Long-term growth, feed efficiency, enteric methane emission, and blood metabolite responses to in utero hyperthermia in Holstein heifers.

Dairy producers are experiencing production and animal welfare pressures from the increasing frequency and severity of heat stress events due to global climate change. Offspring performance during the preweaning and lactating periods is compromised when exposed to heat stress during late gestation (in utero). However, knowledge of the lingering effects of in utero heat stress on yearling dairy heifers is limited. Herein, we investigated the long-term effects of in utero heat stress on heifer growth, feed efficiency, and enteric methane emissions in postpubertal heifers. During the last 56 d of gestation, 38 pregnant cows carrying heifer calves were exposed to either heat stress (IUHT; n = 17) or artificial cooling (IUCL; n = 21). At 18 ± 1 mo of age, the resulting IUCL and IUHT heifers were enrolled in the present 63-d study. Heifers were blocked by weight and randomly assigned to 3 pens with Calan gates. Body weights were recorded on 3 consecutive days at the start and end of the trial and used to calculate ADG. Body condition score, hip width, body length, and chest girth were measured at the start and end of the study. All heifers were fed a TMR comprised of 46.6% oatlage, 44.6% grass/alfalfa haylage, 7.7% male-sterile corn silage, 0.3% urea, and 0.8% mineral/vitamin supplement (on a DM basis). The TMR and refusal samples were obtained daily, composited weekly, and dried to calculate DMI. During the study, each pen had access to a GreenFeed unit for 8 ± 1d to measure CH4 and CO2 gas fluxes. During the last 3 d of measuring CH4 and CO2 fluxes, fecal samples were collected, composited by animal, dried, and analyzed to calculate NDF, OM, and DM digestibility. On the last day of fecal sampling, blood samples were also collected via coccygeal venipuncture, and GC time-of-flight MS analysis was performed. Residual feed intake (RFI; predicted DMI - observed DMI), and feed conversion efficiency (FCE; DMI/ADG) were calculated to estimate feed efficiency. No differences were found in initial or final BW, hip width, chest girth, or BCS; however, IUCL heifers were longer in body length compared with IUHT heifers. Dry matter intake, ADG, RFI, and FCE were similar between IUHT and IUCL heifers. In utero heat-stressed and IUCL heifers produced similar amounts of CH4 and CO2, and no differences were found in the number of GreenFeed visits or latency to approach the GreenFeed. The concentrations of 6 blood metabolites involved in lipogenic pathways were different between in utero treatments. In conclusion, in utero heat stress does not seem to have long-term effects on feed efficiency or methane emissions during the postpubertal growing phase; however, IUCL heifers maintained a body-length advantage over their IUHT counterparts and differed in concentrations of several candidate metabolites that encourage further exploration of their potential function in key organs, such as the liver and mammary gland.

In utero hyperthermia in late gestation derails dairy calf early-life mammary development.

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.

In vitro effects of 5-Hydroxy-L-tryptophan supplementation on primary bovine mammary epithelial cell gene expression under thermoneutral or heat shock conditions.

Serotonin (5-HT) is an autocrine-paracrine molecule within the mammary gland regulating homeostasis during lactation and triggering involution after milk stasis. Exposure of dairy cows to hyperthermia during the dry period alters mammary gland involution processes leading to reduced subsequent yields. Herein, primary bovine mammary epithelial cells (pBMEC) under thermoneutral (TN, 37 °C) or heat shock (HS, 41.5 °C) conditions were cultured with either 0, 50, 200, or 500 µM 5-Hydroxy-L-tryptophan (5-HTP; 5-HT precursor) for 8-, 12- or 24-h. Expression of 95 genes involved in 5-HT signaling, involution and tight junction regulation were evaluated using a Multiplex RT-qPCR BioMark Dynamic Array Circuit. Different sets of genes were impacted by 5-HTP or temperature, or by their interaction. All 5-HT signaling genes were downregulated after 8-h of HS and then upregulated after 12-h, relative to TN. After 24-h, apoptosis related gene, FASLG, was upregulated by all doses except TN-200 µM 5-HTP, and cell survival gene, FOXO3, was upregulated by HS-50, 200 and 500 µM 5-HTP, suggesting 5-HTP involvement in cell turnover under HS. Supplementing 5-HTP at various concentrations in vitro to pBMEC modulates the expression of genes that might aid in promoting epithelial cell turn-over during involution in dairy cattle under hyperthermia.

Effects of maternal gestational diet, with or without methionine, on muscle transcriptome of Bos indicus-influenced beef calves following a vaccine-induced immunological challenge.

Maternal nutrition during gestation can cause epigenetic effects that translate to alterations in gene expression in offspring. This 2-year study employed RNA-sequencing technology to evaluate the pre- and post-vaccination muscle transcriptome of early-weaned Bos indicus-influenced beef calves born from dams offered different supplementation strategies from 57 ± 5 d prepartum until 17 ± 5 d postpartum. Seventy-two Brangus heifers (36 heifers/yr) were stratified by body weight and body condition score and assigned to bahiagrass pastures (3 heifers/pasture/yr). Treatments were randomly assigned to pastures and consisted of (i) no pre- or postpartum supplementation (NOSUP), (ii) pre- and postpartum supplementation of protein and energy using 7.2 kg of dry matter/heifer/wk of molasses + urea (MOL), or (iii) MOL fortified with 105 g/heifer/wk of methionine hydroxy analog (MOLMET). Calves were weaned on d 147 of the study. On d 154, 24 calves/yr (8 calves/treatment) were randomly selected and individually limit-fed a high-concentrate diet until d 201. Calves were vaccinated on d 160. Muscle biopsies were collected from the same calves (4 calves/treatment/day/yr) on d 154 (pre-vaccination) and 201 (post-vaccination) for gene expression analysis using RNA sequencing. Molasses maternal supplementation led to a downregulation of genes associated with muscle cell differentiation and development along with intracellular signaling pathways (e.g., Wnt and TGF-ß signaling pathway) compared to no maternal supplementation. Maternal fortification with methionine altered functional gene-sets involved in amino acid transport and metabolism and the one-carbon cycle. In addition, muscle transcriptome was impacted by vaccination with a total of 2,396 differentially expressed genes (FDR ≤ 0.05) on d 201 vs. d 154. Genes involved in cell cycle progression, extracellular matrix, and collagen formation were upregulated after vaccination. This study demonstrated that maternal supplementation of energy and protein, with or without, methionine has long-term implications on the muscle transcriptome of offspring and potentially influence postnatal muscle development.

Histological and transcriptomic analysis of adipose and muscle of dairy calves supplemented with 5-hydroxytryptophan.

In mammals, peripheral serotonin is involved in regulating energy balance. Herein, we characterized the transcriptomic profile and microstructure of adipose and muscle in pre-weaned calves with increased circulating serotonin. Holstein bull calves (21 ± 2 days old) were fed milk replacer supplemented with saline (CON, 8 mL/day n = 4) or 5-hydroxytryptophan (5-HTP, 90 mg/day, n = 4) for 10 consecutive days. Calves were euthanized on d10 to harvest adipose and muscle for RNA-Sequencing and histological analyses. Twenty-two genes were differentially expressed in adipose, and 33 in muscle. Notably, Interferon gamma inducible protein-47 was highly expressed and upregulated in muscle and adipose (avg. log FC = 6.5). Enriched pathways in adipose tissue revealed serotonin's participation in lipid metabolism and PPAR signaling. In muscle, enriched pathways were related to histone acetyltransferase binding, Jak-STAT signaling, PI3K-Akt signaling and cell proliferation. Supplementation of 5-HTP increased cell proliferation and total cell number in adipose and muscle. Adipocyte surface area was smaller and muscle fiber area was not different in the 5-HTP group. Manipulating the serotonin pathway, through oral supplementation of 5-HTP, influences signaling pathways and cellular processes in adipose and muscle related to endocrine and metabolic functions which might translate into improvements in calf growth and development.

Effects of feeding an immunomodulatory supplement to heat-stressed or actively cooled cows during late gestation on postnatal immunity, health, and growth of calves.

Heat stress during late gestation negatively affects the physiology, health, and productivity of dairy cows as well as the calves developing in utero. Providing cows with active cooling devices, such as fans and soakers, and supplementing cows with an immunomodulating feed additive, OmniGen-AF (OG; Phibro Animal Health Corporation), improves immune function and milk yield of cows. It is unknown if maternal supplementation of OG combined with active cooling during late gestation might benefit the developing calf as well. Herein we evaluated markers of innate immune function, including immune cell counts, acute phase proteins, and neutrophil function, of calves born to multiparous dams in a 2 × 2 factorial design. Dams were supplemented with OG or a bentonite control (NO) beginning at 60 d before dry off and exposed to heat stress with cooling (CL) or without active cooling (HT) during the dry period (∼46 d). At birth, calves were separated from their dams and fed 6.6 L of their dams' colostrum in 2 meals. Calf body weight and rectal temperature were recorded, and blood samples were collected at birth (before colostrum feeding) and at 10, 28, and 49 d of age. Calves born to either CL dams or OG dams were heavier at birth than calves born to HT or NO dams, respectively. Concentrations of serum amyloid A were higher in the blood of calves born to OG dams relative to NO and for HT calves relative to CL calves. In addition, calves born to cooled OG dams had greater concentrations of plasma haptoglobin than calves born to cooled control dams. Neutrophil function at 10 d of age was enhanced in calves born to cooled OG dams and lymphocyte counts were higher in calves born to OG dams. Together these results suggest that adding OG to maternal feed in combination with active cooling of cows during late gestation is effective in mitigating the negative effects of in utero heat stress on postnatal calf growth and immune competence.

Maternal dietary vitamin D carry-over alters offspring growth, skeletal mineralisation and tissue mRNA expressions of genes related to vitamin D, calcium and phosphorus homoeostasis in swine.

Maternal dietary vitamin D carry-over effects were assessed in young pigs to characterise skeletal abnormalities in a diet-induced model of kyphosis. Bone abnormalities were previously induced and bone mineral density (BMD) reduced in offspring from sows fed diets with inadequate vitamin D3. In a nested design, pigs from sows (n 23) fed diets with 0 (-D), 8·125 (+D) or 43·750 (++D) µg D3/kg from breeding through lactation were weaned and, within litter, fed nursery diets arranged as a 2×2 factorial design with 0 (-D) or 7·0 (+D) µg D3/kg, each with 95 % (95P) or 120 % (120P) of P requirements. Selected pigs were euthanised before colostrum consumption at birth (0 weeks, n 23), weaning (3 weeks, n 22) and after a growth period (8 weeks, n 185) for BMD, bone mechanical tests and tissue mRNA analysis. Pigs produced by +D or ++D sows had increased gain at 3 weeks (P<0·05), and at 8 weeks had increased BMD and improved femur mechanical properties. However, responses to nursery diets depended on maternal diets (P<0·05). Relative mRNA expressions of genes revealed a maternal dietary influence at birth in bone osteocalcin and at weaning in kidney 24-hydroxylase (P<0·05). Nursery treatments affected mRNA expressions at 8 weeks. Detection of a maternal and nursery diet interaction (P<0·05) provided insights into the long-term effects of maternal nutritional inputs. Characterising early stages of bone abnormalities provided inferences for humans and animals about maternal dietary influence on offspring skeletal health.