Prenatal heat stress during late gestation exerts long-term effects on growth and productivity of the dairy calf. Further, direct exposure to heat stress during the preweaning period impairs calf thermoregulation and performance. We examined the effects of heat stress abatement during the prenatal period, postnatal period, or both on calf performance. We hypothesized that calves exposed to pre- and postnatal heat stress abatement would perform most optimally in terms of thermoregulation, growth, and health responses when compared with calves that are heat-stressed at any time in the pre- or postnatal periods. Holstein calves born to heat-stressed (HT) or cooled (CL) dams during late gestation (44 ± 5 d; prenatal HT or CL) were exposed to heat stress or cooling postnatally for 56 d (postnatal HT or CL), resulting in 4 treatments: HT-HT, HT-CL, CL-HT, and CL-CL; n = 12/treatment. Calves were administered 4 L of pooled colostrum and after 2 d of age allotted 10 L/d milk replacer and up to 3 kg/d concentrate in automatic feeder group pens (n = 6/pen). Postnatal cooling was achieved by 2 fans (average wind speed 2 m/s). Thermoregulatory responses (respiration rate and heart rate; rectal, body, and skin temperature), feed intake, growth parameters including average daily gain and medication events were recorded, and blood samples were collected weekly. Thermoregulatory responses were lower in postnatal CL calves compared with postnatal HT. In the afternoon, HT-HT calves had the highest respiration rate and rectal temperature, HT-CL calves had the lowest respiration rate, and CL-HT calves had the lowest heart rate compared with the other treatment groups. Prenatal CL calves weighed more at birth and weaning with a tendency for greater average daily gain compared with prenatal HT calves, whereas postnatal CL calves had increased milk replacer and concentrate intake and a tendency for reduced fever, infection, and total medication events relative to postnatal HT. Prenatal HT calves were esophageal tube fed more often than prenatal CL. Blood hematocrit and 24-h serum IgG concentration were greater in prenatal CL calves relative to prenatal HT. Prenatal heat stress abatement improves weight gain, hematocrit, and immunoglobulin transfer, whereas postnatal heat stress abatement modulates thermoregulatory responses, feed intake, and calf health. This study is the first to characterize the combined effects of pre- and postnatal heat stress or active cooling on the dairy calf.

Uterine diseases and heat stress (HS) are major challenges for the dairy cow. Heat stress alters host immune resilience, making cows more susceptible to the development of uterine disease. Although HS increases the incidence of uterine disease, the mechanisms by which this occurs are unclear. We hypothesize that evaporative cooling (CL) to alleviate HS in prepartum cows has carry-over effects on postpartum innate immunity. Nulliparous pregnant Holstein heifers were assigned to receive either forced CL that resulted in cool conditions (shade with water soakers and fans; n = 14) or to remain under HS conditions (barn shade only; n = 16) for 60 d prepartum. Postpartum, all cows were housed in a freestall barn equipped with shade, water soakers, and fans. Respiratory rate and rectal temperature during the prepartum period were greater in HS heifers compared with CL heifers, indicative of HS. Although milk production was decreased in HS cows compared with CL cows, the incidence of uterine disease and content of total or pathogenic bacteria in vaginal mucus on d 7 or d 21 postpartum was not affected by treatment. Whole blood was collected on d 21 and subjected to in vitro stimulation with lipopolysaccharide. Lipopolysaccharide-induced accumulation of IL-1β, IL-10, and MIP-1α was greater in blood collected from HS cows compared with CL cows. Our results imply that prepartum HS during late pregnancy has carry-over effects on postpartum innate immunity, which may contribute to the increased incidence of uterine disease observed in cows exposed to prepartum HS.

Summary: Pregnant, nulliparous heifers that were heat stressed during late gestation produced heifer calves that were negatively affected in terms of growth and metabolism, immune function, and thermoregulation. These in utero heat-stressed heifer calves suffered negative outcomes during the postnatal period, which was evidenced by decreased apparent efficiency of absorption of immunoglobulin G after colostrum feeding, decreased serum immunoglobulin G concentrations throughout the preweaning period, and increased surface temperatures at weaning.

Prenatal hyperthermia has immediate and long-term consequences on dairy cattle growth, immunity, and productivity. While changes in the molecular architecture are reported in the mature mammary gland (MG), any influence on early-life mammary development is unknown. Herein, we characterize the impact of late-gestation in utero heat stress on heifer mammary gross and cellular morphology at early-life developmental stages (i.e., birth and weaning). During summer, pregnant dams were exposed to environmental heat stress (shade of a free-stall barn) or offered active cooling (shade, fans, and water soakers) for 54 ± 5 d before parturition (avg. temperature–humidity index = 79). Heifer calves born to these dams were either in utero heat-stressed (IU-HT; n = 36) or in utero cooled (IU-CL; n = 37) and were managed as a single cohort thereafter. A subset of heifers was euthanized at birth (d0; n = 8/treatment; 4.6 ± 2.3 h after birth) and after weaning (d63; n = 8/treatment; 63.0 ± 1.5 d) to harvest the whole MG. An ultrasound of rear mammary parenchyma (MPAR) was taken prior to d63 and correlated to harvested MPAR cross-sectional area and weight. Portions of mammary fat pad (MFP) and MPAR were preserved for compositional and histological analysis, including ductal structure number and cross-sectional area, connective tissue area, and adipocyte number and cross-sectional area. Cellular proliferation in MPAR was assessed via Ki-67 immunohistochemistry. Relative to IU-CL heifers, the MGs of IU-HT heifers were shorter in length at d0 and d63 (P ≤ 0.02). There were moderate correlations between d63 ultrasound and harvest measures. The IU-HT heifers had reduced MG and MFP mass at d0 and d63 (P ≤ 0.05), whereas MPAR mass was reduced only at d0 (P = 0.01). IU-HT heifers had greater MPAR protein and DNA content at d63 (P ≤ 0.04), but there were no MFP compositional differences (P ≥ 0.12). At d0, IU-HT heifers had fewer MPAR ductal structures (P ≤ 0.06), but there were no differences at d63. Yet, MPAR luminal and total ductal structure cross-sectional areas of IU-HT heifers were reduced at both d0 and d63 (P ≤ 0.01). The MFP adipocytes of IU-HT heifers were smaller at d0 (P ≤ 0.01), but differences were not detected at d63. The IU-HT heifers had diminished MPAR total, stromal, and epithelial cellular proliferation at both d0 and d63 (P < 0.01). Prenatal hyperthermia derails dairy calf early-life mammary development with potential carry-over consequences on future synthetic capacity. Late-gestation in utero heat stress in dairy cattle negatively affects the mammary microstructure and milk yield at maturity, but investigation into early-life windows of mammary development is needed to fully characterize the lifelong consequences of intrauterine heat stress on the mammary gland (MG). The present study quantified mammary gross morphology and mammary fat pad and parenchyma composition, tissue microstructure, and cellular proliferation at birth and after weaning from heifers exposed to late-gestation prenatal hyperthermia. The whole MGs and fat pads of in utero heat-stressed heifers are lighter across early life relative to in utero cooled heifers. The mammary parenchyma is smaller at birth with stunted ductal development and cellular proliferation at birth and after weaning. These impairments may limit later mammary epithelial development and impact long-term productivity.

The monoamine serotonin (5-hydroxytryptamine, 5-HT) has been reported to inhibit milk protein gene expression and increase mammary epithelial cell (MEC) tight junction permeability after milk stasis. We hypothesized that increasing serotonin synthesis and signaling within the mammary epithelium before milk stasis would increase systemic and local involution markers, and downregulate the expression of milk protein and tight junction during involution, leading to more efficient tissue growth during the redevelopment phase. Herein, we examined the outcomes of increasing local mammary 5-HT synthesis before milk stasis on involution biomarkers, mammary gland microstructure, and gene and protein expression during the dry period. Multiparous Holstein cows were administered intramammary infusions (via the teat canal) of sterile water (CON, 4 mL/teat, n = 7) or 5-hydroxy-l-tryptophan (5-HTP, serotonin precursor, 20 mg/teat, n = 7) once daily for 5 d before dry-off (d 0). Blood, milk, and mammary secretions were collected and analyzed for components and metabolites. Mammary secretions were collected 12 h after the last milking and on d 1 to 4 during the dry period at 1200 h. Mammary gland biopsies were performed on d 4 (i.e., involution phase) and d 36 (i.e., redevelopment phase) of the dry period for histological and molecular evaluation. Milk protein and tight junction gene expression was quantified via real-time PCR. Hematoxylin and eosin staining, immunohistochemistry (Ki67), and immunofluorescence (serotonin, cleaved caspase 3) were performed to visualize tissue microstructure and to quantify serotonin intensity and cell turnover. Data were analyzed in SAS (SAS Institute Inc.) using 2-way ANOVA. After d 0, mammary secretions of 5-HTP cows had increased concentrations of 5-HT, lactoferrin, and bovine serum albumin. On d 1, 5-HTP cows had greater α-lactalbumin concentrations in plasma relative to CON. Serotonin intensity was increased in the mammary tissue of 5-HTP cows on d 4, relative to CON. On d 4, milk protein and tight junction gene expression was downregulated, MEC number was reduced, and cleaved caspase 3 protein was greater in mammary tissue of 5-HTP cows, relative to CON. On d 36, milk protein genes were upregulated, and the lumen:outer alveolar area and Ki67-positive cells were increased in the mammary tissue of 5-HTP cows, relative to CON. Amplifying serotonin signaling in the mammary epithelium before milk stasis at dry-off achieves greater apoptosis, leading to a reduction in MEC, allowing for greater cell proliferation, which results in more MEC during the redevelopment phase preceding the onset of lactation.

Late-gestation in utero heat stress derails organ development and growth, leading to reduced first-lactation milk yield. Yet, carry-over effects of in utero heat stress on peripubertal heifers remain unexplored. This study investigated the long-term effects of in utero heat stress on body growth, mammary gland (MG) development, and ovarian function in peripubertal heifers. Dry, pregnant cows were housed in freestall barns and provided only shade or shade plus evaporative cooling for the last 54 ± 5 d of gestation during a subtropical summer. The resulting in utero cooled (IUCL, n = 21) and in utero heat-stressed heifers (IUHT, n = 19) were raised as a single cohort until 12 mo of age. Body weight, hip height (HH), chest girth (CG), body length (BL), withers height (WH), head circumference (HC), and MG macrostructure was recorded monthly from 3 to 12 mo. Once puberty was confirmed with 2 consecutive weeks of plasma progesterone (P₄) concentrations >1 ng/mL, heifers were assigned to a protocol for the synchronization of estrus (D-7: GnRH + CIDR, D-2: PGF_2α, D-1: PGF_2α + CIDR removal). Blood and MG biopsies were collected on d 0 (high estradiol [E₂]/low P₄) and d 13 (low E₂/high P₄) to quantify mammogenic hormones and MG microstructure. Ovarian ultrasounds were conducted on d 0, 9, and 13 to analyze the cross-sectional area and volume of the dominant follicle and corpus luteum. Data were analyzed in SAS. A treatment by month interaction was observed for BW and CG, whereby IUHT heifers were lighter (3, 7, and 8 mo) and had smaller CG (3, 4, and 8 mo). Additionally, IUHT heifers had shorter HH (114.8 vs. 117.5 ± 0.6 cm; IUHT vs. IUCL), BL (100.6 vs. 102.2 ± 0.5 cm), and WH (108.3 vs. 110.4 ± 0.6 cm) and smaller HC (74.9 vs. 75.8 ± 0.3 cm). The distance between the teats and width of the MG did not differ, but teat lengths were shorter in IUHT heifers (front teats: 25.5 vs. 28.3 ± 0.6 mm; rear teats: 22.1 vs. 24.4 ± 0.6 mm). At 12 mo, MG length was shorter in IUHT than IUCL heifers (78.5 vs. 85.5 ± 1.4 cm). Fewer proliferating (0.38 vs. 0.85 ± 0.10%) and ERα-positive cells (10.1 vs. 12.8 ± 0.7%) were observed in IUHT heifers. In the MG fat pad, IUHT heifers had more adipocytes, particularly on d 13 (47.7 vs. 33.9 ± 3.3 cells). Although systemic P₄ was similar, E₂ concentrations were reduced in IUHT heifers on d 0 (3.8 vs. 5.0 ± 0.3 pg/mL). Additionally, IUHT heifers had a smaller follicle diameter and volume (diameter: 13.5 vs. 15.5 ± 0.6 mm; volume: 1,468.0 vs. 2,208.5 ± 262.0 mm³) and a smaller corpus luteum diameter and volume (diameter: 22.2 vs. 26.9 ± 0.7 mm; volume: 6,170.4 vs. 10,506.0 ± 617.7 mm³). Overall, exposure to in utero heat stress had lasting implications, leading to a disruption in the crosstalk between the MG and ovaries during the peripubertal phase. The observed effects during peripuberty may provide insight into the reduced lactation performance at maturity for in utero heat-stressed heifers.