Rumen-protected methionine supplementation during the transition period under artificially induced heat stress: impacts on cow-calf performance.

Dairy cows experiencing heat stress (HS) during the pre-calving portion of the transition period give birth to smaller calves and produce less milk and milk protein. Supplementation of rumen-protected methionine (RPM) has been shown to modulate protein, energy, and placenta metabolism, making it a potential candidate to ameliorate HS effects. We investigated the effects of supplementing RPM to transition cows under HS induced by electric heat blanket (EHB) on cow-calf performance. Six weeks before expected calving, 53 Holstein cows were housed in a tie-stall barn and fed a control diet (CON, 2.2% Met of MP) or a CON diet supplemented with Smartamine®M (MET, 2.6% Met of MP, Adisseo Inc., France). Four weeks pre-calving, all MET and half CON cows were fitted with an EHB. The other half of the CON cows were considered thermoneutral (TN), resulting in 3 treatments: CONTN (n = 19), CONHS (n = 17), and METHS (n = 17). Respiratory rate (RR), skin temperature (ST), and rectal temperature (RT) were measured thrice weekly and core body temperatures recorded bi-weekly. Post-calving body weights (BW) and BCS were recorded weekly, and DMI was calculated and averaged weekly. Milk yield was recorded daily and milk components were analyzed every third DIM. Biweekly AA and weekly nonesterified fatty acids (NEFA), ß-hydroxybutyrate (BHB), insulin, and glucose were measured from plasma. Calf birth weight and 24 h growth, thermoregulation, and hematology profile were measured and apparent efficiency of absorption (AEA) of immunoglobulins was calculated. Data were analyzed using the MIXED procedure of SAS with 2 preplanned orthogonal contrasts: CONTN vs. the average of CONHS and METHS (C1) and CONHS vs. METHS (C2). Relative to TN, EHB cows had increased RT during the post-calving weeks and increased RR and ST during the entire transition period. Body weight, BCS, DMI, and milk yield were not impacted by the EHB or RPM. However, protein % and SNF were lower in CONHS, relative to METHS cows. At calving, METHS dams had higher glucose concentrations, relative to CONHS, and during the post-calving weeks, the EHB cows had lower NEFA concentrations than TN cows. Calf birthweight and AEA were reduced by HS, while RR was increased by HS. Calf withers height tended to be shorter and RT were lower in CONHS, compared with MTHS heifers. Overall, RPM supplementation to transition cows reverts the negative impact of HS on blood glucose concentration at calving and milk protein % in the dams and increases wither height while decreasing RT in the calf.

Review: Phenotypic and molecular evidence of inter- and trans-generational effects of heat stress in livestock mammals and humans.

Internal and external factors can change an individual's phenotype. A significant external threat to humans and livestock is environmental heat load, a combination of high ambient temperatures and humidity. A heat stress response occurs when an endothermal animal is exposed to a heat load that challenges its' thermoregulation capacity. With the ongoing climate change trends, the incidence of chronically elevated temperatures causing heat stress is expected to rise, posing an even greater risk to the health and survival of all species. Heat stress is generally related to adverse effects on food intake, health, and performance in mammal livestock species and humans. Evidence from epidemiological and experimental studies of humans and livestock demonstrated that exposing pregnant females to heat stress affects the phenotype of the newborn in various ways. For instance, in utero heat stress is related to lower BW at birth and changes in metabolic and immune functions in the newborn. In cows, the effects of heat stress on the performance of the offspring last for three or four generations, suggesting intergenerational effects. The molecular mechanism orchestrating these effects of heat stress may be epigenetic regulation, as various epigenetic mechanisms control genome reprogramming. Epigenetic modifications are attached to DNA and histone proteins and can influence how specific genes are expressed, resulting in phenotypic changes. Epigenetic modifications can be triggered in response to environmental heat stress without altering the DNA sequence. Heat stress insults during critical periods of organ development (i.e., fetal exposure) can trigger epigenetic modifications that impact health and productivity across generations. Thus, epigenetic changes caused by extreme temperatures can be passed down to the offspring if the mother is exposed to the insult during pregnancy. Understanding the phenotypic and molecular consequences of maternal heat stress, including the carry-over lingering effects on the resulting progeny, is necessary to develop effective mitigation strategies and gain translational knowledge about the fundamental processes leading to intergenerational and transgenerational inheritance. This review examines the phenotypic and molecular evidence of how maternal exposure to extreme heat can affect future generations in several species, including humans, swine, sheep, goats, and cattle. The current knowledge of the molecular mechanisms involved in intergenerational and transgenerational epigenetic inheritance will also be presented and discussed.

Late-gestation heat stress in Holstein dams programs in utero development of daughter's germline, triggering skin and hair morphology adaptations of granddaughters.

Homeostasis and thermoregulation are influenced by the interplay of hair coat and skin characteristics. Our previous work indicated that hair and skin adaptations, triggered by in utero heat stress, affect thermoregulation in postnatal life. Herein, we investigate multigenerational carry-over effects of late-gestation heat stress on hair and skin characteristics beyond the first generation. Pregnant Holstein dams (F0, grand-dams) were heat stressed (HT, shade, n = 41) or provided active cooling (CL, shade, fans, and water soakers, n = 41) for the last 56 d of gestation during summer (temperature-humidity index ≥68). The first generation of heifers (F1, daughters) who were in utero heat stressed (HTF1, n = 36) or not (CLF1, n = 37) were born and raised as a cohort from birth to first calving. Thirty second-generation heifers (F2, granddaughters), born to HTF1 (HTF2, n = 12) and CLF1 (CLF2, n = 18), were raised as a cohort from birth to 70 d of age. Hair samples and skin biopsies from HTF2 and CLF2 were collected on postnatal d 70 (n = 6/group). Hair samples were subdivided into topcoat and undercoat, and skin tissue was fixed for ~18 h in 10% formalin, dehydrated, paraffin-embedded, sectioned, and stained with hematoxylin and eosin to visualize morphology. Variables analyzed included hair length and diameter; stratum corneum cross-sectional area and thickness; epidermis thickness; sweat gland depth, number, cross-sectional area, and average size; and sebaceous gland number, cross-sectional area, and average size. Measurements were performed using the ImageJ software and analyzed using PROC MIXED in SAS (SAS Institute Inc.). Hair length and diameter tended to be shorter and thicker in HTF2, compared with CLF2. The HTF2 skin had smaller stratum corneum cross-sectional area and tended to a thinner epidermis. to CLF2, HTF2 skin had more but smaller sebaceous glands, whereas no differences in sweat glands were observed. In summary, we report phenotypic alterations in hair and skin characteristics of granddaughters. Whether these adaptations grant improved postnatal thermoregulatory ability for the granddaughters remains to be investigated.

Unraveling the immune and metabolic changes associated with metritis in dairy cows.

The objective was to unravel the peripartum immune and metabolic changes associated with metritis in Holstein cows. Holstein cows (n = 128) had blood collected at -14, 0, 3, and 7 d relative to parturition (DRP). Flow cytometry was used to evaluate blood leukocyte counts, proportions, and activation. Total cells, live cells, single cells, monocytes (CD172α+/CD14+), polymorphonuclears (CD172α+/CD14-/SSChigh), B-cells (CD21+/MHCII+), CD4+ T-cells (CD4+), CD8+ T-cells (CD8+), and γδ T-cells (γδTCR+) were evaluated. Both CD62L and CD11b were used as markers of cell activation. Major histocompatibility complex class II was used as a marker of antigen presentation in monocytes. A Milliplex Bovine Cytokine/Chemokine 08-plex kit was used to evaluate plasma concentrations of IFN-γ, IL-1α, IL-1ß, IL-4, IL-6, IL-8, IL-10, and tumor necrosis factor-α. The body weight (BW) change prepartum was calculated as the difference between calving BW and prepartum BW divided by the number of days between measurements. Plasma fatty acids (FA) were measured at -14 and 0 DRP using untargeted gas chromatography with time-of-flight mass spectrometry. Data were analyzed by ANOVA for repeated measures. Cows that developed metritis (n = 57) had greater prepartum BW, prepartum BW loss, and greater FA concentrations at calving. Plasma FA at calving was positively correlated with IL-1ß. Cows that developed metritis had persistent systemic inflammation, which was demonstrated by greater B-cell activation, greater pro-inflammatory cytokine concentrations, and greater cell damage pre- and postpartum. Postpartum, we observed greater polymorphonuclear cell activation and extravasation but lesser monocytes and CD4+ T-cells activation and extravasation, which suggests postpartum immune tolerance. Greater prepartum adiposity in cows that developed metritis may lead to systemic inflammation pre- and postpartum and immune tolerance postpartum, which may lead to failure to prevent bacterial infection, and development of puerperal metritis.

Amplifying local serotonin signaling prior to dry-off hastens mammary gland involution and redevelopment in dairy cows.

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.

Methods for detecting heat stress in hutch-housed dairy calves in a continental climate.

Dairy calves exposed to solar radiation, elevated ambient temperature, and humidity are at risk of impaired welfare and productivity. Initial detection of thermal discomfort requires determination of optimal heat stress indicators and thresholds. Such values have recently been established in calves in chronic, subtropical, and acute continental environments but not in continuous, temperate conditions. Herein, the objectives were to determine associations between animal-based and environmental heat stress indicators and establish environmental breakpoints for hutch-raised dairy calves during a continental summer. From June to August, dairy calves (n = 63; 14 to 42 d of age) were individually hutch-housed and managed according to the dairy standard operating procedures in Arlington, Wisconsin. Calf respiration rates (RR), rectal temperatures (RT), shaved or unshaved skin temperatures (ST), and hutch internal and external air speed were measured thrice weekly at 0700 and 1400 h after a 15 min hutch restriction. Environmental indices including dry bulb temperature (Tdb), black globe temperature, and relative humidity were measured every 15 min, averaged hourly, and used to calculate temperature-humidity index (THI) using 8 different equations (THI1-8). Correlation and linear regression models were used to determine relationships within and between animal-based and environmental indicators. Environmental breakpoints were established using segmented regression models to estimate THI and Tdb thresholds for abrupt changes in animal responses. There were strong, positive correlations between animal-based indicators and Tdb or THI1-8, with the strongest association observed between unshaved ST and Tdb (r = 0.80). The linear regression of animal-based indicators with the best fit included Tdb or Tdb plus relative humidity and air speed. The threshold at which RR and RT began to rise was at a THI of 69 for both or at a Tdb of 21.0 or 21.5°C, respectively. No threshold was established for ST. Together, these outcomes indicate that Tdb is an appropriate measurement to detect thermal discomfort for calves in a temperate summer climate and individual hutch housing. Monitoring of calves is warranted before ambient temperature reaches 21.0°C, corresponding to RR of 40 breaths per minute and RT of 38.5°C, to promote calf comfort and reduce the risk of hyperthermia-related welfare and productivity consequences.

Effect of in utero exposure to hyperthermia on postnatal hair length, skin morphology, and thermoregulatory responses.

Skin and hair coat play important functions in maintaining homeostasis and thermoregulation for cattle, which can affect all modes of heat loss. Our objective was to investigate the effect of hyperthermia experienced in utero during late gestation on postnatal hair length, skin properties, and thermoregulation. Pregnant dams were heat stressed (n = 41) or actively cooled (n = 41) for the last ∼56 d of gestation and gave birth to heifers that were in utero heat stressed (IUHT) or in utero cooled (IUCL), respectively. Hair samples and skin tissue biopsies were collected from neck and rump locations at birth (d 0), 1 wk after weaning (d 63), and at 12 mo. Hair samples were also obtained at 4 and 8 mo. Skin tissue was stained with hematoxylin and eosin to visualize morphology. Hair length (short and long hairs, undercoat and topcoat, respectively), stratum corneum (SC) area, SC thickness, epidermis thickness, sweat gland (SWT) number, SWT cross-sectional area, SWT average size, sebaceous gland (SEB) number, SEB cross-sectional area, SEB average size, and sweat gland depth were assessed. Respiration rate, skin temperature, sweating rate, and rectal temperature was measured weekly from d 7 to 63. Additionally, thermoregulatory patterns were measured every 4 h over a 36-h interval beginning 4 d after weaning. Data were analyzed using PROC MIXED in SAS with a main effect of in utero treatment with location and time points analyzed separately. No difference in hair parameters were detected at d 0 or 12 mo. At d 63, IUHT heifers had longer average hair length (14.8 vs. 13.8 ± 0.2 mm, standard error), shorter undercoats (9.3 vs. 10.4 ± 0.3 mm), longer topcoats (19.6 vs. 17.1 ± 0.3 mm), and a greater difference between topcoat and undercoat (10.1 vs. 7.0 ± 0.4 mm). At 4 mo, IUHT heifers had longer average hair lengths (26.1 vs. 22.2 ± 1.0 mm) and longer topcoats (36.9 vs. 33.9 ± 1.1 mm), and at 8 mo, IUHT had longer average hair lengths (17.9 vs. 16.2 ± 0.6 mm), relative to IUCL. At d 0, IUHT heifers had more (13 vs. 9 ± 2 glands) but smaller average sized SEB (neck: 1,636 vs. 2,238 ± 243 µm2; rump: 2,100 vs. 3,352 ± 379 µm2) and reduced SC area (79,243 vs. 169,419 ± 13,071 µm2). At d 63, IUHT had fewer SEB (11 vs. 15 ± 2 glands), smaller SWT (0.16 vs. 0.23 ± 0.02 mm2), fewer SWT (16 vs. 23 ± 4 glands), and deeper SWT (0.5 vs. 0.4 ± 0.03 mm). At 12 mo, IUHT had greater distance from the skin surface to the most superficial SWT (0.016 vs. 0.015 ± 0.0004 mm), shorter distance to the deepest SWT (0.031 vs. 0.033 ± 0.001 mm), and smaller SWT (81.1 vs. 108.9 ± 10.8 µm2), relative to IUCL. When measured both weekly and hourly, IUHT heifers had higher rectal temperature and sweating rate. Overall, in utero hyperthermia triggers long-lasting hair and skin adaptations, possibly leading to differences in postnatal thermoregulation.

Flow cytometry panels for immunophenotyping dairy cattle peripheral blood leukocytes.

Many aspects of the bovine immune system remain poorly characterized, which poses an obstacle to improving dairy cow health. Herein, we describe two flow cytometry panels that included antibodies against CD8α, CD4, TCR-δ, CD172α, CD14, MHCII, CD21, CD62L, and CD11b. These panels were used to characterize the phenotype of leukocyte subpopulations from the peripheral blood of 30-day old Holstein calves and Holstein cows at 260 d of gestation and calving. No leukocyte subset differences were found between the pre- and post-partum cows. However, calf leukocytes presented a higher proportion of CD3+ lymphocytes, γδ T-cells, CD8+ γδ T-cells, and monocytes when compared with mature cows. Conversely, cow lymphocytes had a higher proportion of CD4+ and CD8+ T-cells, and B-cells than calf lymphocytes. The proportion of CD4+ T-cells and B-cells expressing CD62L was greater in calves than in cows, while cow B-cells expressed greater levels of CD11b than calf B-cells. In contrast, calf polymorphonuclear cells (PMN) and monocytes expressed greater levels of CD11b compared to cows. Moreover, calf monocytes expressed higher levels of MHCII compared with those of cows. Collectively, our data provides a resource to better understand the bovine immune system as well as immune-related diseases that affect dairy cattle.

Programming effects of late gestation heat stress in dairy cattle.

The final weeks of gestation represent a critical period for dairy cows that can determine the success of the subsequent lactation. Many physiological changes take place and additional exogenous stressors can alter the success of the transition into lactation. Moreover, this phase is pivotal for the final stage of intrauterine development of the fetus, which can have negative long-lasting postnatal effects. Heat stress is widely recognised as a threat to dairy cattle welfare, health, and productivity. Specifically, late gestation heat stress impairs the dam's productivity by undermining mammary gland remodelling during the dry period and altering metabolic and immune responses in early lactation. Heat stress also affects placental development and function, with relevant consequences on fetal development and programming. In utero heat stressed newborns have reduced birth weight, growth, and compromised passive immune transfer. Moreover, the liver and mammary DNA of in utero heat stressed calves show a clear divergence in the pattern of methylation relative to that of in utero cooled calves. These alterations in gene regulation might result in depressed immune function, as well as altered thermoregulation, hepatic metabolism, and mammary development jeopardising their survival in the herd and productivity. Furthermore, late gestation heat stress appears to exert multigenerational effects, influencing milk yield and survival up to the third generation.

Late gestation hyperthermia: epigenetic programming of daughter's mammary development and function.

Exposure to stressors during early developmental windows, such as prenatally (i.e., in utero), can have life-long implications for an animal's health and productivity. The mammary gland starts developing in utero and, like other developing tissues and organs, may undergo fetal programming. Previous research has implicated factors, such as prenatal exposure to endocrine disruptors or alterations in maternal diet (e.g., maternal over or undernutrition), that can influence the developmental trajectory of the offspring mammary gland in postnatal life. However, the direct links between prenatal insults and future productive outcomes are less documented in livestock species. Research on in utero hyperthermia effects on early-life mammary development is scarce. This review will provide an overview of key developmental milestones taking place in the bovine mammary gland during the pre- and postnatal stages. We will showcase how intrauterine hyperthermia, experienced by the developing fetus during the last trimester of gestation, derails postnatal mammary gland development and impairs its synthetic capacity later in life. We will provide insights into the underlying histological, cellular, and molecular mechanisms taking place at key postnatal developmental life stages, including birth, weaning and the first lactation, that might explain permanent detriments in productivity long after the initial exposure to hyperthermia. Collectively, our studies indicate that prenatal hyperthermia jeopardizes the normal developmental trajectory of the mammary gland from fetal development to lactation. Further, in utero hyperthermia epigenetically programs the udder, and possibly other organs critical to lactation, yielding a less resilient and less productive cow for multiple lactations.

Effect of late-gestation heat stress in nulliparous heifers on postnatal growth, passive transfer of immunoglobulin G, and thermoregulation of their calves.

Youngstock such as nulliparous pregnant dairy heifers are not typically considered for active cooling, as they are thought to be more heat tolerant than mature cows. Recently, the benefits of heat stress abatement in pregnant heifers were studied, but the effect of in utero hyperthermia on the calf is still unknown. Herein, we aimed to investigate the effects of late-gestation heat stress in nulliparous heifers on the growth, immune, and thermoregulatory responses of their calves. Pregnant nulliparous dams were randomly selected for either active cooling (CL; fans and soakers; n = 15) or heat stress (HT; n = 16) 60 d before expected calving. After birth, respiration rate, rectal temperature, skin temperature, and sweating rate were obtained from their heifer calves at 24 h and on d 14, 28, 42, and 56. Blood samples and body weights were both collected for measurement of total protein and hematocrit and calculation of average daily gains, respectively. Gestation length was shorter in HT heifers (272 vs. 276 ± 1.28 d) relative to CL heifers. Birth weights, weaning weights, body weights up to d 56, and average daily gain from birth to weaning were not different between in utero HT (IUHT; n = 13) and in utero CL (IUCL; n = 12) calves. Apparent efficiency of absorption of immunoglobulin G tended to be lower in IUHT calves (26.3 vs. 42.7 ± 9.0%), and serum IgG concentrations from birth to d 56 were significantly lower in IUHT calves relative to IUCL calves (22.0 vs. 32.4 ± 4.47 g/L). Postnatal respiration rate, rectal temperature, sweating rate, total protein, and hematocrit were not different between in utero treatments. There was a tendency for IUHT calves to have lower skin temperature at 24 h (34.9 vs. 36.9 ± 1.05°C), and skin temperature tended to be elevated in IUHT calves at d 56 (29.6 vs. 27.9 ± 1.05°C). In summary, the lack of heat stress abatement during late gestation reduces gestation length of nulliparous heifers. Additionally, providing active cooling to nulliparous heifers during the prepartum period confers immune benefits to their offspring, although it does not translate to growth improvements.

Late-gestation heat stress abatement in dairy heifers promotes thermoregulation and improves productivity.

Multiparous, nonlactating pregnant cows are negatively affected by heat stress, but the effect of heat stress on more thermotolerant pregnant heifers has received less attention. Our objective was to characterize the effect of late-gestation heat abatement on thermoregulatory responses and subsequent milk production of nulliparous Holstein heifers. Pregnant heifers, blocked by body condition score (BCS) and predicted transmitting ability (PTA) for milk, were enrolled in either heat stress (HT, shade of freestall barn; n = 16) or cooling (CL, shade of freestall barn, water soakers, and fans; n = 15) environments during the last 60 d of pregnancy (~8 weeks). Rectal temperature (RT; thermometer), respiration rate (RR; breaths/min), sweating rate (SR; VapoMeter, Delfin Technologies, Kuopio, Finland), and skin temperature (ST; infrared thermometer) were measured thrice weekly from enrollment to calving. Vaginal temperature (VT; i-button intravaginal device) was measured every 10 min for 7 consecutive days at wk -8, -6, -4, and -2 relative to calving and averaged hourly. Daily thermoregulatory patterns assessed by SR and ST, were measured every 4 h over a 36-h time interval at wk -6, -4, and -2 relative to calving. Upon calving, milk, protein, and fat yields were recorded twice daily for 15 wk. The average temperature-humidity index (Hobo Pro temperature probe, Onset Computer Corporation, Pocasset, MA) in the barn during the precalving period was 77 (minimum of 72, maximum of 82). Only heifers that gave birth to a female calf (CL = 12, HT = 14) were included in the statistical analysis. In the precalving period, CL heifers had lower RR (44.3 vs. 60.0 ± 1.6 breaths/min), RT (38.7 vs. 38.8 ± 0.04°C), unshaved ST (34.7 vs. 35.3 ± 0.17°C), and unshaved SR (19.0 vs. 35.2 ± 1.9 g/m2h), relative to HT heifers. Additionally, VT was lower in CL heifers during wk -4, and -2, specifically during early morning and early afternoon hours. When measured over a 36-h time interval, ST and SR were lower in CL heifers, when compared with HT heifers for all weeks. Notably, ST was reduced overnight and SR was reduced during the daytime. Cooled heifers had higher milk yield (35.8 vs. 31.9 ± 1.4 kg/d), when compared with HT heifers. Similar to multiparous cows, our data indicate that actively cooling heifers in late pregnancy is effective in promoting thermoregulation and results in elevated milk production postcalving.

Peripheral serotonin regulates glucose and insulin metabolism in Holstein dairy calves.

Peripheral serotonin regulates energy metabolism in several mammalian species, however, the potential contribution of serotonergic mechanisms as metabolic and endocrine regulators in growing dairy calves remain unexplored. Objectives were to characterize the role of serotonin in glucose and insulin metabolism in dairy calves with increased serotonin bioavailability. Milk replacer was supplemented with saline, 5-hydroxytryptophan (90 mg/d), or fluoxetine (40 mg/d) for 10-d (n = 8/treatment). Blood was collected daily during supplementation and on days 2, 7, and 14 during withdrawal. Calves were euthanized after 10-d supplementation or 14-d withdrawal periods to harvest liver and pancreas tissue. 5-hydroxytryptophan increased circulating insulin concentrations during the supplementation period, whereas both treatments increased circulating glucose concentration during the withdrawal period. The liver and pancreas of preweaned calves express serotonin factors (ie, TPH1, SERT, and cell surface receptors), indicating their ability to synthesize, uptake, and respond to serotonin. Supplementation of 5-hydroxytryptophan increased hepatic and pancreatic serotonin concentrations. After the withdrawal period, fluoxetine cleared from the pancreas but not liver tissue. Supplementation of 5-hydroxytryptophan upregulated hepatic mRNA expression of serotonin receptors (ie, 5-HTR1B, -1D, -2A, and -2B), and downregulated pancreatic 5-HTR1F mRNA and insulin-related proteins (ie, Akt and pAkt). Fluoxetine-supplemented calves had fewer pancreatic islets per microscopic field with reduced insulin intensity, whereas 5-hydroxytryptophan supplemented calves had increased islet number and area with greater insulin and serotonin and less glucagon intensities. After the 14-d withdrawal of 5-hydroxytryptophan, hepatic mRNA expression of glycolytic and gluconeogenic enzymes were simultaneously downregulated. Improving serotonin bioavailability could serve as a potent regulator of endocrine and metabolic processes in dairy calves.

Methods for assessing heat stress in preweaned dairy calves exposed to chronic heat stress or continuous cooling.

Although dairy calves are more thermotolerant relative to mature cows, they are still susceptible to heat stress, as demonstrated by elevated physiological responses and reduced feed intake under high ambient temperature and relative humidity. However, indicators of heat stress have not been well-characterized in calves. Herein, we evaluated associations between environmental and thermoregulatory and productive animal-based indicators of heat stress in dairy calves exposed to chronic heat stress or continuous cooling in a subtropical climate. Holstein calves were exposed to heat stress (HT; shade of barn, n = 24) or continuous cooling (CL; shade of barn plus 2 fans, n = 24) from 2 to 42 d of age. Environmental indices, including ambient temperature, relative humidity, temperature-humidity index (THI), and wind speed, and animal-based indices, including respiration (RR), heart rate (HR), rectal (RT), and skin temperature (ST) were recorded thrice daily (0900, 1300, and 1900 h). Milk replacer (MI) and grain intakes were recorded daily from 15 to 42 d of age. Using segmented regression models, we then estimated THI thresholds for significant changes in physiological responses. We found a strong, positive correlation between animal-based indicators (except for HR, MI, and grain intakes) and ambient temperature and THI, with the highest correlation obtained with ST and THI (r ≥ 0.72). Ambient temperature and ST and ambient temperature or THI and MI were the only correlations that differed between treatments. The coefficient of determination (R2) obtained from regression analyses to model animal-based indicators was substantially improved by the inclusion of environmental indicators, with the greatest improvement achieved with THI. Overall, continuous cooling by fans promoted calf heat loss as CL calves had lower RR, RT, ST, and higher feed intake compared with HT calves. Temperature-humidity index breakpoints could be determined for RT (THI = 67), RR (THI = 65), and MI (THI = 82) in HT calves, and only for RR (THI = 69) in CL calves. Skin temperature variables had no detectable breakpoints in either treatment due to the strong linear relationship to THI. Collectively, our results suggest that ST is appropriate to estimate chronic heat stress and that THI is the best environmental indicator of heat stress in dairy calves raised in a shaded, subtropical environment. At a practical level, calves should be closely monitored when THI reaches 65 to 69 to minimize the risk of heat stress-related impairments.

Increasing serotonin bioavailability alters gene expression in peripheral leukocytes and lymphoid tissues of dairy calves.

Dairy calves are born with a naïve immune system, making the pre-weaning phase a critical window for immune development. In the U.S., 40-60% of dairy farms feed milk replacer to pre-weaned calves, which are devoid of bioactive factors with immunological roles. Serotonin is a bioactive factor with immunoregulatory properties naturally produced by the calf and present in milk. Human and rodent immune cells express the serotonin machinery, but little is known about the role of serotonin in the bovine immune system. Supplementing milk replacer with 5-hydroxytryptophan (serotonin precursor) or fluoxetine (reuptake inhibitor) increases serotonin bioavailability. We hypothesized that increased serotonin bioavailability promotes serotonergic signaling and modulates the expression of immune related genes in peripheral leukocytes and immune-related tissues of dairy calves. The present experiment targeted candidate genes involved in serotonin production, metabolism, transport, signaling and immune regulation. We established that bovine peripheral leukocytes express all known serotonin receptors, and can synthesize, uptake and degrade serotonin due to the expression of serotonin metabolism-related genes. Indeed, we showed that increasing serotonin bioavailability alters gene expression of serotonin receptors and immune-related genes. Further research will determine whether manipulation of the serotonin pathway could be a feasible approach to bolster dairy calves' immune system.

Late-gestation heat stress impairs daughter and granddaughter lifetime performance.

Records of late-gestation heat stress studies conducted over 10 consecutive years in Florida were pooled and analyzed to test the hypothesis that maternal hyperthermia during late gestation impairs performance of the offspring across multiple generations and lactations, ultimately impeding the profitability of the US dairy sector. Dry-pregnant multiparous dams were actively cooled (CL; shade of a freestall barn, fans and water soakers, n = 196) or not (HT; shade only, n = 198) during the last 46 d of gestation, concurrent with the entire dry period. After data mining, records of 156 daughters (F1) that were born either to CL (CLF1, n = 77) or HT dams (HTF1, n = 79) and 45 granddaughters (F2) that were born either to CLF1 (CLF2, n = 24) or HTF1 (HTF2, n = 21) were used in the analysis. Life events and daily milk yield for 3 lactations of daughters and granddaughters were obtained. Milk yield, reproductive performance, and productive life data were analyzed using MIXED and GLIMMIX procedures, and lifespan was analyzed using PHREG and LIFETEST procedures of SAS (SAS Institute Inc., Cary, NC). Milk production of HTF1 was reduced in their first (2.2 kg/d), second (2.3 kg/d), and third lactations (6.5 kg/d) compared with CLF1. More HTF1 were culled before first calving, and the productive life and lifespan of HTF1 were reduced relative to CLF1 (4.9 and 11.7 mo, respectively). The granddaughters (HTF2) born to HTF1 produced less milk in their first lactation (1.3 kg/d) relative to granddaughters (CLF2) born to CLF1. More HTF2 were culled before first breeding relative to CLF2; however, productive life and lifespan were not different between HTF2 and CLF2 animals. An economic analysis was then performed based on the number of heat stress days, dry cows per state, and the aforementioned impairments on daughters' lifespans and milk production. Collectively in the United States, the economic losses for additional heifer rearing cost, reduced productive life, and reduced milk yield of the F1 offspring were estimated at $134, $90, and $371 million per year, respectively. In summary, late-gestation heat stress exerts carryover effects on at least 2 generations. Providing heat abatement to dry-pregnant dams is important to rescue milk loss of the dam and to prevent losses in their progeny.

Carry over effects of late-gestational heat stress on dairy cattle progeny.

The impacts of late gestation heat stress on the dam and her subsequent lactation are well-recognized. However, more recent research has demonstrated the long-lasting and severe negative consequences on the in-utero heat-stressed progeny. Dairy calves born to late gestation heat-stressed dams weigh less at birth and up to one year of age and have compromised metabolism and immune function. In-utero programming of these offspring may coordinate alterations in thermoregulation, mammary development, and milk synthetic capacity at different developmental windows. Thus, prenatally heat-stressed dairy heifers will produce less milk across multiple lactations and have a lower herd survival rate, potentially negatively impacting the U.S. dairy economy. Dry period heat stress abatement strategies should be considered not only for the productivity and welfare of the pregnant dam but also for the developing calf.

Late gestation heat stress in dairy cows: Effects on dam and daughter.

In dairy cattle, the final weeks before parturition are physiologically challenging and an important determinant of subsequent production performance. External stressors should be carefully managed during this period to avoid adding strain on the animals. Late-gestation heat stress impairs productivity in the dam and exerts transgenerational effects on progeny. Physiological responses are complex and detriments to performance are multifaceted. Late-gestation heat stress blunts mammary gland involution in the first half of the dry period and impairs cell proliferation as calving approaches. Moreover, cows that were exposed to prepartum heat-stress exhibit reduced adipose tissue mobilization and a lower degree of insulin resistance during early lactation. Prepartum heat exposure also depresses immune function and evidence links this decrease to altered prolactin signaling under heat stress. Placental functions are also impaired as reflected in a higher cotyledon mass but lower maternal circulating estrone sulfate concentrations, potentially resulting in lower nutrient supply and reduced calf birth weight. In addition, calves born to heat-stressed dams show impaired immune function and therefore higher disease susceptibly. Novel evidence reported that intrauterine heat stress alters the methylation profile of liver and mammary DNA, which may also contribute to the poorer performance during adulthood of calves exposed to heat stress in utero. Understanding the contribution of all altered biological systems during late-gestation heat stress can be used as a basis for improving cow management during the dry period. This article provides a review of the impacts of late-gestation heat stress and of the emerging understanding of the biological mechanisms that underlie the observed impairments of performance.

Pre- and postnatal heat stress abatement affects dairy calf thermoregulation and performance.

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.

Short communication: Effects of mammary biopsy in the dry period on activity and feeding behavior of dairy cows.

In dairy cattle, mammary biopsies are commonly used to study development and function of the mammary gland. The objective of this study was to investigate changes in activity and feeding patterns following the mammary biopsy procedure. Pregnant, nonlactating Holstein dairy cows (20 d before expected calving date) were exposed to either (1) a biopsy procedure, in which a mammary tissue sample (60 × 4 mm in diameter) was obtained from cows (n = 9) using a biopsy tool from the rear left quarter, following administration of a sedative (xylazine, 20 µg/kg of body weight) and local anesthetic (3 mL of lidocaine), or (2) a sham procedure, in which cows (n = 8) were removed from the pen and restrained for a similar duration of time as for the biopsy procedure. Behavior of cows was monitored for 5 d, beginning on the day following biopsy (approximately 14 h after the procedure). Cows were fitted with accelerometers to record daily lying time, lying bout frequency, and lying side. Daily individual feed intake was recorded using the Calan Broadbent feeding system, and feeding time and meal characteristics were determined from a subset of cows (n = 6 per treatment) using a change-of-state data logger to record the times the cows were accessing the feed bunk. Total daily lying time did not differ between treatments [13.9 h/d; standard error (SE) = 0.56], but biopsied cows had more frequent, shorter lying bouts on the biopsied side on d 1 following the procedure (6.67 vs. 4.25 bouts/d, SE = 1.03, and 70.0 vs. 97.0 min/bout, SE = 8.6; left vs. right side), whereas control cows had no side preference. We found no effects of treatment on feed intake and feeding time but, on the first day after treatment, biopsied cows had meals that were more frequent (7.2 vs. 4.6 meals/d; SE = 0.93) and tended to be shorter (28.2 vs. 60.9 min/meal; SE = 11.8) than control cows. In conclusion, we did not detect effects of mammary biopsy on feed intake or lying time during our time frame of observation, but activity patterns were altered, which could be indicative of increased overall restlessness and specific pain in the biopsied quarter.