Characterization of breeding success and litter characteristics in the New Mexico Alcohol Research Center moderate drinking paradigm

Abstract

Rodent models of prenatal alcohol exposure are a powerful tool for studying causal factors of prenatal exposure effects. Under the New Mexico Alcohol Research Center, we combined a voluntary drinking-in-the-dark (DID) paradigm with a Volumetric Drinking Monitor (VDM) system to quantify maternal ethanol (EtOH) intake, drinking duration, and drinking patterns throughout gestation and determined whether these variables altered breeding success or early offspring development. Female C57BL/6J mice received daily 2-h access to either 20% EtOH or water using the VDM system. Following a two-week acclimation period, breeding was initiated while daily drinking continued throughout gestation until parturition. Breeding success, litter characteristics, and early growth were evaluated from 176 dam-sire pairings across three breeding cohorts, yielding over 800 offspring for demographic analyses. Maternal EtOH exposure did not affect pregnancy success, litter loss, litter size, pup weight at postnatal day 10, offspring sex distribution, or weaning weight. Average daily gestational EtOH consumption and duration of preconception EtOH exposure did not predict offspring outcomes. Although EtOH-exposed dams consistently exhibited front-loading behavior, neither front-loading status nor front-loading intensity was associated with litter characteristics or early offspring growth. Moderate voluntary maternal EtOH consumption, duration of exposure, or drinking pattern did not drive litter characteristics that may affect the early-life environment (pup number and sex) or early growth markers (P10 and weaning weight). These data indicate that differences in the early postnatal environment are not likely to be responsible for long-term behavioral and neurobiological phenotypes reported in offspring produced using this paradigm.

1. Introduction

Since the first reports that developmental alcohol exposure could have severe and long-lasting consequences on the neurobehavior of offspring (Jones, 1975; Jones & Smith, 1973), there has been a consistent focus on identifying how alcohol affects the developing fetus. Even 50 years later, prenatal alcohol exposure (PAE) remains common (Day et al., 2002; Green et al., 2009; Thomas et al., 1998), with recent studies reporting that between 5 and 10% women binge drink (Ethen et al., 2009) and as many as one-third of women drink at some point during pregnancy (Gosdin et al., 2022). A growing consensus demonstrates that even moderate alcohol intake during pregnancy leads to lasting cognitive impairments. These impairments, categorized as Fetal Alcohol Spectrum Disorders (FASDs), are often not diagnosed until late childhood or early adolescence and are characterized by impairments in executive function, working memory, response inhibition, and behavioral flexibility (Green et al., 2009; Mattson et al., 1999; Streissguth et al., 1991). Rodent models have become an important tool for studying the developmental effects of alcohol exposure, particularly as studies in human patients and rodent models suggest a congruent effect of blood alcohol concentration (BAC) on behavioral outcomes across species (Driscoll et al., 1990).

Preclinical models are powerful tools for studying causal factors of prenatal exposure effects. The use of genetically homogeneous populations of inbred mice can circumvent the issue of genetic heterogeneity in epidemiological studies, making it more feasible to parse the effects of environmental factors. Critically, environmental insults, such as alcohol, can be strictly timed and given in exact quantities, enabling the discovery of sensitive time windows and thresholds of harmful doses. The impacts of PAE in mouse models can be tested on multiple levels, including neuroanatomical, neurochemical, neuronal, and behavioral, enabling the discovery of the effects of certain environmental insults on the whole organism (Marquardt & Brigman, 2016).

The New Mexico Alcohol Research Center (NMARC) was founded in 2014 with the goal of advancing our understanding of the teratogenic consequences of PAE. As part of its aim to develop more effective diagnostic approaches and neurobehavioral, educational, and pharmacotherapeutic interventions for individuals with FASD, the center consolidated PAE mouse production into a Scientific Core to standardize it. After repeated refinement of the initial bottle-based drinking-in-the-dark (DID) procedure, the NMARC added a Volumetric Drinking Monitor (VDM) system to our DID model in 2024 to more accurately quantify both total ethanol intake and drinking patterns in dams. Using this paradigm, we demonstrated that maternal drinking patterns—including the timing, rate, and distribution of ethanol intake during gestation—can be more predictive of offspring outcomes than total ethanol consumption alone (Myrick et al., 2024). By continuously monitoring drinking behavior throughout pregnancy, that study identified associations between specific maternal drinking patterns and offspring neurobehavioral phenotypes, including motor performance, locomotor activity, and sensory processing. Notably, cumulative gestational ethanol exposure was not itself associated with these outcomes, highlighting the importance of characterizing detailed drinking behaviors rather than relying solely on total intake (Myrick et al., 2024). These findings suggest that variation in maternal drinking patterns may also influence reproductive outcomes, breeding success, and offspring viability. To address this question, the current study leverages the same NMARC moderate ethanol exposure paradigm to examine how average ethanol consumption, drinking duration, and drinking patterns relate to breeding success, litter characteristics, and postnatal growth across 176 dams and more than 800 offspring.

2. Methods

Animals:

8-week-old male and female C57BL/6J mice were obtained from The Jackson Laboratories (JAX) to be used as breeders to produce prenatally alcohol exposed (EtOH) and control (Water) subjects. The three breeding rounds consisted of 176 dam/sire pairings (VDM1 = 64; VDM2 = 48; VDM3 = 64). Upon arrival, mice were single housed in 6 × 10 × 5 inch amber cages with wire top food hoppers and standard water bottles. Mice were acclimated to the room for two weeks under a reverse 12-h light/dark cycle (8 a.m. lights on/8 p.m. lights off) before study initiation. All animals had ad lib access to LabDiet 5LOD (LabDiet, Gray Summit, MO), a mouse chow previously shown to support reliable EtOH intake (Myrick et al., 2024). Water was always available except for 2-h a day in EtOH exposed mice (see below). All procedures were approved by the University of New Mexico HSC Institutional Animal Care and Use Committee and conformed to the Guidelines for the Care and Use of Mammals in Neuroscience and Behavioral Research (National Research Council, 2003).

Drinking-in-the-dark:

Animals were provided either 20% EtOH or Water via a VDM system (Columbus Instruments, Columbus OH) using standard DID procedures (Linsenbardt & Boehm, 2014; Myrick et al., 2024). Briefly, 3 h into the dark cycle, standard water bottles were replaced daily with specialized sippers for 2 h. This resulted in a total of 720 10-s drinking data bins collected (7200 s * 60 s = 120 min) per mouse daily. Water for all sipper drinking solutions came from the same source within the UNM HSC Animal Resource Facility as used for standard housing water bottles (Fig. 1A).

Fig. 1. Experimental Timeline and maternal ethanol exposure does not alter time-to-pregnancy.

Dams underwent room acclimation, Drinking-in-the-Dark (DID) acclimation, breeding, gestation, and postnatal assessments (A). Daily ethanol access was provided beginning with DID acclimation and terminated following parturition. Dams were weighed daily after sire pairing to assess conception-related weight gain to retroactively determine a successful breeding round. Litters were assessed at postnatal day 10 (P10) for litter size and weight and at weaning (~P23) for sex determination and weights. Time-to-pregnancy did not differ between Water (H₂O) or Ethanol (EtOH) dams (B).

Breeding:

Following a two-week DID acclimation period, each female was housed with a male for 3 h immediately after daily sipper removal each day for three consecutive days. Females were removed from male cages at least an hour before lights on. Dams were reintroduced to sires every 5 days following the initial pairing for one 3-h session until the dam’s weight increased by 25%. Following birth, dates were back-calculated to determine the breeding round and sire that led to pregnancy. Dams who gained ≥25% of their total weight and then decreased ≥10% of that weight were determined to have reabsorbed the pregnancy. 15 days following confirmation of pregnancy, dams were monitored twice daily for presences of a litter and each litter born recorded. Daily drinking sessions were continued for the entirety of gestation and terminated as soon as a dam gave birth (Fig. 1A).

Litter Characterization:

Litters were left undisturbed for ten days, at which time litter size was determined. In addition, any pups found dead were recorded, as well as any instances of full litter cannibalization. Litters were weighed on postnatal day 10 (P10), and individual pups were weighed at weaning (approximately P23). The numbers of male and female offspring were determined at weaning. A unique, non-descriptive identifier was assigned to each offspring at weaning before distribution to individual projects. All records associated with an individual mouse were maintained by the Scientific Core. This information included: dam identification, maternal ethanol consumption, maternal weight gain, litter characteristics (pup and sex numbers, litter weight), weaning date, offspring information (weights, notes regarding health), any recorded environmental variations, and assignment of mice to investigators (Fig. 1A).

Statistical Analyses:

All analyses were conducted in R (version 4.5.2, 2025–10-31) using RStudio (version 2026.1.1.403). Statistical significance was determined with a Bonferroni-corrected significance threshold to account for multiple hypothesis testing. With 26 comparisons, the alpha level was adjusted to α_corrected = 0.05/26, making p < 0.002 required for significance. Data were visually assessed for outliers and distributional assumptions before analysis. Normality was assessed using Q-Q plots and Shapiro-Wilk tests when appropriate. Homogeneity of variances was assessed via residual-versus-fitted plots. Statistical tests used are described in the appropriate Results sections. Results are reported as mean ± standard deviation unless otherwise noted. Estimated effect sizes and 95% confidence intervals are reported where applicable.

3. Results

Since 2024, the Scientific Core has conducted 3 breeding rounds using the VDM System (NMARC VDM1–3), yielding over 800 offspring. Round size varied based on the investigator’s need, but incorporated either 48 or 64 dams.

VDM1 (NMARC19):

63 dams (1 dam was lost prior to the study start) were paired across 8 breeding rounds, for a total of 54 pregnancies. Of confirmed pregnancies, 46 litters were weaned for a total of 316 pups. Average litter size was 6.59 ± 2.29 pups, with EtOH exposed dams averaging 6.65 ± 2.04 pups per litter and Water exposed averaging 6.54 ± 2.53 pups. Of weaned litters, on average 3.26 ± 1.57 pups were sexed male and 3.61 ± 1.67 were female. The average gestational intake for EtOH exposed dams was 3.24 ± 0.82 g/kg. Descriptive breeding and offspring metrics are summarized in Table 1.

Table 1.

Summary of breeding outcomes and offspring characteristics across 3 volumetric drinking monitor (VDM) cohorts.

Characteristic	VDM1 (NMARC19)	VDM2 (NMARC20)	VDM3 (NMARC21)
Study Design
Dams enrolled	63a	46b	64
H2O dams	31	23	32
EtOH dams	32	23	32
Maximum breeding rounds	8	6	5
Breeding Outcomes
Total pregnancies	54	36	44
H2O pregnancies	28	18	23
EtOH pregnancies	26	18	21
Weaned litters	46	36	39
H2O weaned litters	24	18	20
EtOH weaned litters	22	18	19
Total offspring	316	256	251
Offspring Characteristics
Overall litter size (mean ± SD)	6.59 ± 2.29	7.31 ± 1.85	6.53 ± 1.70
H2O litter size (mean ± SD)	6.54 ± 2.53	7.44 ± 1.85	6.61 ± 1.73
EtOH litter size (mean ± SD)	6.65 ± 2.04	7.17 ± 1.89	6.45 ± 1.70
Male offspring per litter (mean ± SD)	3.26 ± 1.57	4.00 ± 1.39	3.46 ± 1.57
Female offspring per litter (mean ± SD)	3.61 ± 1.67	3.11 ± 1.62	2.97 ± 1.53
Mean gestational EtOH intake (g/kg/day)c	3.24 ± 0.82	3.18 ± 0.77	3.04 ± 1.00

Note.

^aOne dam was lost before study initiation.

^bTwo dams were lost before study initiation.

^cCalculated from ethanol-exposed dams only.

VDM2 (NMARC20):

46 dams (2 dams were lost prior to study start) were paired across 6 breeding rounds, for a total of 36 pregnancies. Of confirmed pregnancies, 36 litters were weaned for a total of 256 pups. Average litter size was 7.31 ± 1.85 pups, with EtOH exposed dams averaging 7.17 ± 1.89 pups per litter and Water exposed averaging 7.44 ± 1.85 pups. Of weaned litters, on average 4.00 ± 1.39 pups were sexed male and 3.11 ± 1.62 were female. The average gestational intake for EtOH exposed dams was 3.18 ± 0.77 g/kg. Descriptive breeding and offspring metrics are summarized in Table 1.

VDM3 (NMARC21):

64 dams were paired across 5 breeding rounds, for a total of 44 pregnancies. Of confirmed pregnancies, 39 litters were weaned for a total of 251 pups. Average litter size was 6.53 ± 1.70 pups, with EtOH-exposed dams averaging 6.45 ± 1.70 pups per litter and water-exposed averaging 6.61 ± 1.73 pups. Of weaned litters, on average, 3.46 ± 1.57 pups were sexed male and 2.97 ± 1.53 were female. The average gestational intake for EtOH-exposed dams was 3.04 ± 1.00 g/kg. Descriptive breeding and offspring metrics are summarized in Table 1.

PAE impact on breeding and offspring:

Overall, over 800 total pups were produced from 176 dam-sire pairings. 62 offspring were used in experiments prior to weaning, and 15 pups were lost prior to weaning, leaving 823 offspring for demographic analysis. Kaplan-Meier survival analysis revealed that treatment did not significantly affect time-to-pregnancy, measured as percentage of pregnancies confirmed per breeding round (log-rank χ²(1) = 0.93; p = 0.34; Fig. 1B). Cox proportional hazards regression similarly indicated no effect of treatment (HR = 0.86, p = 0.37). Total proportion of dams confirmed as pregnant did not significantly differ between Water and EtOH groups (83.7% vs 74.7%, respectively; 2 proportion Z test; z = 1.62, p = 0.20; Fig. 2A). Treatment did not significantly affect litter loss, measured by reabsorption (EtOH = 0/65, H₂O = 3/72; Fisher’s exact test, p = 0.25; Fig. 2B), offspring cannibalization (EtOH = 2/65, H₂O = 2/72; Fisher’s exact test, p = 1.00; Fig. 2B) or litters exhibiting single pup loss (EtOH = 7/65, H₂O = 8/72; Fisher’s exact test, p = 1.00; Fig. 2B). Treatment had no significant effect on litter size at P10 (Welch’s t (128) = 0.52, p = 0.60; EtOH = 6.84 ± 1.70, H2O = 7.00 ± 1.79; Fig. 2C). Average pup weight at P10 was normalized at the litter level and natural log-transformed to satisfy Welch’s t-test normality assumptions. Welch’s t-test revealed a nonsignificant effect of treatment on average pup weight at P10 (Welch’s t(122) = −1.87, p = 0.063; Fig. 2D). Similarly, treatment did not significantly affect offspring sex distribution (EtOH: 188F/208M; H₂O: 206F/221M; 2 proportion Z test; z = 0.02, p = 0.88; Fig. 2E). A linear mixed-effects model revealed a main effect of sex, with male offspring weighing more than females at weaning (β = 0.571 g, t(112.4) = 5.33; p < 0.001); however, neither EtOH exposure (β = 0.079 g; t(148.5) = 0.36; p = 0.717) nor the interaction of sex and treatment (β = −0.067 g; t(112.2) = −0.44, p = 0.660) significantly affected offspring weight at weaning (Fig. 2F).

Fig. 2. Maternal ethanol exposure does not influence pregnancy outcomes, litter characteristics, or offspring growth.

Dams exposed to either water (H₂O) or Ethanol (EtOH) did not differ from each other for pregnancy rate (A), litter loss outcomes (B), litter size at postnatal day 10 (P10; C), average pup weight at P10 (D), offspring sex distribution (E), or average pup weight at weaning (F). Male offspring weighed more than female offspring at weaning, regardless of treatment. Data in panels A and F are presented as means with 95% confidence intervals. Panels C, D, and F display individual litter/offspring observations. ***p < 0.001, main effect of Sex.

Impact of average gestational EtOH consumption on offspring:

We next analyzed how maternal average daily drinking behavior affected pup outcomes. Linear regression testing showed that average EtOH consumption across gestation did not predict litter size (β = −0.003 pups per g/kg EtOH consumed; t(61) = −0.014, p = 0.989, R² < 0.001; Fig. 3A). Similarly, average pup weight at P10 was not predicted by average EtOH consumption during gestation (β = 0.125 g per g/kg EtOH consumed; t(57) = 1.80; p = 0.077; R² = 0.054; Fig. 3B). Sex distribution of litters was not significantly predicted by maternal EtOH consumption, as a binomial generalized linear model did not reveal any association between average maternal EtOH consumption and offspring sex ratio (β = −0.097; z = −0.856; p = 0.392; Fig. 3C). Finally, we tested whether average maternal EtOH consumption during gestation predicted offspring weight at weaning for male and female pups using a linear mixed-effects model with dam identity included as a random effect. The linear mixed model revealed no association between average maternal EtOH consumption and offspring weaning weights (β = −0.004; t (73.35) = −0.023; p = 0.982). Additionally, this relationship did not differ significantly between male and female offspring (β = 0.198, t (51.94) = 1.422; p = 0.161; Fig. 3D).

Fig. 3. Average daily ethanol consumption does not influence litter characteristics or offspring growth.

Variations in average daily ethanol consumption among ethanol (EtOH) exposed dams were not associated with litter size at postnatal day 10 (P10; A), average pup weight at P10 (B), offspring sex distribution (C), or average pup weight at weaning (D). Individual litter (A, C) and offspring (B, D) observations displayed as scatterplot points with regression lines and 95% confidence intervals.

Impact of time-to-conception and drinking duration on offspring:

We next assessed if increased duration of pre-conception EtOH exposure, estimated by breeding round at successful conception, was associated with pup outcomes. Treatment was included in all models to account for potential confounding factors such as dam age and stress associated with multiple sire pairings. Because breeding rounds 7 and 8 in VDM-1 contained very few litters, sensitivity analyses were performed using data from breeding rounds 1 through 6. Restricting the dataset did not alter the interpretation of any model, indicating that the primary findings were not driven by late sparse breeding rounds. Using linear regression, breeding round did not predict litter size at P10 (β = 0.279 pups per breeding round; t(124) = 1.82; p = 0.071). Similarly, neither treatment (β = −0.075, p = 0.902) nor the interaction of treatment and breeding round (β = −0.021, p = 0.919) significantly influenced litter size (Fig. 4A). Additionally, linear regression revealed that breeding round did not predict average pup weight at P10 (β = −0.063 g per breeding round; t(119) = −1.31; p = 0.194). There were no effects of treatment (β = −0.254, p = 0.171) or the interaction of treatment and breeding round (β = −0.039, p = 0.536; Fig. 4B). Binomial generalized linear modeling was used to assess the offspring sex ratio, which was not associated with the number of breeding rounds (β = 0.045; z = 0.602; p = 0.547). Mirroring other analyses, treatment (β = 0.240; p = 0.421) and interaction of treatment and breeding round (β = −0.079; p = 0.418) were not significant (Fig. 4C). Finally, linear mixed-effects modeling was used to assess whether treatment and/or breeding round predicted sex-specific weaning weight, while controlling for dam identity as a random effect. Breeding round did not predict offspring weaning weight (β = 0.099 g per breeding round; t(150.93) = 0.846; p = 0.399). No effects of treatment (β = 0.111; p = 0.804), treatment-by-breeding round interaction (β = −0.020; p = 0.896), the breeding round-by-sex interaction (β = −0.007; p = 0.930), or the treatment-by-breeding round-by-sex interaction (β = −0.003; p = 0.977) were detected (Fig. 4D).

Fig. 4. Duration of preconception does not influence litter characteristics or offspring growth.

Breeding round of successful conception was not associated with litter size at postnatal day 10 (P10; A), average pup weight at P10 (B), offspring sex distribution (C), or average pup weight at weaning (D) in either Water (H₂O) or Ethanol (EtOH) exposed dams. Individual litter (A, C) and offspring (B, D) observations displayed as scatterplot points with regression lines and 95% confidence intervals.

Impact of front-loading behavior on offspring:

Implementation of the VDM system enables assessment of maternal drinking patterns. One such pattern previously shown to impact pup outcomes, front-loading, was defined as consumption of 12.5% or more of total daily EtOH intake within the first 15 min of access (Myrick et al., 2024). EtOH drinking dams reliably developed front-loading behavior, with 92.06% identified as front-loading after two-weeks of DID acclimation. In contrast, only 8.96% of Water drinking controls developed front-loading behavior. EtOH dams also frontloaded more than Water controls, with an average of 26.92% of total drinking volume in the first 15 min versus 15.79% in controls. To assess the impact of front-loading on offspring outcomes, we first assessed whether EtOH exposed dams who were identified as front-loaders had different offspring outcomes from those who were not front-loaders. Front-loading behavior did not predict litter size at P10 (Linear regression; β = 0.262 pups; t(61) = 0.329; p = 0.743; R² = 0.002), average pup weight at P10 (Linear regression; β = −0.175 g; t(59) = −0.493; p = 0.624; R² = 0.004), offspring sex ratio (Binomial generalized linear model; β = 0.089; z = 0.243; p = 0.808) nor offspring weaning weights (Linear mixed-effects modeling; β = −0.110 g; t(74.10) = −0.199; p = 0.843). Additionally, linear mixed-effects modeling revealed no interaction between front-loading behavior and offspring sex (β = −0.016 g; t (51.92) = −0.036; p = 0.972).

We next analyzed whether front-loading intensity, quantified as the proportion of total EtOH intake consumed during the first 15 min of access, was associated with offspring outcomes among front-loading EtOH dams. Linear regression revealed that front-loading intensity did not predict litter size at P10 (β = −1.880 pups; t(56) = −0.767; p = 0.447; R² = 0.010; Fig. 5A). Additionally, linear regression showed that front-loading intensity did not predict average pup weight at P10 (β = 0.363 g; t(54) = 0.322; p = 0.749; R² = 0.002; Fig. 5B). Front-loading intensity was not predictive of offspring sex ratio in a binomial generalized linear model (β = −0.214; z = −0.187; p = 0.852; Fig. 5C). Finally, linear mixed-effects modeling demonstrated that front-loading intensity did not predict offspring weaning weight (β = −0.685; t(70.84) = −0.380; p = 0.705). Additionally, there was no interaction of weaning weights and offspring sex (β = −1.762; t(48.34) = −1.193; p = 0.239) or any differences by sex (β = 0.982; t(48.57) = 2.331; p = 0.024; Fig. 5D).

Fig. 5. Intensity of front-loading behavior does not influence litter characteristics or offspring growth.

Among ethanol (EtOH) exposed dams classified as front-loaders, front-loading intensity was not associated with litter size at postnatal day 10 (P10; A), average pup weight at P10 (B), offspring sex distribution (C), or average pup weight at weaning (D). Individual litter (A, C) and offspring (B, D) observations displayed as scatterplot points with regression lines and 95% confidence intervals.

4. Discussion

In the current analysis of three large breeding rounds conducted by the NMARC Scientific Core, we found no evidence that alcohol drinking had a significant impact on breeding success in dam/sire pairings. EtOH drinking dams did not differ from water controls on the overall rate of breeding success, or rate of success on subsequent dam/sire pairings. In addition, alcohol drinking did not significantly impact litter loss by reabsorption, cannibalization, or individual pup loss. Alcohol consumption also did not affect litter characteristics, as indicated by the average number of pups per litter or the weight of pups at P10 or at weaning. Similarly, alcohol-exposed litters did not differ in makeup from water controls, either in percentage of female vs. male offspring or weight of either sex at weaning, although on average, male pups of both treatments weighed significantly more than female pups at weaning. Taken together, these data suggest that previously reported changes in PAE offspring observed in adolescence and adulthood are unlikely to be due to gross changes in litter composition or pup size either during the third-trimester equivalent or at weaning (Myrick et al., 2024). Even with current VDM approaches, average daily intake during gestation varies among EtOH-exposed dams, suggesting that the total EtOH amount may alter litter characteristics and, therefore, the early postnatal environment. Therefore, we tested whether total EtOH consumption during gestation altered litter characteristics. Our results found that total average gestational intake did not significantly predict any litter features, including pup number, weight at P10, sex breakdown, or weight by sex at weaning. Given the need to conduct repeated dam/sire pairings, offspring from later breeding rounds were produced by dams with progressively longer preconception ethanol exposure. To examine the impact of this, we analyzed all our litter and pup characteristics by the breeding round in which pregnancy was confirmed. We showed that breeding round had no impact on pup number, weight at P10, sex ratio, or weaning weight for either sex. While total consumption time may have significant effects on later pup behavior, these changes are likely not due to the postnatal litter environment or early pup growth. Additionally, we found that the breeding success rate did not differ by treatment as breeding rounds increased, with EtOH and water control animals showing similar rates. We also show that breeding success did not significantly increase after 6 breeding rounds, suggesting that continued pairings are not beneficial.

Drinking-in-the-dark limited-access models have been used for decades to examine the impact of moderate-to-high developmental ethanol exposures on numerous systems and behavioral domains. Prior to the founding of the NMARC, studies using a precursor of the current VDM model (4-h limited access to 10% EtOH+ 0.066% (w/v) saccharin using traditional bottle drinking) showed that moderate PAE led to impairments in hippocampal-dependent delay fear conditioning and visuospatial discrimination (Allan et al., 2003; Brady et al., 2012a; Caldwell et al., 2014) am that PAE blunted increases in hippocampal neurogenesis seen under enriched housing conditions (Choi et al., 2005).

Following the establishment of the NMARC model, further studies showed that the moderate limited-access saccharine bottle-drinking exposure led to impairments in multiple domains of executive control including: impaired behavioral flexibility and altered corticostriatal activity and coordination (Marquardt et al., 2020) which were rescued via circuit-specific optogenetic stimulation (Licheri et al., 2025); altered cue-induced reinstatement (Olguin et al., 2019); impaired cognitive control and altered parietal power in PAE mice during choice (Olguin et al., 2023); and deficits in visuospatial learning and altered dentate granule cell integration (Kajimoto et al., 2013), spontaneous activity (Kajimoto et al., 2016) and response to enrichment as previously reported (Gustus et al., 2020). Concurrent studies examining putative mechanisms of these changes revealed that the moderate PAE model alters the expression and function of OFC GABAergic interneurons (Kenton et al., 2020), and decreases NMDA-evoked current density in OFC pyramidal neurons of male mice (Licheri et al., 2021). The NMARC model has also been shown to alter immune sensitization, with PAE offspring demonstrating sensitization of glial cells and increased production of their products, such as proinflammatory cytokines (Noor et al., 2017; Sanchez et al., 2017; Noor S, 2018). Moderate PAE has also been found to increase susceptibility to developing allodynia, with even minor nerve injury during adulthood.

Our current results suggest that the numerous behavioral, morphological, and functional impacts observed in PAE offspring from earlier bottle drinking, and now the VDM approach in the NMARC, cannot be attributed to EtOH-induced changes in the early postnatal litter environment or to gross changes in pup weight or growth. In addition, our breeding analysis suggests that EtOH does not significantly impact overall breeding success or rate of breeding success across dam/sire pairings. This agrees with early work from the NMARC demonstrating that our PAE model does not alter dam-pup interactions, as measured by time on the nest and pup retrieval time (Brady et al., 2012b). However, current ongoing NMARC breeding rounds are incorporating maternal behaviors, including pup-retrieval times and nest-building, to more clearly capture these features in our VDM approach.

Our newly implemented VDM approach decreases the total availability of alcohol daily (from 4 to 2 h) and also eliminates the need for saccharine in the alcohol and control fluids. This is a particularly important development, as we have recently shown that maternal saccharine intake can change the value of high-sugar rewards in offspring (Chandrasekaran et al., 2023). Recent VDM model studies examining maternal drinking suggest that average consumption and, critically, dam drinking pattern, can have effects on offspring (Myrick et al., 2024). Specifically, total alcohol consumption was found to drive decreases in locomotor activity in alcohol exposed females, whereas large intake at the start of the drinking session, or frontloading, drove a weight gain delay in male alcohol-exposed subjects that allowed them to perform better than their larger control peers (Myrick et al., 2024). These data suggest that, in addition to average drinking time, the pattern of drinking may be a critical factor in later pup outcomes. In the current study, we show that EtOH-drinking dams reliably develop front-loading behaviors over two weeks of drinking before breeding. Although the non-frontloading group comprised less than 10% of the total EtOH dams analyzed, we found no evidence that frontloading status affected any measured litter characteristic. In addition, our analysis of litter outcomes based on frontloading intensity found that the percentage of total drinking done in the first 15 min did not predict litter size, weights at P10, or weaning or sex distribution of pups. Together, these data suggest that, similar to total gestational intake, frontloading behavior’s impact on later pup growth and behaviors is not due to litter factors, initial pup size, or growth at weaning.

Over the previous 20+ years, researchers in the New Mexico Alcohol Research Center and its precursors have worked to refine and improve a limited-access moderate PAE model for examining the long-term impacts of alcohol exposure on offspring. Our current data show that EtOH consumption does not significantly impair breeding success, whether measured by average daily intake or total drinking time. Importantly, these factors do not grossly alter litter size, sex of offspring, size at P10 or pup growth till weaning. These data suggest that the early postnatal environment and pup growth are unlikely to account for the numerous effects reported in PAE offspring from early adolescence through adulthood. Ongoing work will provide further characterization of dam-pup interactions and examine how maternal drinking patterns may affect these early-life factors.

Data availability statement

All data presented in this manuscript is available upon request.