (Smith et al., 2023). The first aim of this study was to determine concentrations of pro- and anti-inflammatory cytokines in purebred Bos indicus beef cattle plasma samples on producer operations before FTAI. Furthermore, Ault et al. (2019a) stated that relative abundances of bacteria appear to shift during estrus synchronization and may influence fertility. Therefore, an additional objective was to evaluate the fluctuations in vaginal bacteria relative abundance associated with the 7-day CO-Synch synchronization protocol in purebred Bos indicus beef cattle.

The immune system will respond to microbial invasion by releasing cytokines or chemokines into the bloodstream to heal or mitigate the effects of infection (Cavaillon, 2001; Murphy et al., 2022). In addition, cytokines are essential for pregnancy establishment and pregnancy maintenance (Yockey and Iwasaki, 2019). In the current study, the pro-inflammatory cytokine IFNγ was observed to be greater in Cows compared to Heifers. A greater concentration of pro-inflammatory cytokines in the reproductive tract is typically associated with uterine involution (Balkwill, 2006; Galvao et al., 2011), which potentially explains the greater concentrations in Cows, who have undergone the involution process, as compared to Heifers in the current study. The pleiotropic cytokine IP10 also had greater concentrations measured in Cows than in Heifers as well as a parity-by-status interaction for Open Cows. Interferon gamma-induced protein 10 is regulated by IFNγ signaling and nuclear factor kappa B (NF-κB) activity, thus it makes sense that both IFNγ and IP10 concentrations would be greater in Cows compared to Heifers. Pleiotropic cytokines can display both pro- and anti-inflammatory behavior and stimulate a wide variety of cells (Zhang and An, 2007). As a biomarker, IP10 has been shown as an indicator of infection prior to onset of clinical symptoms in humans (Moreno et al., 2022; Wang et al., 2023). Collectively, greater IP10 concentrations in Open Cows prior to AI could indicate inflammation hindering the establishment of a pregnancy. The anti-inflammatory IL1F5 was also observed in greater concentration in Cows than in Heifers; and in one study, it was found to have a correlation with bacteria that induce cell inflammation (Smith et al., 2023). Additionally, IL1F5 inhibits the activation of NF-κB, which is important for regulating pro-inflammatory immune responses (Abdulrahman Alrabiah et al., 2021). Therefore, the increased concentrations of pro-inflammatory immune responses (i.e., IFNγ) would also be associated with an increase in an anti-inflammatory immune response (i.e., IL1F5) in Cows.

Concentrations of the anti-inflammatory cytokine IL13 tended to be greater in concentration for Open females compared to Pregnant females. Ma et al. (2013) and Dickinson et al. (2016) concluded that IL13 inhibits cell breakdown of old and damaged cell components. Interleukin 13 will also increase the production of an IL1 receptor antagonist, which can bind with IL1 and reduce its pro-inflammatory properties. Typically, IL13 is involved in downregulating pro-inflammatory cytokine and chemokine production (de Vries, 1998); however, some studies investigating the pathogenesis of asthma and the lungs have demonstrated that IL13 can induce chemokine responses (Zhu et al., 2002). Interestingly, concentrations of the chemokine MIP1b were greater in Open cattle than in Pregnant cattle. The increased concentrations of MIP1b may be attributed to its function as a macrophage-attracting chemokine that plays a role in controlling macrophage influx to tissues, such as the vagina (von Stebut et al., 2003). In addition, cytokine concentrations did not differ by day of the synchronization protocol in the current study, which is different from a previous study by Smith et al. (2023), where it was noted that hormone changes were associated with alterations in cytokine concentrations by day in plasma, vaginal flushes, and uterine flushes.

Bacterial phyla such as Firmicutes, Bacteroidetes, Proteobacteria, Actinobacteria, and Tenericutes have been discovered to dominate the reproductive tract of beef cattle which aligns with the findings of the current study (Swartz et al., 2014; Clemmons et al., 2017; Messman et al., 2020; Pickett et al., 2022). Additionally, some studies have shown that bacterial abundances will shift due to hormonal alterations in Bos taurus females (Ault et al., 2019a, Ault et al., 2019b; Poole et al., 2023). In Bos indicus cattle specifically, some studies have shown that Firmicutes, Proteobacteria, and Bacteroidetes are frequently detected (Laguardia-Nascimento et al., 2015; Giannattasio-Ferraz et al., 2019). The current study identified that the relative abundance of Firmicutes was more abundant at CIDR IN than CIDR OUT. These results coincide with Ault et al. (2019b), where Bos taurus females subjected to an estrus synchronization protocol had decreased relative abundances of Firmicutes two days prior to AI (synonymous with CIDR OUT in the current study). In addition, the relative abundance of Firmicutes increased from CIDR OUT to AI DAY, where removal of the CIDR device and administration of a PGF_2α injection caused the corpus luteum to undergo luteolysis and P4 concentrations to decrease. More specifically, as P4 decreased the relative abundance of Firmicutes appeared to increase. Notably, the tendency for Cows to have greater relative abundances of Firmicutes compared to Heifers was also observed in a study completed by Moore et al. (2017). Oppositely, the relative abundances of Proteobacteria and Bacteroidetes were reduced on CIDR IN compared to CIDR OUT days. The shift in the relative abundance of Proteobacteria was also correlated with P4 (Ault et al., 2019b), further demonstrating that as P4 concentrations increase, Proteobacteria relative abundance increases. Interestingly, an increased abundance of Bacteroidetes and decreased relative abundance of Proteobacteria has been found in Bos taurus metritic cows (Jeon et al., 2015), although no incidences of uterine inflammation were noted in this study.

The genus of Butyrvibrio, within phylum Firmicutes, was seen in greater relative abundance in the vagina of Cows compared to Heifers. Furman et al. (2020) reported that Butyrvibrio had greater abundance in the rumen of Bos taurus calves that were delivered vaginally versus via cesarean section, indicating this may be a common genus found in the vaginal microbiome. Though not typically categorized as pathogenic, Butyrivibrio has been previously identified within the uterus of Bos taurus Open cows (Clemmons et al., 2017; Ault et al., 2019b). Ruminococcus, Blautia, and Mogibacterium, all genera under the phylum of Firmicutes, had greater relative abundance at CIDR IN day than CIDR OUT day. This indicates that the introduction of a CIDR may have caused shifts in the vaginal microbiome. Such alterations to the microbiome have been shown to cause a rise in vaginal pH which may then influence fertility (Dias, 2022). While Butyrvibrio and Mogibacterium have been associated with compromised fertility (Clemmons et al., 2017; Ault et al., 2019a), no differences by fertility outcomes were observed in this study. The genus Mannheimia, under the phylum of Proteobacteria, had greatest relative abundance on CIDR OUT day and is known to be pleomorphic, which means it can be irregular and vary in its form (Laishevtsev, 2020). The occurrence of Mannheimia in the vagina of Bos indicus beef cows is not surprising as it has also been found in the vagina of dairy cows (Bicalho et al., 2017), but the association between the greater relative abundance of Mannheimia and its relation to CIDR OUT day needs to be further assessed.

In conclusion, cytokine concentrations may be associated with parity or fertility outcomes. This study also suggests that bacterial relative abundances can shift through an estrus synchronization protocol in Bos indicus cattle. Additional research may evaluate hormone concentrations with or without the introduction of a foreign object (e.g., CIDR) on bacterial shifts. Future research is necessary to determine the factors that can positively affect the immune system and vaginal microbiome to improve reproductive efficiency in Bos indicus cattle.

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/supplementary material.

The animal studies were approved by Texas A&M University Institutional Animal Care and Use Committee. The studies were conducted in accordance with the local legislation and institutional requirements. Written informed consent was obtained from the owners for the participation of their animals in this study.

MS: Investigation, Methodology, Writing – original draft, Writing – review & editing. DS: Methodology, Writing – review & editing. BM: Methodology, Writing – review & editing. KH-B: Methodology, Writing – review & editing. ES: Methodology, Writing – review & editing. RP: Conceptualization, Formal Analysis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Writing – review & editing.