Carry-over effects of maternal late-gestation heat stress on granddaughter's growth and mammary gland development.

Maternal (F0) exposure to late-gestation heat stress reduces their daughter's (F1) mammary gland fat pad mass (FP), parenchyma (PAR) mass, and epithelial cell proliferation when evaluated at birth and weaning, and go on to produce less milk in their first lactation. Herein, we investigated the effect of maternal late-gestation heat stress on whole-body growth and mammary development of their granddaughters (F2). Multiparous F0 cows had access to heat abatement (n = 41, shade, and active cooling via fans and water soakers) or not (n = 41, shade only) for the last 56 d of gestation during a subtropical summer. Consequently, the F1 daughters, born to F0 cows, were heat-stressed (HTF1, n = 36) or cooled (CLF1, n = 37) in utero during the last 2 mo of gestation. All F1 heifers were raised as an identically managed cohort until first calving. The F2 granddaughters, born to HTF1 (HTF2, n = 12) or CLF1 (CLF2, n = 17), were raised as an identically managed cohort until 70 d of age. Dry matter intake (DMI), body weight, hip height, wither height, chest girth, head circumference, mammary gland teat length, and left-right and front-rear teat distances were measured. Average daily gain (ADG) was calculated for the pre-weaned period (0-49 d). Mammary ultrasounds were performed on d 21, 49, and 70 (n = 9/group) on the rear left and right quarters to quantify PAR and FP areas. Mammary biopsies were collected for histological evaluation of epithelial structures (H&E staining), and to quantify cells positive for ERα (estrogen receptor, α subunit), cell proliferation (Ki67), and apoptosis (TUNEL). Heifer growth from birth to d 49 was similar between CLF2 and HTF2 for all parameters evaluated. Distances between teats and teat length were not different between groups. On d 70, CLF2 tended to have a greater average PAR (right and left quarters) relative to HTF2. Although the left FP was smaller in HTF2 relative to CLF2, the average FP was not different. The lumenal and non-lumenal epithelial structures in the PAR of HTF2 were significantly smaller than those of CLF2. In addition, HTF2 had a reduced percentage of proliferating cells in the epithelial and stromal compartments and a greater percentage of apoptotic cells, particularly in the stroma. The percentage of ERα positive cells was significantly reduced in HTF2. In summary, although HTF2 heifer's DMI was similar and they grew at the same rate as CLF2 heifers throughout the pre-weaning phase, their mammary glands had smaller PAR areas with fewer epithelial structures characterized by reduced cell turnover and lower ERα expression. These early changes in the microstructure and cellular turnover of the mammary gland may partly explain the reduction in lactation performance relative to CLF2 counterparts at maturity.

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 pre-weaning and lactating periods is compromised when exposed to heat stress during late gestation (in utero). However, knowledge of the lingering impacts 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 post-pubertal 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 (BW) were recorded on 3 consecutive days at the start and end of the trial and used to calculate average daily gain (ADG). Body condition score (BCS), 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 (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 gas chromatography time-of-flight mass spectrometry 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 post-pubertal 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.

Programming effects of intrauterine hyperthermia on adrenal gland development.

Maternal heat stress during late pregnancy can lead to intrauterine hyperthermia and affect fetal hypothalamic-pituitary-adrenal axis development and function. Herein, we investigated the effects of chronic environmental heat stress exposure of Holstein cows in the last 2 mo of gestation on their offspring's adrenal gland histomorphology and transcriptome. Cows in their last 54 ± 5 d of gestation were either heat-stressed (i.e., housed under the shade of a free stall barn) or provided heat-stress abatement via active cooling (i.e., via water soakers and fans) during a subtropical summer (Temperature-Humidity Index >68). Respiration rate (RR) and skin temperature (ST) were elevated in heat-stressed dams relative to the cows with access to heat abatement (23 bpm and 2 ◦C higher for RR and ST, respectively). Heifers born to heat-stressed cows experienced heat stress in utero (HS), while heifers born to actively cooled cows did not (CL). The adrenal gland was harvested from 6 heifers per group that were euthanized at birth (d 0; n = 12) or one week after weaning (d 63; n = 12). Circulating cortisol was measured from blood samples collected weekly throughout the pre-weaning period. At d 63, heifers that experienced HS while developing in utero had heavier adrenal glands, with a greater total tissue surface area and thickness of the zona glomerulosa (ZG), fasciculata (ZF), and reticularis (ZR), compared with CL heifers. In addition, the adrenal gland of in utero HS heifers had less cells in the ZG, more and larger cells in the ZF and larger cells in the ZR, relative to CL heifers. Although no changes in circulating cortisol were observed through the pre-weaning period, the transcriptomic profile of the adrenal tissue was altered by fetal exposure to hyperthermia. Both at birth and on d 63, approximately 30 pathways were differentially expressed in the adrenal glands of in utero HS heifers relative to CL. These pathways were associated with immune function, inflammation, prolactin signaling, cell function, and calcium transport. Upstream regulators significantly activated or inhibited in the adrenal glands of heifers exposed to intrauterine hyperthermia were identified. Maternal exposure to heat stress during late gestation caused an enlargement of their offspring's adrenal glands by inducing ZG and ZF cell hypertrophy, and caused gene expression changes. These phenotypic, histological, and molecular changes in the adrenal gland might lead to alterations in stress, immune, and metabolic responses later in life.

Intravenous infusion of 5-hydroxytryptophan to mid-lactation Holstein cows transiently affects milk production and circulating amino acid concentrations.

In dairy cows, the lactating mammary glands synthesize serotonin, which acts in an autocrine-paracrine manner in the glands and is secreted into the periphery. Serotonin signaling during lactation modulates nutrient metabolism in peripheral tissues such as adipose and liver. We hypothesized that the elevation of circulating serotonin during lactation would increase nutrient partitioning to the mammary glands, thereby promoting milk production. Our objective was to elevate circulating serotonin via intravenous infusion of the serotonin precursor 5-hydroxytryptophan (5-HTP) to determine its effects on mammary supply and extraction efficiency of AA, and milk components production. Twenty-two multiparous mid-lactation Holstein cows were intravenously infused with 5-HTP (1 mg/kg body weight) or saline, in a crossover design with two 21-d periods. Treatments were infused via jugular catheters for 1 h/d, on d 1 to 3, 8 to 10, and 15 to 17 of each period, to maintain consistent elevation of peripheral serotonin throughout the period. Milk and blood samples were collected in the last 96 h of each period. Whole-blood serotonin concentration was elevated above saline control for 96 h after the last 5-HTP infusion. Dry matter intake was decreased for cows receiving 5-HTP, and on average they lost body weight over the 21-d period, in contrast to saline cows who gained body weight. Milk production and milk protein yield were lower in cows receiving 5-HTP during the 3 infusion days, but both recovered to saline cow yields in the days after. Although milk fat yield exhibited a day-by-treatment interaction, no significant difference occurred on any given day. Milk urea nitrogen concentration was lower in 5-HTP cows on the days following the end of infusions, but not different from saline cows on infusion days. Meanwhile, plasma urea nitrogen was not affected by 5-HTP infusion. Circulating concentrations of AA were overall transiently decreased by 5-HTP, with concentrations mostly returning to baseline within 7 h after the end of 5-HTP infusion. Mammary extraction efficiency of AA was unaffected by 5-HTP infusion. Overall, both lactation performance and circulating AA were transiently reduced in cows infused with 5-HTP, despite sustained elevation of circulating serotonin concentration.

Prepartum heat stress in dairy cows increases postpartum inflammatory responses in blood of lactating dairy cows.

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-1ß, 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.

ADSA Foundation Scholar Award: Early-life exposure to hyperthermia: Productive and physiological outcomes, costs, and opportunities.

Global rising temperature is a considerable threat to livestock production and an impediment to animal welfare. In fact, the 5 warmest years on record have occurred since 2016. Although the effect of heat stress on lactating cattle is well recognized and extensively studied, it is increasingly evident that rising temperatures will affect dairy cattle of all ages and lactation states. However, the extent and consequences of this effect are less understood and often overlooked in the literature and dairy industry. Early-life experiences, such as exposure to hyperthermia, can have life-long implications for health and productivity. This review highlights the body of work surrounding the effects of heat-stress exposure in young dairy cattle, including the prenatal fetus (in utero), postnatal calves (preweaning), and growing heifers, which are all categories that are typically not considered for heat-stress abatement on farm. Insights into the physiological and molecular mechanisms that might explain the adverse phenotypic outcomes of heat-stress exposure at different stages of development are also discussed. The estimated economic loss of in utero hyperthermia is addressed, and the ties between biological findings and opportunities for the application of cooling management interventions on farm are also presented. Our research highlights the importance of heat-stress abatement strategies for dry-pregnant cows to ensure optimal multigenerational productivity and showcases the benefits of cooling neonatal calves and growing heifers. Understanding the implications of heat stress at all life stages from a physiological, molecular, economic, and welfare perspective will lead to the development of novel and refined practices and interventions to help overcome the long-lasting effects of climate change in the dairy industry.

Effect of heat stress during the early and late dry period on mammary gland development of Holstein dairy cattle.

Dry period heat stress impairs subsequent milk yield. Our objective was to evaluate the effect of heat stress or cooling during the early and late dry period on mammary gland gene expression and microstructure. Cows were dried off ∼45 d before expected parturition and randomly assigned to 1 of 2 treatments: heat stress (HT, n = 39) or cooling (CL, n = 39) during the first 21 d of the dry period. On d 22, cows were switched or remained on HT and CL and this yielded 4 treatments: heat stress during the entire dry period (HTHT, n = 18); cooling during the entire dry period (CLCL, n = 20); HT for the first 21 d dry, then CL until calving (HTCL, n = 21); or CL for the first 21 d dry, then HT until calving (CLHT, n = 19). Data were analyzed in 2 periods: first 21 d dry (early dry period) and from 22 d until calving (late dry period) and analyzed using PROC MIXED or GLM in SAS (SAS Institute Inc., Cary, NC). Mammary biopsies (5-8 cows/treatment) were collected at -3, 3, 7, 14, and 25 d relative to dry-off to evaluate mammary gland gene expression and histology [i.e., cellular apoptosis (terminal deoxynucleotidyl transferase dUTP nick end labeling) and proliferation (Ki67)]. Mammary alveoli number and connective tissue were visualized by hematoxylin and eosin and Mason's trichrome staining, respectively. During the early dry period, CL upregulated expression of CASP3, IGF1R, HSP90, HSF1, BECN1, ATG3, ATG5, and PRLR-LF relative to HT. However, in the late dry period, CLHT treatment upregulated expression of CASP3, CASP8, HSP70, HSP90, PRLR-LF, STAT5, CSN2, and ATG3 relative to CLCL. During the early dry period, cows exposed to HT had reduced mammary and stroma cell apoptosis and proliferation relative to CL. In addition to these findings, cows exposed to HT had lower connective tissue 3 d after dry-off relative to CL. However, in the late dry period, HTHT cows had higher connective tissue relative to CLCL. Also, in the early dry period, cows exposed to HT had greater alveoli number relative to CL, and HT decreased expression of genes related to autophagy and apoptosis in the early dry period, consistent with a delay in involution with HT. Thus, cows exposed to HT have extended involution with delayed apoptosis and autophagy signaling. Also, HT compromises mammary gland cell proliferation and leads to higher connective tissue later in the dry period. These results provide evidence that heat stress impairs overall mammary gland turnover during the dry period, which then affects secretory activity and productivity in the next lactation.

Effect of heat stress during early, late, and entire dry period on dairy cattle.

Cooling during the entire dry period abates the negative effects of heat stress postpartum, yet the temporal relationship of cooling (i.e., early or late dry period) to performance is unknown. We evaluated the effect of heat stress early, late, and for the entire dry period on subsequent performance. Cows were selected based on mature-equivalent milk yield and dried off 45 d before expected calving. Cows were blocked by parity, previous 305-d mature equivalent milk yield, and body weight (BW) and randomly assigned to cooling (shade, fans, and soakers; CL) or heat stress (shade; HT). Treatments included CL (n = 20) or HT (n = 18) during the entire dry period, HT during the first 3 wk dry and then CL until calving (HTCL, n = 21), or CL during the first 3 wk dry period and then HT until calving (CLHT, n = 19). Heat stress increased rectal temperature (RT; CL, 38.8; HT, 39.1 ± 0.04°C) and respiration rate (RR; CL, 52.9; HT, 70.5 ± 1.9 breaths/min) during the early dry period. In the late dry period, HT increased RT and RR relative to CL cows (RT = CL, 38.7; HT, 39.1; CLHT, 39.1; HTCL, 38.9 ± 0.05°C; RR = CL, 47; HT, 64; CLHT, 66; HTCL, 53 ± 2.1 breaths/min). During the early dry period, HT decreased dry matter intake (CL, 11.8; HT, 10.5 ± 0.35 kg/d) but dry matter intake did not differ among treatments during late dry period (HT, 10.7; HTCL, 11.1; CL, 11.2; CLHT, 10.1 ± 0.55 kg/d). Cows exposed to prepartum cooling during the entire dry period had increased dry matter intake compared with cows exposed to heat stress during the late dry period (CL vs. CLHT, 11.2 ± 0.55 and 10.1 ± 0.55 kg/d, respectively). Heat stress at any time reduced gestation length compared with cows under prepartum cooling during the entire dry period (CL, 277 vs. HT, 274; CLHT, 273; and HTCL, 274 ± 1.17 d). Dry period length decreased by approximately 4 d if cows were exposed to HT at any time. During the early dry period, HT decreased BW, whereas CL increased BW relative to that at dry-off (CL, 6.9; HT, -9.4 ± 3.7 kg). In the late dry period, we detected no differences in BW gain among treatments, but cows exposed to prepartum cooling for the entire dry period tended to have increased BW gain compared with HT and HTCL. Prepartum cooling during the early or late dry period alone partially rescued milk yield only in the first 3 wk of lactation (CL, 32.9; HT, 26.6; CLHT, 29.7; HTCL, 30.7 ± 1.37 kg/d). Cooling for the entire dry period increased milk yield up to 30 wk into lactation compared with all other treatments. Thus, HT at any time during the dry period compromises performance of cows after calving.

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.

Effect of nutritional immunomodulation and heat stress during the dry period on subsequent performance of cows.

Heat stress in dairy cows during the dry period impairs milk yield in the next lactation. Feeding OmniGen-AF (OG; Phibro Animal Health Corp., Teaneck, NJ) to lactating cows during heat stress may increase dry matter intake (DMI) and lowers respiration rate (RR) and rectal temperature (RT), but the effects in dry cows are not known. We hypothesized that OG supplementation before, during, and after the dry period (approximately 160 d total) would overcome the effects of heat stress and improve cow performance in the next lactation. Cows were randomly assigned to OG or control (placebo) treatments for the last 60 d in milk (DIM), based on mature-equivalent milk yield in the previous lactation. Cows were dried off 45 d before expected calving and randomly assigned to heat stress (HT) or cooling (CL) treatments. Thus, cows received dietary supplementation during late lactation before they were exposed to either CL or HT. After dry-off, treatment groups included heat stress with placebo (HT, only shade, 56 g/d of placebo, n = 17), HT with OG supplementation (HTOG, 56 g/d of OG, n = 19), cooling with placebo (CL, shade, fans, and soakers, 56 g/d of placebo, n = 16), and CL with OG supplementation (CLOG, 56 g/d of OG, n = 11). After parturition, all cows were kept under the same CL system and management, and all cows continued to receive OG or control treatment until 60 DIM. Cooling cows during the dry period reduced afternoon RT (CL vs. HT; 38.9 ± 0.05 vs. 39.3 ± 0.05°C) and RR (CL vs. HT; 45 ± 1.6 vs. 77 ± 1.6 breaths/min). Respiration rate was also decreased by OG supplementation under HT conditions (HTOG vs. HT; 69.7 ± 1.6 vs. 77.2 ± 1.6 breaths/min). An interaction was observed between OG supplementation and HT; HTOG cows tended to have lower morning RT compared with HT cows. During the dry period, OG reduced DMI relative to control cows. Birth weight was greater in calves from CL cows (CL vs. HT; 40.6 ± 1.09 vs. 38.7 ± 1.09 kg). No differences were detected among treatments in hematocrit, total protein, and body condition score. Cows offered CLOG, CL, and HTOG treatments had greater body weight during the dry period (794.9 ± 17.9, 746.8 ± 16.7, and 762.9 ± 14.9 kg, respectively) than HT cows (720 ± 16.2 kg). Gestation length was approximately 4 d longer for CL cows compared with HT cows. Cows offered CLOG, CL, and HTOG treatments produced more milk (41.3 ± 1.6, 40.7 ± 1.6, and 40.5 ± 1.6 kg/d, respectively) than HT treatment (35.9 ± 1.6 kg/d). Body weight after parturition and DMI were evaluated up to 60 DIM and averaged 661.5 ± 15.8 and 19.4 ± 0.7 kg/d, respectively, with no differences observed among treatments. These results confirm that exposure of dry cows to heat stress negatively affects milk yield in the subsequent lactation. Active cooling of dry cows and OG supplementation can reduce the negative effects of heat stress in the dry period on subsequent performance.

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.

Androgen receptor DNA methylation regulates the timing and androgen sensitivity of mouse prostate ductal development.

Androgen receptor (AR) signaling initiates mouse prostate development by stimulating prostate ductal bud formation and specifying bud patterns. Curiously, however, prostatic bud initiation lags behind the onset of gonadal testosterone synthesis by about three days. This study's objective was to test the hypothesis that DNA methylation controls the timing and scope of prostate ductal development by regulating Ar expression in the urogenital sinus (UGS) from which the prostate derives. We determined that Ar DNA methylation decreases in UGS mesenchyme during prostate bud formation in vivo and that this change correlates with decreased DNA methyltransferase expression in the same cell population during the same time period. To examine the role of DNA methylation in prostate development, fetal UGSs were grown in serum-free medium and 5 alpha dihydrotestosterone (DHT) and the DNA methylation inhibitor 5'-aza-2'-deoxycytidine (5AzadC) were introduced into the medium at specific times. As a measure of prostate development, in situ hybridization was used to visualize and count Nkx3-1 mRNA positive prostatic buds. We determined that inhibiting DNA methylation when prostatic buds are being specified, accelerates the onset of prostatic bud development, increases bud number, and sensitizes the budding response to androgens. Inhibition of DNA methylation also reduces Ar DNA methylation in UGS explants and increases Ar mRNA and protein in UGS mesenchyme and epithelium. Together, these results support a novel mechanism whereby Ar DNA methylation regulates UGS androgen sensitivity to control the rate and number of prostatic buds formed, thereby establishing a developmental checkpoint.

DNA methylation of E-cadherin is a priming mechanism for prostate development.

In prostate and other epithelial cancers, E-cadherin (CDH1) is downregulated inappropriately by DNA methylation to promote an invasive phenotype. Though cancer frequently involves a reawakening of developmental signaling pathways, whether DNA methylation of Cdh1 occurs during organogenesis has not been determined. Here we show that DNA methylation of Cdh1 mediates outgrowth of developing prostate ducts. During the three-day gestational window leading up to and including prostate ductal initiation, Cdh1 promoter methylation increases and its mRNA and protein abundance decreases in epithelium giving rise to prostatic buds. DNA methylation is required for prostate specification, ductal outgrowth, and branching morphogenesis. All three endpoints are impaired by a DNA methylation inhibitor, which also decreases Cdh1 promoter methylation and increases Cdh1 mRNA and protein abundance. A CDH1 function-blocking antibody restores prostatic identity, bud outgrowth, and potentiates epithelial differentiation in the presence of the DNA methylation inhibitor. This is the first study to mechanistically link acquired changes in DNA methylation to the normal process of prostate organogenesis. We propose a novel mechanism whereby Cdh1 promoter methylation restricts Cdh1 abundance in developing prostate epithelium to create a permissive environment for prostatic bud outgrowth. Thus, DNA methylation primes the prostate primordium to respond to developmental cues mediating outgrowth, differentiation and maturation of the ductal network.

Liver functional genomics in beef cows on grazing systems: novel genes and pathways revealed.

The adaptation of the liver to periods of negative energy balance is largely unknown in beef cattle on grazing systems. We evaluated liver transcriptome throughout gestation and early lactation of purebred and crossbred beef cows [Angus, Hereford, and their F1 crossbreeds (CR)], grazing high or low herbage allowances (HA) of native grasslands (4 and 2.5 kg dry matter/kg body wt annual mean; n = 16) using an Agilent 4 × 44k bovine array. A total of 4,661 transcripts were affected by days [272 ≥ 2.5-fold difference, false discovery rate (FDR) ≤ 0.10] and 47 pathways were altered during winter gestation (-165 to -15 days relative to calving), when cows experienced decreased body condition score, decreased insulin, and increased nonesterified fatty acid concentrations. Gluconeogenesis and fatty acid oxidation pathways were upregulated, while cell growth, DNA replication, and transcription pathways were downregulated (FDR ≤ 0.25). We observed only small changes in the liver transcriptome during early lactation (+15 to +60 days). A total of 225 genes were differentially expressed (47 ≥ 2-fold difference, FDR ≤ 0.10) between HA. The majority of those were related to glucose and pyruvate metabolism and were upregulated in high HA, reflecting their better metabolic status. Two genes were upregulated in CR cows, but 148 transcripts (74 ≥ 2-fold change difference, FDR ≤ 0.10) were affected by the HA and cow genotype interaction. The transcriptional changes observed indicated a complex and previously unrecognized, hepatic adaptive program of grazing beef cows in different nutritional environments. Novel target candidate genes, metabolic pathways, and regulatory mechanisms were reported.

Exploring the contribution of mammary-derived serotonin on liver and pancreas metabolism during lactation.

During lactation, the murine mammary gland is responsible for a significant increase in circulating serotonin. However, the role of mammary-derived serotonin in energy homeostasis during lactation is unclear. To investigate this, we utilized C57/BL6J mice with a lactation and mammary-specific deletion of the gene coding for the rate-limiting enzyme in serotonin synthesis (TPH1, Wap-Cre x TPH1FL/FL) to understand the metabolic contributions of mammary-derived serotonin during lactation. Circulating serotonin was reduced by approximately 50% throughout lactation in Wap-Cre x TPH1FL/FL mice compared to wild-type mice (TPH1FL/FL), with mammary gland and liver serotonin content reduced on L21. The Wap-Cre x TPH1FL/FL mice had less serotonin and insulin immunostaining in the pancreatic islets on L21, resulting in reduced circulating insulin but no changes in glucose. The mammary glands of Wap-Cre x TPH1FL/FL mice had larger mammary alveolar areas, with fewer and smaller intra-lobular adipocytes, and increased expression of milk protein genes (e.g., WAP, CSN2, LALBA) compared to TPH1FL/FL mice. No changes in feed intake, body composition, or estimated milk yield were observed between groups. Taken together, mammary-derived serotonin appears to contribute to the pancreas-mammary cross-talk during lactation with potential implications in the regulation of insulin homeostasis.

Effects of thermal stress on calf welfare.

Cold and heat stress present welfare challenges for dairy calves. The consequences of thermal stress on biological functioning have been well documented, and many housing and management strategies have been evaluated to mitigate those detrimental impacts. In cold weather, mitigation strategies have largely focused on nutritional interventions or limiting heat loss with resources such as bedding or jackets. In hot weather, heat abatement strategies such as supplemental shade, increased environmental air exchange through passive ventilation, and forced air movement through mechanical ventilation have been evaluated. Recently in Wisconsin's continental climate, our group evaluated how 2 aspects of calf welfare-the needs for thermal comfort and social contact (i.e., pair or group housing vs. individual housing)-may align or conflict in winter and summer, respectively. In both seasons, calves pair-housed in outdoor hutches preferred social proximity. When 2 calves shared a hutch, the heat load was greater than for a single calf, which may be beneficial for thermal comfort in winter. In summer, the potential detriments from the additional heat load of 2 calves was mitigated with passive hutch ventilation, which calves preferred. Nonetheless, knowledge gaps remain regarding the impacts of thermal stress on calves' affective states, and much remains unknown about their preferences and motivations for specific thermal stress mitigation resources. Future research to address these gaps could improve our understanding of calf welfare and inform best practices for calf management.

Actively ventilating calf hutches using solar-powered fans: Effects on hutch microclimate and calf thermoregulation.

Although active ventilation via fans is an effective and widely adopted heat abatement method for use with adult dairy cattle, it has yet to be investigated in outdoor hutch-housed dairy calves despite most US calves being raised in such systems. We investigated a solar-powered fan system for outdoor calf hutches and its effect on hutch microclimate and calf thermoregulation. During summer, a 3 × 3 Latin square was replicated 4 times (n = 12 preweaning heifers) with 4-d exposure periods to minimally (CON; rear windows closed), passively (PASS; rear windows opened), or actively (ACT; solar-powered fan, activated at dry bulb temperature [Tdb] > 21°C) ventilated hutch systems. Hutch microclimate and calf thermoregulation were evaluated either continuously (Tdb, humidity, rectum surface temperature, and behavior) or after a daily 30-min inside restriction (air speed, air particle number, noise level, respiration, and sweating rate, and skin and rectal temperature). Active ventilation substantially increased hutch air speed relative to PASS and CON (1.76 vs. 0.19 vs. 0.05 m/s). However, PASS hutches had the lowest INT Tdb (27.2 vs. 26.4 vs. 27.8°C), whereas ACT INT Tdb was reduced at 0900 and 1000 h relative to CON but not PASS. Similarly, ACT reduced calf respiration rates and lowered rectum surface temperature at 0800 and 0900 h when compared with CON but not PASS. The lack of strong ACT influence on calf outcomes over PASS could partially be explained by the decreased proportion of time ACT calves spent inside their hutch (48.7 vs. 67.3 vs. 64.1% of each hour). Overall, ACT improved hutch microclimate and calf responses relative to CON but not PASS. Either ACT or PASS ventilation may be sufficient to provide heat abatement to continental hutch-housed calves.

Artificial shade as a heat abatement strategy to grazing beef cow-calf pairs in a subtropical climate.

Grazing livestock in subtropical and tropical regions are susceptible to prolonged exposition to periods of extreme environmental conditions (i.e., temperature and humidity) that can trigger heat stress (HS). Currently, there is limited information on the effects of HS in the cow-calf sector globally, including in the southern U.S., as well as on mitigation strategies that could be implemented to improve animal well-being and performance. This study evaluated the impact of artificial shade (SHADE vs. NO SHADE) and breed (ANGUS vs. BRANGUS) on performance of pregnant-lactating cows, nursing heifers, and their subsequent offspring. Twenty-four Angus and 24 Brangus black-hided cows [579 ± 8 kg body weight (BW); approximately 85 d of gestation] and their nursing heifers (approximately 174 d of age) were randomly allocated to 12 'Pensacola' bahiagrass pastures (Paspalum notatum Flüggé; 1.3 ha, n = 4 pairs/pasture), with or without access to artificial shade [NO SHADE BRANGUS (NSB), NO SHADE ANGUS (NSA), SHADE BRANGUS (SB), and SHADE ANGUS (SA)] for 56 d that anticipated weaning during the summer season in Florida. Body condition score (BCS) of cows, blood samples, and BW of cow-calf pairs were obtained every 14 d during the 56-d experimental period until weaning. Following weaning (d 56), treatments were ceased, and cows and weaned heifers were managed alike. Weaned heifers were randomly allocated to 4 pens (n = 12/pen) equipped with GrowSafe feed bunks for 14 d to assess stress responses during weaning via plasma haptoglobin. An effect of SHADE × BREED interaction was detected for cow ADG, BW change, final BW, and final BCS, where SB had the greatest ADG, BW change, final BW, and final BCS. On d 14, SA cows had the greatest concentrations of insulin whereas on d 28 NSB had the lowest concentrations, NSA the greatest, and SA and SB being intermediate. On d 56, SA tended to have the greatest plasma insulin concentrations and SB the lowest. Weight gain per area (kg/ha) tended to be 11.4 kg/ha greater in SHADE vs. NO SHADE pastures. Pre-weaning calf ADG tended to be 0.14 kg greater for SHADE vs. NO SHADE calves. Weaning weight and BW at 14-d post-weaning were lesser for NSB vs. NSA, SA, and SB, whereas no differences in postweaning ADG or haptoglobin were observed. Effects of SHADE × BREED × day interaction was detected on plasma concentrations of IGF-1, in which NSA heifers had the lowest concentrations on weaning day. Gestation length was greater for SHADE vs. NO SHADE cows, but with no impacts on subsequent calf birth and weaning weight. In summary, providing artificial shade to pregnant-lactating beef cows increased body weight gain of nursing heifers and Brangus cows, while no impact on Angus dams were observed. The provision of artificial shade during the first trimester of gestation did not alter growth performance of the subsequent offspring at birth and weaning even though gestation length was longer.

Serotonin alters the response to a glucose challenge in lactating cows.

Energy partitioning in lactating cows affects milk production, feed efficiency, and body reserves, with the latter having health implications for the transition into the following lactation. One molecule likely involved in the regulation of energy partitioning is serotonin. The objective of this experiment was to explore how increasing circulating serotonin, by intravenous infusion of the serotonin precursor 5-hydroxytryptophan (5-HTP), affects metabolic responses to a glucose challenge in midlactation cows as a means to manipulate energy partitioning. We intravenously infused Holstein cows with 5-HTP (1 mg/kg bodyweight dissolved in saline, n = 11) or saline alone as control (n = 9) over 1 h/day for 3 days. Cows were fasted overnight on day 2. On day 3, fasted cows were given an intravenous bolus of glucose (0.092 g/kg bodyweight). Blood samples were collected for the following 120 min for metabolic and hormonal analysis. Infusion of 5-HTP elevated circulating concentrations of serotonin and free fatty acids, reduced the concentration of insulin and amino acids, and did not affect the concentration of glucose and glucagon before the glucose challenge. Surrogate insulin sensitivity indices indicated improved insulin sensitivity in 5-HTP cows, but due to the unique metabolism of lactating ruminants, these index changes may instead reflect effects in insulin-independent glucose disposal, like milk synthesis. Challenging 5-HTP-treated cows with a glucose bolus reduced the insulin spike and blunted the decrease in free fatty acids, compared to saline cows, without changing glucose dynamics. Overall, these results suggest that serotonin stimulates insulin-independent glucose disposal, requiring less insulin to maintain normoglycemia.

Untargeted Metabolomic Analysis of Lactation-Stage-Matched Human and Bovine Milk Samples at 2 Weeks Postnatal.

Epidemiological data demonstrate that bovine whole milk is often substituted for human milk during the first 12 months of life and may be associated with adverse infant outcomes. The objective of this study is to interrogate the human and bovine milk metabolome at 2 weeks of life to identify unique metabolites that may impact infant health outcomes. Human milk (n = 10) was collected at 2 weeks postpartum from normal-weight mothers (pre-pregnant BMI < 25 kg/m2) that vaginally delivered term infants and were exclusively breastfeeding their infant for at least 2 months. Similarly, bovine milk (n = 10) was collected 2 weeks postpartum from normal-weight primiparous Holstein dairy cows. Untargeted data were acquired on all milk samples using high-resolution liquid chromatography-high-resolution tandem mass spectrometry (HR LC-MS/MS). MS data pre-processing from feature calling to metabolite annotation was performed using MS-DIAL and MS-FLO. Our results revealed that more than 80% of the milk metabolome is shared between human and bovine milk samples during early lactation. Unbiased analysis of identified metabolites revealed that nearly 80% of milk metabolites may contribute to microbial metabolism and microbe-host interactions. Collectively, these results highlight untargeted metabolomics as a potential strategy to identify unique and shared metabolites in bovine and human milk that may relate to and impact infant health outcomes.