Repeated low-dose lipopolysaccharide exposure during the periconception period on ewe immune responses and lamb performance

Abstract

Subclinical inflammatory challenges caused by gram-negative bacterial endotoxin lipopolysaccharide (LPS) may disrupt reproductive physiology and influence offspring development; however, the effects of repeated low-dose exposure during the periconception period remain unclear. Therefore, this study evaluated the effects of repeated low-dose LPS exposure on maternal immune and endocrine responses, reproductive performance, and offspring growth and development. Rambouillet ewes (n = 30; 2–8 yr; 61 ± 6 kg BW) were stratified by body weight and age and assigned to receive subcutaneous injections of saline (CON; n = 10), 1.5 µg/kg BW LPS (LOW; n = 10), or 3.0 µg/kg BW LPS (HIGH; n = 10) on d 5, 10, and 15 of a synchronized estrous cycle prior to breeding. Rectal temperature, serum cortisol, and complete blood count (CBC) parameters were measured before and after LPS administration, with repeated measurements collected through 24 h following treatment. Reproductive performance was evaluated after a 35-d breeding season, and lamb hematology, growth, morphometric traits, and wool characteristics were assessed from birth through weaning (d 60) and repeated-measures outcomes were analyzed using mixed-effects models. Maternal LPS exposure increased rectal temperature and serum cortisol on day 5 (treatment × hour, P ≤ 0.05). Cortisol responses remained elevated across repeated exposures (treatment × hour, P ≤ 0.05), but rectal temperatures did not differ on subsequent LPS challenge days. Total white blood cell, and lymphocyte concentrations decreased 2–4 h after treatment and increased at 24 h on d 5 (treatment × hour, P ≤ 0.05). Red blood cell and platelet parameters were minimally affected by treatment. Maternal LPS exposure did not affect conception rate, pregnancy rate, or lamb production traits. However, offspring from LOW-treated ewes had greater total white blood cell, lymphocyte, and neutrophil concentrations at birth and increased neutrophil concentrations at d 14 (P ≤ 0.05), but these differences were not maintained through weaning. No treatment effects were detected for lamb growth, morphometric development, or wool characteristics. Collectively, these results indicate that repeated low-dose maternal LPS exposure during the periconception period alters maternal immune responses and is associated with transient alterations in neonatal white blood cell profiles without affecting reproductive performance or growth outcomes.

Repeated low-dose lipopolysaccharide exposure during the periconception period induced febrile and hematological responses in ewes without affecting reproductive performance or lamb growth. However, altered lamb blood cell profiles at birth suggest that early-life immune development may be influenced despite normal production outcomes.

Introduction

Culling of breeding females can lead to substantial economic costs in animal production systems due to the need for replacement animals (Bascom and Young 1998). In sheep production systems, reproductive failure and health-related disorders are major contributors to reduced ewe longevity and increased rates of culling (McLaren et al. 2020). While the detrimental effects of overt clinical disease on reproductive performance are well documented, low-grade or subclinical inflammatory challenges frequently go undetected and untreated. Endotoxin exposure disrupts ovarian function by impairing granulosa cell steroidogenic pathways by decreasing aromatase and β-catenin expression (Ferranti et al. 2022), decreasing estradiol and progesterone concentrations (Sheldon et al. 2009), disrupting the preovulatory estradiol rise and subsequent LH and FSH surges (Battaglia et al. 2000), and reducing LH pulse frequency and delaying ovulation (Suzuki et al. 2001).

Lipopolysaccharide (LPS) is an endotoxin found on the outer membrane of gram-negative bacteria that can induce inflammatory responses through activation of Toll-like receptor 4 (TLR4). Following release from gram-negative bacteria, LPS is transported systemically by lipopolysaccharide-binding protein and activates the TLR4/myeloid differentiation protein 2 receptor complex (Mazgaeen and Gurung 2020; Ferranti et al. 2022) subsequently initiating a pro-inflammatory signaling cascade (Castro et al. 2026). Because ovarian granulosa cells express this receptor complex, they can respond directly to LPS exposure (Herath et al. 2007; Magata et al. 2014). Lipopolysaccharide has been detected in follicular fluid from slaughterhouse ovaries, where elevated concentrations are associated with reduced estradiol concentrations and decreased expression of key steroidogenic enzymes (Magata et al. 2014). Additionally, follicular LPS accumulation and in vitro LPS exposure impair oocyte developmental competence and embryonic development (Forrest et al. 2022; Castro et al. 2024).

While the effects of LPS on ovarian function are well characterized, systemic immune activation during the periconception period may also influence reproductive processes and subsequent offspring outcomes. A recent study demonstrated that repeated low-dose LPS exposure during the periconception period induced febrile, cortisol, and acute phase responses, while altering inflammatory gene expression in peripheral blood leukocytes (PBLs) in ewes (Austin et al. 2024). However, because PBLs are a mixed white blood cell population, it remains unclear whether these responses reflected changes in gene expression, shifts in white blood cell population composition, or both.

In addition to these maternal responses, immune activation during the periconception period has been proposed as a critical determinant of offspring developmental programming, and maternal inflammatory signaling may influence fetal development even without direct fetal exposure to LPS (Ashdown et al. 2006). In sheep, gestational LPS exposure resulted in male lambs with increased sperm abnormalities during early puberty (Henrique et al. 2020). Furthermore, repeated subclinical LPS exposure during the periconception period has been associated with reduced subsequent lamb performance, including decreased lamb birth weight and a tendency for reduced 60-d adjusted weaning weights, despite limited effects on overall pregnancy outcomes (Austin et al. 2024). However, the effects of maternal LPS exposure before breeding on offspring immune response, growth, morphometric development, and wool characteristics remain unknown.

Therefore, the objective of this study was to evaluate the effects of repeated low-dose LPS exposure during the periconception period on maternal endocrine responses, circulating white blood cell dynamics assessed by complete blood count analysis, reproductive outcomes, and offspring growth and development. We hypothesized that repeated low-dose LPS challenges during the periconception period would alter maternal immune and endocrine function and impair early-life growth and development in lambs.

Materials and methods

All procedures were approved by the New Mexico State University Institutional Animal Care and Use Committee (IACUC #2209000058). Ewes were housed at the New Mexico State University West Sheep Unit (Las Cruces, NM).

Animals and treatments

Thirty Rambouillet ewes (2–8 yr of age; 61 ± 6 kg BW) were maintained in a single dry lot pen (11 × 23 m) under ambient conditions and fed 1.6 kg/ewe per day of alfalfa hay with ad libitum access to water, shade, mineral supplement, and salt. Ewes were stratified by age and body weight and assigned to one of three treatments: saline control (CON; n = 10), low-dose lipopolysaccharide (LPS; 1.5 μg/kg BW; LOW; n = 10), or high-dose LPS (3.0 μg/kg BW; HIGH; n = 10). Two ewes from the LOW group were removed due to CIDR retention and an adverse response to LPS, resulting in final group sizes of CON (n = 10), LOW (n = 8), and HIGH (n = 10; total n = 28). Estrus was synchronized using a 5-d controlled internal drug release device (CIDR; Eazi-Breed; Zoetis, Kalamazoo, MI), followed by two intramuscular injections of prostaglandin F₂α (5 mg; Lutalyse; Zoetis Inc., Kalamazoo, MI) administered at 0 and 4 h relative to CIDR removal. Presumed estrus occurred 48 h after CIDR removal and was designated as day 0. Ewes received treatment injections on days 5, 10, and 15 relative to estrus to target small, medium, and large follicles, respectively. Lipopolysaccharide (Escherichia coli O55: B5; Sigma-Aldrich, Milwaukee, WI) was reconstituted to 1 mg/mL in sterile saline and administered subcutaneously (3 mL total volume) as saline (0 μg/kg BW), 1.5 μg/kg BW, or 3.0 μg/kg BW. Dose selection was based on previous studies demonstrating that subcutaneous LPS administration at 2–3 μg/kg BW induces a mild inflammatory response without severe clinical symptoms (Ferranti et al. 2022; Austin et al. 2024). On d 16, ewes were exposed to a fertile Rambouillet ram for a 35-d breeding season. Using a digital thermometer (Walgreen Corporation, Deerfield, IL), baseline rectal temperatures were recorded at presumed estrus (d 0) to exclude ewes with preexisting hyperthermia. Rectal temperatures were also measured at 0, 1, 2, 4, 6, 9, and 12 h following LPS administration on d 5 and 15.

Blood collection and analyses

Blood samples (∼10 mL) were collected via jugular venipuncture into serum (VACUETTE; Greiner Bio-One, Kremsmünster, Austria) and EDTA-treated tubes (Monoject; Cardinal Health, Dublin, OH) on d − 7, −2, 0, 2, 5, 10, and 15 relative to estrus. On d 5 and 15, serum samples were collected at 0, 1, 2, 4, 6, 9, and 12 h following LPS administration to evaluate endocrine responses. On day 10, a single serum sample was collected. EDTA-treated blood samples and rectal temperatures were collected at 0, 2, 4, 12, and 24 h on treatment days.

Serum samples were centrifuged (1,500 × g for 15 min at 4 °C), aliquoted, and stored at −30°C until analysis. Serum progesterone concentrations were measured on days 0, 5, 10, and 15, and cortisol was evaluated on d 5 and 15 using radioimmunoassay as previously described (Castanon et al. 2012; Forrest et al. 2022). Intra-assay CV for progesterone was 7.3%, and cortisol assays had intra-assay CV of 11.6% and 13.8%, with an inter-assay CV of 1.6%. Whole blood was used for complete blood count (CBC) analysis at 0, 2, 4, 12, and 24 h on d 5, 10, and 15 in a subset of ewes (CON, n = 5; LOW, n = 5; HIGH, n = 5) using a hematology analyzer (VetScan HM5; Zoetis Inc., Kalamazoo, MI).

Lamb collection and measurements

A total of 44 lambs were recorded at birth, including 15 lambs from saline-treated ewes (CON), 12 lambs from ewes receiving 1.5 µg LPS/kg BW (LOW), and 17 lambs from ewes receiving 3.0 µg LPS/kg BW (HIGH). Thirty-six of these lambs remained through weaning (d 60), including 14 lambs from vehicle-treated ewes, 8 lambs from LOW LPS-treated ewes, and 14 lambs from HIGH LPS-treated ewes. Lambs were sampled on d 0, 7, 14, 28, and 60. Blood samples were collected via jugular venipuncture into EDTA-treated tubes for CBC analysis.

Body weight and morphometric measurements, including heart girth, body length, wither height, hind leg circumference, and head dimensions, were recorded using standardized anatomical landmarks. Wool samples were collected from the left mid-side region and submitted for fiber analysis (Hettinger Research Extension Center, North Dakota State University, Hettinger, ND).

Statistical analysis

Statistical analyses were conducted using SAS (version 9.4; SAS Institute Inc., Cary, NC). Maternal repeated-measures variables were analyzed using the MIXED procedure. Models for serum progesterone, cortisol, rectal temperature, and CBC parameters included treatment, day or hour, and their interactions as fixed effects, with ewe as a random effect. An autoregressive [AR(1)] covariance structure was used for serum progesterone and cortisol, where a compound symmetry covariance structure was used for rectal temperature and maternal CBC parameters. Binary reproductive outcomes were analyzed using the GLIMMIX procedure with a binomial distribution and logit link function. Offspring repeated-measures variables, including lamb CBC parameters and body measurements, were analyzed using the MIXED procedure with treatment, day, sex when applicable, and relevant interactions included as fixed effects. Repeated measures for lamb CBC and morphometric traits were modeled using an autoregressive [AR(1)] covariance structure. Lamb birth weight adjusted 60-d weaning weight, and adjusted average daily gain were analyzed as non-repeated variables using the MIXED procedure. The ewe was considered the experimental unit for maternal measurements. Offspring measurements were averaged by ewe when arising from twins. Least squares means are reported. Pairwise comparisons were used to evaluate treatment differences within day or hour, and reported P-values are unadjusted. Statistical significance was declared at P ≤ 0.05, and tendencies were discussed when 0.05 < P ≤ 0.10.

Results and discussion

Serum progesterone confirms successful estrus synchronization

Serum progesterone concentrations varied across the estrous cycle (Figure 1; day, P < 0.0001), consistent with normal luteal development following synchronization (Bartlewski et al. 1999). No overall effect of treatment (P = 0.177) or treatment × day interaction (P = 0.138) was detected. When evaluated within day, a treatment effect was observed on d 15 (P = 0.0016). Pairwise comparisons indicated lower progesterone concentrations in LOW-treated ewes compared with CON and HIGH on d 15. Numerically, progesterone concentrations were 2.70 ± 0.49, 4.71 ± 0.44, and 4.73 ± 0.44 ng/mL for LOW, CON, and HIGH. Despite this reduction, progesterone concentrations remained within a range consistent with maintenance of luteal function and support of pregnancy in ewes, where concentrations of approximately 2–3 ng/mL are sufficient to sustain normal reproductive outcomes (Diskin and Niswender 1989). Collectively, these findings indicate that repeated low-dose LPS exposure during the periconception period had limited effects on circulating progesterone, supporting successful estrus synchronization and a consistent hormonal environment across all treatment groups.

Figure 1.

Serum progesterone concentrations during the synchronized estrous cycle. Ewes (n = 28) were synchronized using a 5-d CIDR protocol with 2 injections of PGF₂α before treatment administration. Presumed estrus occurred on day 0, and ewes received a subcutaneous injection of saline (CON), 1.5 µg LPS/kg BW (LOW), or 3.0 µg LPS/kg BW (HIGH) on days 5, 10, and 15 of the estrous cycle. Serum progesterone concentrations were measured on days 0, 5, 10, and 15. Although no treatment × day interaction was detected, LOW-treated ewes had lower progesterone concentrations on day 15, with a within-day pairwise difference observed among treatments. Values are least squares means ± SEM. Asterisks (*) indicate within-day treatment differences (P ≤ 0.05); these comparisons were not protected by a significant treatment × day interaction.

Repeated LPS exposure reduces the febrile response over time

Baseline rectal temperature (d 0) did not differ among treatments. Following LPS administration on d 5, rectal temperature increased (hour, P < 0.0001), with a treatment × hour interaction detected (P < 0.05; Figure 2A). Peak responses occurred at 4 h, where rectal temperature was greater in HIGH and LOW compared with CON (40.15 ± 0.12 and 40.03 ± 0.13 vs. 39.34 ± 0.12 °C, respectively). In contrast, on d 10 and 15 rectal temperature was affected by hour (P < 0.0001) but no treatment effect or treatment × hour interaction was detected, and no differences among treatments were observed within any hour (Figure 2B). The pronounced response following the initial challenge and diminished responses after subsequent exposures are consistent with previous observations in sheep subjected to repeated low-dose LPS challenge (Austin et al. 2024). Thus, the present findings indicate that repeated exposure reduced the magnitude of the febrile response over time, consistent with physiological adaptation to repeated endotoxin exposure (O’Reilly et al. 1988; Ferranti et al. 2022).

Figure 2.

Rectal temperature and serum cortisol responses to LPS challenge. Rectal temperature (A, B) and serum cortisol concentrations (C, D) were evaluated in control and LPS-treated ewes on day 5 (A, C) and day 15 (B, D) of the estrous cycle. Ewes (n = 28) received a subcutaneous injection of saline (CON), 1.5 µg LPS/kg BW (LOW), or 3.0 µg LPS/kg BW (HIGH). Rectal temperature was measured at 0, 1, 2, 4, 6, 9, and 12 h, and serum cortisol was measured at 0, 2, 4, 6, and 12 h following LPS challenge. On day 5, LPS increased rectal temperature, with HIGH and LOW greater than CON at 2–h (P ≤ 0.05). No treatment differences in rectal temperature were detected on day 15. Cortisol concentrations were increased in HIGH and LOW compared with CON from 2 to 6 h on day 5 and day 15 (P ≤ 0.05). Values are least squares means ± SEM. Asterisks (*) indicate differences among treatments within hour (P ≤ 0.05).

Endocrine stress responses remain active despite reduced febrile response

In contrast to the blunted febrile response, serum cortisol concentrations increased following LPS challenge on both d 5 and 15 (Figure 2C, D). On d 5, cortisol was affected by treatment (P = 0.0057), hour (P < 0.0001), and a treatment × hour interaction (P = 0.0013). Baseline concentrations were similar among treatments, but increased following LPS administration, with peak responses observed between 2 and 6 h. At 4 h, cortisol concentrations were greater in HIGH and LOW compared with CON (35.72 ± 4.78 and 28.33 ± 5.35 vs. 12.30 ± 4.78 ng/mL, respectively; Figure 2C). Similarly, on d 15, cortisol was affected by treatment (P = 0.006), hour (P < 0.0001), and a treatment × hour interaction (P = 0.005), with increased concentrations in LPS-treated ewes relative to CON between 2 and 6 h (Figure 2D). No differences among treatments were detected at 0 or 12 h on either day.

Persistence of the cortisol response despite attenuation of the febrile response indicates that repeated LPS exposure did not eliminate physiological responsiveness. Instead, different components of the response appeared to adapt differently across repeated challenges. A similar dissociation between diminished febrile responses and maintained cortisol responses has been reported in ewes following repeated low-dose LPS administration (Austin et al. 2024). Collectively, these findings support partial adaptation to repeated endotoxin exposure rather than complete tolerance.

Repeated LPS exposure alters white blood cell dynamics across challenge days

Total white blood cell (WBC), lymphocyte, and neutrophil concentrations exhibited time-dependent responses to LPS challenge that differed across sampling days (Figure 3). On d 5, WBC, lymphocyte, and neutrophil concentrations were affected by hour (P < 0.0001), with treatment × hour interactions detected (P ≤ 0.036) and no main effect of treatment. At early time points (2–4 h), WBC and lymphocyte concentrations were lower in LPS-treated ewes relative to CON (Figure 3A, C). In contrast, at 24 h, WBC, lymphocyte, and neutrophil concentrations were greater in LOW and HIGH compared with CON (P ≤ 0.05; Figure 3A, C, E). This early decrease followed by increased circulating cell concentrations at 24 h is consistent with the characteristic temporal redistribution of leukocytes reported following LPS challenge in sheep (Yates et al. 2011; Cadaret et al. 2021). Because WBC production is not immediate, increases observed at 24 h likely reflect redistribution of existing cells rather than de novo production. Neutrophil percentages were greater and lymphocyte percentages were reduced in HIGH-treated ewes at 12 and 24 h, consistent with a transition toward a neutrophil-dominated inflammatory response.

Figure 3.

White blood cell responses to LPS challenge during the estrous cycle. Total white blood cell (WBC) concentrations (A, B), lymphocyte concentrations (C, D), and neutrophil concentrations (E, F) were evaluated in response to LPS challenge on day 5 (A, C, E) and day 15 (B, D, F) of the estrous cycle. Ewes (n = 28) received a subcutaneous injection of saline (CON), 1.5 µg LPS/kg BW (LOW), or 3.0 µg LPS/kg BW (HIGH). Whole blood samples for complete blood count (CBC) analysis were collected from a subset of ewes (n = 5 per treatment) at 0, 2, 4, 12, and 24 h following LPS challenge. On day 5, WBC concentrations tended to be lower in LPS-treated ewes at 2–4 h, whereas lymphocyte concentrations were reduced (P ≤ 0.05), followed by increased WBC, lymphocyte, and neutrophil concentrations at 24 h compared with CON (P ≤ 0.05). On day 15, responses shifted to later time points, with increased WBC and neutrophil concentrations in HIGH-treated ewes at 12 and 24 h (P ≤ 0.05), a tendency for increased lymphocyte concentrations at 12 h, and increased lymphocyte concentrations at 24 h (P ≤ 0.05). Values are least squares means ± SEM. Asterisks (*) indicate differences among treatments within hour (P ≤ 0.05), and tendencies are denoted at 0.05 < P ≤ 0.10.

On d 10, WBC, lymphocyte, and neutrophil concentrations were affected by hour (P ≤ 0.002), with no differences among treatments. Treatment × hour interactions were observed for WBC percentages, with lymphocyte percentages increased at 2 h and decreased by 4 h in LOW-treated ewes, while neutrophil percentages exhibiting the opposite pattern. Because absolute WBC, lymphocyte, and neutrophil concentrations did not differ among treatments, these percentage changes likely reflect a transient change in relative leukocyte composition rather than a sustained alteration in circulating cell numbers.

On d 15, WBC, lymphocyte, and neutrophil concentrations were affected by hour (P ≤ 0.009), with treatment × hour interactions detected (P ≤ 0.03) and no main effect of treatment. At later time points, responses were primarily driven by HIGH-treated ewes, where total WBC and neutrophil concentrations were greater compared with CON at 12 and 24 h (P ≤ 0.05; Figure 3B, F), and lymphocyte concentrations were greater at 24 h (P ≤ 0.05; Figure 3D). These findings indicate that repeated LPS exposure altered the timing and magnitude of circulating leukocyte responses across challenge days. Previous work using this model detected increased TLR4 and IL-8 expression in PBLs following repeated LPS exposure (Austin et al. 2024). The present CBC data complement those findings by demonstrating concurrent changes in circulating leukocyte populations, although neither study identifies the contribution of individual cell populations to the previously observed gene-expression responses.

Red blood cell and platelet parameters remain largely stable during LPS challenge

Red blood cell (RBC) count, hemoglobin concentration, and hematocrit percentage were affected by hour across d 5, 10, and 15 (P ≤ 0.003), with no effects of treatment and no treatment × hour interactions detected (Figure 4). Although some within-hour differences were observed, these included greater hematocrit percentage in LOW and HIGH compared with CON at multiple time points on d 5 (Figure 4E), and increased RBC count and hematocrit in HIGH-treated ewes at early time points (2–4 h) on d 15 (Figure 4B, F), with a tendency for increased hemoglobin at 2 h (0.05 < P ≤ 0.10; Figure 4D). However, these differences were not protected by a treatment × hour interaction. Thus, repeated low-dose LPS exposure had little evidence of a sustained effect on RBC mass or oxygen-carrying capacity. Similar transient changes in RBC-related measures have been reported following acute LPS challenge in sheep (Yates et al. 2011).

Figure 4.

Red blood cell parameters in response to LPS challenge during the estrous cycle. Red blood cell (RBC) count (A, B), hemoglobin concentration (C, D), and hematocrit percentage (E, F) were evaluated in response to LPS challenge on day 5 (A, C, E) and day 15 (B, D, F) of the estrous cycle. Ewes (n = 28) received a subcutaneous injection of saline (CON), 1.5 µg LPS/kg BW (LOW), or 3.0 µg LPS/kg BW (HIGH). Whole blood samples for complete blood count (CBC) analysis were collected from a subset of ewes (n = 5 per treatment) at 0, 2, 4, 12, and 24 h following LPS challenge. On day 5, hematocrit percentage was greater in LOW and HIGH compared with CON at 0, 2, 12, and 24 h (P ≤ 0.05). On day 15, RBC count was greater in HIGH compared with CON at 2 and 4 h (P ≤ 0.05), with a tendency for increased hemoglobin at 2 h (0.05 < P ≤ 0.10) and greater hematocrit percentage at 2 h (P ≤ 0.05). No treatment × hour interactions were detected; within-hour differences are presented as pairwise comparisons. Values are least squares means ± SEM. Asterisks (*) indicate differences among treatments within hour (P ≤ 0.05), and tendencies are denoted at 0.05 < P ≤ 0.10.

Mean corpuscular volume differed among treatments (P ≤ 0.024); however, this difference was present before LPS administration and therefore was not attributed to treatment. Red blood cell distribution width was affected by hour on d 5 and 10 (P ≤ 0.004), with no treatment effects detected. Collectively, these findings indicate that RBC morphology parameters were not meaningfully altered by repeated endotoxin exposure.

Platelet count was affected by hour across all sampling days (P ≤ 0.004), with no overall treatment effect detected. Within-hour differences were observed, particularly in HIGH-treated ewes on d 10, where platelet counts were greater compared with CON at 4, 12, and 24 h (P ≤ 0.05). These increases likely reflect short-term inflammatory activation rather than sustained changes in platelet production, as platelets actively participate in immune responses through interactions with WBCs and the release of cytokines (Semple et al. 2011). Previous LPS studies in sheep have likewise reported limited or inconsistent platelet responses despite clear systemic and leukocyte responses (Yates et al. 2011; Riquelme et al. 2018). Therefore, leukocyte dynamics appeared to be a more responsive hematological indicator of the repeated low-dose challenge used in the present study.

Reproductive performance is maintained despite LPS-induced inflammation

Ewe reproductive performance and lamb production outcomes are presented in Table 1 and were not affected by maternal LPS treatment. Maternal LPS treatment did not alter first- or second-service conception rates, time to estrus, remounting rate, or overall pregnancy rate (P ≥ 0.96). Lamb production traits were also not affected by maternal treatment. Birth weight (P = 0.43), adjusted 60-d weaning weight (P = 0.35), and average daily gain (P = 0.27) were similar among treatments. Sex influenced birth weight, with male lambs heavier than females (P = 0.005), while twinning rate did not differ among treatments (P = 0.85).

Table 1.

Ewe reproductive performance and lamb production outcomes.

	Treatment1
Parameter	CON	LOW	HIGH	SE	P-value
Reproductive data				–	–
Ewes	10	8	10	–	–
Ewes that lambed	10	8	10	–	–
1st service conception2	90	87.5	90	–	0.98
2nd service conception3	10	12.5	10	–	0.98
Time to estrus (d)4	3.1	2.75	3.1	0.95	0.96
Remounted (%)5	10	12.5	10	–	0.98
Pregnancy rate6	100	100	100	–	–
Production data				–	–
Lamb born (n)	15	12	17	–	–
Twin births (%)	40	37.5	50	–	0.85
Males	8	6	10	–	–
Females	7	6	7	–	–
Birth weight, kg	5.2	4.8	5.3	0.27	0.43
Adj. weaning wt7 kg	21.6	23.7	23.5	1.04	0.35
Adj. ADG8 kg/d	0.24	0.28	0.25	0.01	0.27

Parameter definitions:

¹Ewes received saline (CON), 1.5 µg/kg BW lipopolysaccharide (LOW), or 3.0 µg/kg BW LPS (HIGH).

²Percentage conceiving at first service.

³Percentage conceiving at second service.

⁴Time to estrus defined as interval from ram introduction to initial estrus detection based on first raddle marking.

⁵Percentage of ewes remarked during the breeding season.

⁶Percentage pregnant after a 35-d breeding season.

⁷Adjusted 60-d weaning weight.

⁸Average daily gain calculated from adjusted 60-d weaning weight.

Despite measurable physiological and hematological responses to LPS, reproductive performance was maintained. Differences among studies may partly reflect the source and route of endotoxin exposure. Naturally occurring LPS may originate within the reproductive tract, as occurs with gram-negative uterine disease, or from the gastrointestinal tract following disruption of barrier function, resulting in different patterns of local and systemic exposure (Khafipour et al. 2009; Sheldon et al. 2009; Magata et al. 2014). Experimental models also range from direct exposure of granulosa cells or oocytes in vitro (Herath et al. 2007; Castro et al. 2024) to intravenous administration, which can produce pronounced endocrine and reproductive effects (Battaglia et al. 2000; Suzuki et al. 2001). Consequently, the site, magnitude, and kinetics of LPS exposure likely contribute to variation in reproductive outcomes among studies.

The repeated low-dose subcutaneous challenge used here represents a systemic but comparatively mild exposure. Importantly, Ferranti et al. (2022) demonstrated that repeated subcutaneous LPS administration can increase LPS within dominant follicular fluid, indicating that this route can influence the ovarian environment. Therefore, the absence of impaired fertility in the present study does not necessarily indicate an absence of reproductive exposure or biological responsiveness. Rather, the magnitude and kinetics of the inflammatory challenge may have been sufficient to elicit systemic responses while remaining below the threshold required to measurably impair ovulation, conception, or pregnancy establishment. These findings emphasize that reproductive consequences of LPS exposure likely depend not only on LPS exposure itself, but also on its source, route, dose, and duration.

Maternal LPS exposure alters early life immune profiles in lambs

Across all variables, responses were primarily affected by day (P ≤ 0.006), reflecting normal postnatal changes in circulating immune cell populations following birth. Treatment × day interactions were observed for total WBC and neutrophil concentrations (P ≤ 0.03), with a tendency observed for lymphocytes (P = 0.06; Figure 5), indicating that maternal LPS exposure influenced the temporal pattern of immune cell responses. At birth (d 0), lambs born to LOW-treated ewes had greater total WBC, lymphocyte, and neutrophil concentrations compared with CON and HIGH (P ≤ 0.05; Figure 5A–C). This response was accompanied by reduced lymphocyte percentage in LOW lambs, indicating a greater relative contribution of neutrophils to circulating WBC. Because both lymphocyte and neutrophil counts increased at birth, the lower lymphocyte percentage reflects a shift toward neutrophils rather than a decrease in lymphocytes. Treatment effects were not limited to birth. Neutrophil concentrations were greater in LOW lambs at postnatal d 14 (P ≤ 0.05; Figure 5B), whereas differences in total WBC and lymphocyte concentrations were largely confined to birth. These findings suggest that maternal inflammatory exposure before breeding was associated with altered early-life immune profiles in lambs.

Figure 5.

Hematological parameters of lambs born to ewes exposed to LPS during the periconception period. Total white blood cell (WBC) concentrations (A), neutrophil concentrations (B), lymphocyte concentrations (C), red blood cell (RBC) count (D), hemoglobin concentration (E), and hematocrit percentage (F) in lambs born to ewes challenged with lipopolysaccharide (LPS) during the periconception period. Ewes received a subcutaneous injection of saline (CON), 1.5 µg LPS/kg BW (LOW), or 3.0 µg LPS/kg BW (HIGH) on days 5, 10, and 15 of the estrous cycle prior to breeding. Blood samples were collected from lambs born to these ewes at days 0, 7, 14, 28, and 60 of age (day 0 = birth). (A–C) At birth (day 0), lambs born to LOW-treated ewes exhibited greater WBC, lymphocyte, and neutrophil concentrations compared with CON and HIGH (P ≤ 0.05). Neutrophil concentrations were also greater in LOW lambs on day 14 (P ≤ 0.05). No consistent treatment differences were observed at later time points beyond day 14. (D–F) No effects of maternal treatment were detected for RBC count, hemoglobin concentration, or hematocrit across days of age. Values are least squares means ± SEM. Asterisks (*) indicate differences among treatments within day (P ≤ 0.05), and tendencies are denoted at 0.05 < P ≤ 0.10.

Because LPS challenges occurred during the estrous cycle before ram exposure, the mechanism linking maternal treatment to lamb hematological differences cannot be determined from the present study. One possible explanation is that inflammatory exposure during follicular development altered the oocyte, early embryo, or maternal reproductive environment in a manner that influenced offspring hematological profiles. Previous studies have demonstrated that inflammatory exposure during early development can affect later developmental outcomes; however, the present study did not evaluate epigenetic mechanisms nor functional immune responses. The findings therefore demonstrate an association between maternal periconceptional LPS exposure and altered neonatal hematology but do not establish a specific developmental-programming mechanism or altered immune competence.

Nevertheless, the transient neonatal differences are biologically relevant because they occurred despite unchanged reproductive performance and growth. Gestational immune-challenge studies have similarly shown that maternal inflammatory exposure can influence later offspring immune-related phenotypes, including altered responses to subsequent LPS challenge in cattle (Carroll et al. 2021). Although the timing of exposure differs substantially from the present study, these observations support further investigation of whether the hematological differences observed here correspond to altered immune responsiveness later in life.

In contrast to the treatment effects observed in WBC populations, RBC parameters were not influenced by maternal LPS exposure. Lamb RBC parameters were primarily affected by day (P < 0.0001), reflecting normal postnatal changes in RBC dynamics (Figure 5D–F). No effects of treatment or treatment × day interactions were detected for RBC count, hemoglobin concentration, hematocrit, or RBC morphology parameters (P ≥ 0.08), and platelet differences were limited and inconsistent across sampling days. Thus, among the hematological variables examined, leukocyte populations appeared to be the offspring phenotype most responsive to maternal periconceptional LPS exposure.

Pre-weaning growth and morphometric development are not affected by maternal LPS exposure

Lamb growth and morphometric measurements, including body weight, heart girth, wither height, body length, hind leg circumference, head width, head length, and total body length, increased across days (P < 0.0001), reflecting normal postnatal growth. Sex influenced several traits, with male lambs exhibiting greater heart girth and wither height compared with females (P = 0.03), while head width was also reduced in females. No main effects of maternal treatment were detected for any growth or morphometric variable (P ≥ 0.75), although heart girth and head length exhibited tendencies for treatment × day interactions (P = 0.06 and P = 0.07, respectively). At birth (d 0), heart girth was lower in LOW compared with CON and HIGH (P ≤ 0.05), and on d 14, head length was greater in HIGH compared with CON (P ≤ 0.05). These differences were not maintained across subsequent time points and did not reflect consistent alterations in growth trajectories.

These findings indicate that pre-weaning growth and morphometric development were driven primarily by age and sex rather than maternal inflammatory exposure. This is consistent with previous work demonstrating that lamb growth during early life is largely influenced by age, sex, and birth-related factors, which significantly affect body weight and morphometric traits (Afolayan et al. 2006). Importantly, the absence of broad growth effects occurred alongside detectable differences in neonatal leukocyte profiles, suggesting that responses to maternal inflammatory exposure may be trait-specific rather than expressed as generalized developmental impairment. Other gestational inflammatory models have similarly reported selective offspring effects in the absence of substantial changes in early body weight or growth (Henrique et al. 2020; Johnson et al. 2024). The present findings extend this concept to a periconceptional challenge and indicate that early hematological outcomes may be more sensitive than gross growth measures to this level of maternal inflammatory exposure.

Wool characteristics are minimally influenced by maternal LPS exposure

Wool characteristics were largely unaffected by maternal treatment (Table 2). Average fiber diameter, a primary determinant of wool quality, did not differ among treatments (P = 0.14). Measures of fiber uniformity, including the standard deviation and coefficient of variation of fiber diameter, tended to differ among treatments (P = 0.06), with coefficient of variation lower in HIGH compared with CON, whereas LOW did not differ from either group. Minimum fiber diameter (P = 0.18), maximum fiber diameter (P = 0.17), fibers less than 15 μm (P = 0.14), spinning fineness (P = 0.10), comfort factor (P = 0.31), and fiber curvature (P = 0.55) were not affected by treatment.

Table 2.

Wool fiber characteristics of ewe lamb offspring at weaning (day 60).

Parameter	Control	Low LPS	High LPS	P-value
Mean fiber diameter (µm)1	17.61 ± 0.26	17.89 ± 0.33	17.07 ± 0.33	0.14
Spinning fineness (µm)2	16.88 ± 0.26	17.09 ± 0.33	16.25 ± 0.26	0.10
SD fiber diameter (µm)3	3.36 ± 0.07	3.32 ± 0.09	3.13 ± 0.07	0.06
CV fiber diameter (%)4	19.12 ± 0.26	18.60 ± 0.33	18.19 ± 0.26	0.06
Minimum fiber diameter (µm)5	17.11 ± 0.28	17.40 ± 0.35	16.59 ± 0.28	0.18
Maximum fiber diameter (µm)6	18.53 ± 0.27	18.50 ± 0.27	17.86 ± 0.34	0.17
Fibers <15 µm (%)7	19.71 ± 1.9	15.34 ± 2.4	21.51 ± 1.9	0.14
Curvature (deg/mm)8	86.32 ± 2.0	87.39 ± 2.5	89.41 ± 2.0	0.55
SD curvature9	64.81 ± 1.8	67.69 ± 2.3	67.56 ± 1.8	0.49
Comfort factor (%)10	99.81 ± 0.07	99.92 ± 0.08	99.96 ± 0.07	0.30

Parameter definitions:

¹Mean fiber diameter (µm), average diameter of individual wool fibers.

²Spinning fineness (µm), calculated estimate of fiber fineness accounting for fiber diameter distribution.

³SD fiber diameter (µm), standard deviation of fiber diameter, representing within-sample variability.

⁴CV fiber diameter (%), coefficient of variation of fiber diameter, indicating fiber uniformity.

⁵Minimum fiber diameter (µm), lowest observed fiber diameter within the sample.

⁶Maximum fiber diameter (µm), highest observed fiber diameter within the sample.

⁷Fibers <15 µm (%), proportion of fibers with diameters less than 15 µm.

⁸Fiber curvature (deg/mm), average curvature of wool fibers.

⁹SD curvature, standard deviation of fiber curvature.

¹⁰Comfort factor (%), proportion of fibers ≤30 µm, representing expected skin comfort.

These findings indicate that maternal LPS exposure did not alter primary determinants of wool quality. Although a tendency for reduced variation in fiber diameter was observed in HIGH, this effect was limited to fiber uniformity and was not accompanied by changes in mean fiber diameter, suggesting limited biological impact. Wool follicle development occurs during fetal life and can be influenced by maternal physiological conditions. Previous work has shown that more severe or prolonged physiological stress can impair wool growth and fiber integrity, particularly under conditions associated with elevated cortisol and sustained inflammatory activation (Colditza et al. 2005). Together with the lack of effects on growth and morphometric development, these findings indicate that the magnitude and duration of the maternal inflammatory challenge were insufficient to produce measurable changes in major production-related offspring traits through weaning.

Conclusion

Repeated low-dose LPS exposure during the periconception period elicited clear maternal inflammatory and endocrine responses without compromising reproductive performance or lamb growth. However, altered leukocyte profiles in offspring during the first two weeks of life indicate that even mild maternal inflammation before conception may influence early immune development. The absence of corresponding effects on growth, morphometric development, or wool characteristics suggests that offspring immune function may be more sensitive than production traits to periconceptional inflammatory exposure. Although these immune differences were transient, they provide evidence that maternal inflammatory events before breeding can influence offspring physiology and warrant further investigation into their consequences for later immune competence and health.

Glossary

Abbreviations

BW

body weight

CBC

complete blood count

CIDR

controlled internal drug release device

FSH

follicle-stimulating hormone

IL-8

interleukin-8

LH

luteinizing hormone

LPS

lipopolysaccharide

PBL

peripheral blood leukocyte

RBC

red blood cell

TLR

toll-like receptor 4

WBC

white blood cell

Funding

This work was supported by the USDA National Institute of Food and Agriculture Hispanic Serving Education Grant (2022-77040-37622). The publication fees for this article were supported in part by the NMSU Open Article Fund.

Author contributions

J.F. Dominguez Arellanes (Conceptualization, Methodology, Investigation, Formal analysis, Writing–original draft, Writing—review & editing), K.L. Luna (Investigation, Writing—review & editing), S.C. Gurule (Investigation, Writing—review and editing), S.A. Navarro-Soto (Formal analysis, Writing—review & editing), C.A. Gifford (Formal analysis, Writing—review & editing), R.L. Gibbs (Formal analysis, Writing—review & editing), J.A. Hernandez Gifford (Conceptualization, Methodology, Formal analysis, Funding acquisition, Supervision, Writing—review & editing)

Conflict of interest statement. The authors declare no real or perceived conflicts of interest.