Effects of evaporative cooling and dietary zinc source on heat shock responses and mammary gland development in lactating dairy cows during summer.

The objective of this study was to examine the effects of evaporative cooling and dietary supplemental Zn source on heat shock responses and mammary gland development of lactating dairy cows during summer. Seventy-two multiparous lactating Holstein cows were randomly assigned to 1 of 4 treatments in a 2 × 2 factorial arrangement. Cows were either cooled (CL) or not cooled (NC) and fed diets supplemented with 75 mg of Zn/kg of dry matter (DM) from Zn hydroxychloride (IOZ) or 35 mg of Zn/kg of DM from Zn hydroxychloride plus 40 mg of Zn/kg of DM from Zn-Met complex (ZMC). The 168-d trial included a 12-wk baseline phase when all cows were cooled and fed respective dietary treatments, and a subsequent 12-wk environmental challenge phase when NC cows were deprived of evaporative cooling. Plasma was collected from a subset of cows (n = 24) at 1, 3, 5, 12, 26, 41, 54, 68, 81 d of the environmental challenge to measure heat shock protein (HSP) 70 concentration. Mammary biopsies were collected from another subset of cows (n = 30) at enrollment (baseline samples) and at d 7 and 56 of the environmental challenge to analyze gene expression related to heat shock response, apoptosis and anti-oxidative enzymes, and to examine apoptosis and cell proliferation using immunohistochemistry. Supplemental Zn source did not affect milk yield but NC cows produced less milk than CL cows. Supplemental Zn source had no effect on mammary gene expression of HSP27, 70, and 90 or plasma concentrations of HSP70. The NC cows had greater mammary gene expression of HSP than CL cows. Circulating HSP70 of NC cows gradually increased and was higher at 81 d of environmental challenge compared with CL cows. Relative to IOZ, ZMC cows tended to have lower total mammary cell proliferation but greater mammary apoptosis. There was a tendency of greater TNFRSF1A mRNA expression for ZMC compared with IOZ cows, which may suggest upregulated extrinsic apoptosis. At d 7 of environmental challenge, NC cows had numerically higher mammary apoptosis than CL cows although not statistically significant. The NC cows tended to have greater mRNA expression of CAT and SOD3 regardless of time, and had greater mRNA expression of GPX1 at d 56 and FAS at d 7 of the environmental challenge than CL cows. Relative to CL cows, mammary cell proliferation rate was higher for NC cows at d 56 of the environmental challenge. In conclusion, dietary source of supplemental Zn has substantial effect on mammary cell turnover in lactating dairy cows, and prolonged exposure to heat stress increases mammary cell proliferation.

Maternal heat stress reduces body and organ growth in calves: Relationship to immune status.

Late-gestation heat stress of dairy cows reduces fetal growth and influences postnatal performance and immune status of the offspring. Our first objective was to evaluate the effect of in utero heat stress on overall fetal and organ growth, particularly organs associated with immune function. The second objective was to examine the cellular mechanism of altered passive immunity in neonatal bull calves after in utero heat stress. Specifically, we examined the rate of apoptosis of intestinal cells early in life, as it is associated with gut closure. Dams were dried off approximately 45 d before expected calving and randomly assigned to 1 of 2 treatments: heat stress (HT) or cooling (CL). During the dry period all cows were housed under shade in a freestall barn, where the pen for CL cows was equipped with active cooling, including water soakers and fans, whereas the pen for HT cows had no soakers or fans. Using rectal temperature and respiration rate as indicators, heat stress was severe. Average rectal temperature in HT cows was 39.3°C compared with 39.0°C in CL cows, and HT cows had a respiration rate of 66.7 breaths/min compared with 43.2 breaths/min for CL cows. Bull calves (n = 30) were immediately separated from their dams at birth, weighed, and then killed before colostrum feeding (n = 5/treatment; d 0) or at 1 or 2 d of age following colostrum feeding (n = 5/treatment per day). After slaughter, the small intestine was removed and weighed, and samples from the jejunum were fixed for immunohistochemistry. Birth weight of bulls from HT dams was 1.1 kg lower than that of bulls from CL dams. Thymus, spleen, and heart weights of HT bulls were lower relative to those of CL bulls, whereas liver weight of HT bulls tended to be lower relative to that of CL bulls. Jejunal cell apoptosis decreased with age in both HT and CL calves after birth, mirroring gut closure. However, in utero heat stress increased the apoptotic rate in the jejunum, particularly at birth. We conclude that the chronic exposure to heat strain of HT compared with CL dams in late gestation significantly affected fetal growth and immune tissue development, which may be associated with reduced immune function in early life. Also, late-gestation heat stress increased calves' intestinal apoptosis in the first 2 d of life, which might explain the decreased IgG uptake and limited passive immune competence observed in previous studies.

Effects of acute and chronic heat stress on feed sorting behaviour of lactating dairy cows.

Nutritional strategies to mitigate the negative effects of heat stress on animal welfare and productivity often involve changes in ration formulation. However, cattle commonly sort their ration in favour of certain components, and it is not clear how feed sorting responds to heat stress. This study investigated the association between heat stress and feed sorting behaviour. Lactating Holstein dairy cows (n = 32; parity = 2.8±1.2; mean±SD) were housed in a free stall barn and milked 3×/day. Cows were fed individually using the Calan Broadbent Feeding System and offered ad libitum access to a total mixed ration (containing on a dry matter basis: 3.3% ryegrass hay, 16.5% ryegrass baleage, 24.7% corn silage, 11.1% brewers grains, 19.7% ground corn, 19.8% concentrate and 4.9% protein/mineral supplement), provided 1×/day. Beginning at 186±60 days in milk, cows were exposed to either: heat stress conditions (HT; n = 15) (average temperature-humidity index: 77.6), or evaporative cooling (CL; n = 17), consisting of misters and fans over the freestall and feed bunks. Data were collected during a 4-day baseline period, and two 4-day experimental periods: starting at 10 days after implementing treatments (defined as acute heat stress for HT cows), and at 62 days after implementing treatments (defined as chronic heat stress for HT cows). Daily feed intake and physiological responses to heat stress (body temperature, respiration rate) were recorded. Samples of fresh and refused feed were collected daily from individual cows for particle size analysis. The particle size separator had three screens (19, 8 and 1.18 mm) and a bottom pan, resulting in 4 fractions (long, medium, short and fine particles). Feed sorting was calculated as the actual intake of each particle size fraction expressed as a percentage of the predicted intake of that fraction. During both heat stress periods, HT cows sorted for long particles more than CL cows (105.0% v. 100.6%; SE = 1.1). During acute heat stress, HT cows sorted to a greater extent than CL cows against medium and short particles, whereas sorting of these fractions did not differ during chronic heat stress. Body temperature and respiration rate were associated across treatments with the extent of sorting for long particles and against short particles during acute heat stress. These results suggest that feed sorting is particularly influenced during acute heat stress, and that sorting for longer particles may increase in heat stress.

Effects of heat stress and dietary zinc source on performance and mammary epithelial integrity of lactating dairy cows.

Dietary Zn and heat stress alter gut integrity in monogastric animals. However, effects of Zn on mammary epithelial integrity in heat-stressed lactating dairy cows have not been studied. Multiparous lactating Holstein cows (n = 72) were randomly assigned to 1 of 4 treatments with a 2 × 2 factorial arrangement to study the effects of environment and Zn source on performance and mammary epithelial integrity. Treatments included 2 environments [cooled (CL) or not cooled (NC)] and 2 Zn sources [75 mg/kg of supplemental Zn as Zn hydroxychloride (IOZ) or 35 mg/kg of Zn hydroxychloride + 40 mg/kg of Zn-Met complex (ZMC)]. The experiment was divided into baseline and environmental challenge phases of 84 d each. All cows were cooled during the baseline phase (temperature-humidity index = 72.5), whereas NC cows were not cooled during environmental challenge (temperature-humidity index = 77.7). Mammary biopsies were collected on d 7 and 56 relative to the onset of environmental challenge to analyze gene expression of claudin 1, 4, and 8, zonula occludens 1, 2, and 3, occludin, and E-cadherin and protein expression of occludin and E-cadherin. Deprivation of cooling increased respiration rate (64.8 vs. 73.9 breaths/min) and vaginal temperature (39.03 vs. 39.94°C) and decreased dry matter intake (26.7 vs. 21.6 kg/d). Energy-corrected milk yield decreased for NC cows relative to CL cows (24.5 vs. 34.1 kg/d). An interaction between environment and Zn source occurred for milk fat content as CL cows fed ZMC had lower milk fat percentage than other groups. Relative to CL cows, NC cows had lower concentrations of lactose (4.69 vs. 4.56%) and solids-not-fat (8.46 vs. 8.32%) but a higher concentration of milk urea nitrogen (9.07 vs. 11.02 mg/mL). Compared with IOZ, cows fed ZMC had lower plasma lactose concentration during baseline and tended to have lower plasma lactose concentration during environmental challenge. Plasma lactose concentration tended to increase at 3, 5, and 41 d after the onset of environmental challenge in NC cows relative to CL cows. Treatment had no effect on milk BSA concentration. Cows fed ZMC tended to have higher gene expression of E-cadherin relative to IOZ. Compared with CL, NC cows had increased gene expression of occludin and E-cadherin and tended to have increased claudin 1 and zonula occludens 1 and 2 gene expression in the mammary gland. Protein expression of occludin and E-cadherin was unchanged. In conclusion, removing active cooling impairs lactation performance and affects gene expression of proteins involved in the mammary epithelial barrier, and feeding a portion of dietary zinc as ZMC improves the integrity of the mammary epithelium.

Short communication: Repeated mammary tissue collections during lactation do not alter subsequent milk yield or composition.

Mammary biopsy collection (MB) is a valuable approach for studying mammary gland biology, but it is unclear if repeated MB impair the performance of lactating dairy cows. The objective of this trial was to examine the effect of repeated MB during lactation on udder health, dry matter intake (DMI), and lactation performance of lactating dairy cows. Sixty-four multiparous, mid-lactation Holstein cows were enrolled in a 29-wk trial, and 32 cows were randomly selected for repeated MB. The MB and non-MB (NMB) cows had similar parity (2.6 ± 0.9) and days in milk (96.5 ± 56.3 d) at enrollment. All animals were housed in the same barn and managed in the same manner. Cows were milked 3 times daily with milk yield recorded at each milking. Milk composition was measured weekly and DMI recorded daily. Three MB were performed per cow: 1 wk after enrollment and at 15 and 24 wk. The first and third MB were performed on the left rear quarter, whereas the second MB was on the right rear quarter. The MB were performed based on previously described procedures using a rotating stainless steel cannula with a retractable blade connected to a cordless drill, with appropriate sedation and antiseptic treatment after each MB. After MB, udder health, surgical wound healing, and presence of blood in milk were visually examined at each milking. Blood was cleared from milk 3.86 ± 2.0 d after MB. During the experiment, 4 rear quarters of MB cows and 5 rear quarters from NMB cows were diagnosed and treated for clinical mastitis. No differences were observed in DMI, milk yield, somatic cell score, or milk concentration and yields of fat, protein, lactose, and solids-not-fat between MB and NMB. In conclusion, lactating cows recover rapidly from MB, and repeated MB have no long-term effects on DMI, milk yield and composition, or udder health of lactating dairy cows.

Effects of feeding betaine-containing liquid supplement to transition dairy cows.

Betaine is a natural compound found in sugar beets that serves as a methyl donor and organic osmolyte when fed to animals. The objective was to evaluate the effect of feeding betaine-containing molasses on performance of transition dairy cows during late summer in 2 trials. In early September, cows were randomly assigned to betaine (BET) or control (CON) groups either shortly after dry off (trial 1; n = 10 per treatment) or 24 d before calving (trial 2; n = 8 per treatment) based on parity and previous mature equivalent milk yield. Cows were fed common diets supplemented either with a liquid supplement made of molasses from sugar cane and condensed beet solubles containing betaine [BET, 89.1 g/kg of dry matter (DM)] or a sugar cane molasses-based liquid supplement without betaine (CON) until 8 wk postpartum. The liquid supplements had similar nutrient contents and were fed at a rate of 1.1 and 1.4 kg DM/d for pre- and postpartum cows, respectively. Starting at their entry in the studies, cows were housed in the same freestall barn without a cooling system. After calving, all cows were housed in the same barn cooled by misters and fans and milked thrice daily. Intake was recorded daily and body weight and body condition score were assessed every 2 wk. Milk yield was recorded at each milking and composition was analyzed weekly. Blood samples were collected weekly from a subset of cows to assess concentrations of metabolites and AA. No treatment effects were apparent for DM intake and body weight in the prepartum and postpartum periods. For cows enrolled at dry off, BET supported higher milk yield (45.1 vs. 41.9 kg/d) and fat content (4.78 vs. 4.34%) and elevated plasma concentrations of nonesterified fatty acids and ß-hydroxybutyrate in early lactation compared to CON. However, no differences were observed for milk yield, most milk component contents and yields, and blood metabolites between treatments for cows enrolled during the close-up period. Compared to cows in the CON group, BET cows enrolled during the far-off period tended to have lower plasma concentrations of Met, Thr, and Trp during the pre- and postpartum periods. They also had lower plasma concentrations of Lys and Phe before calving but higher plasma Gly concentration after parturition. In conclusion, feeding a betaine-containing liquid supplement from far-off through early lactation improves lactation performance but increases adipose tissue mobilization and production of ketone bodies in early lactation.

In utero heat stress decreases calf survival and performance through the first lactation.

Calves born to cows exposed to heat stress during late gestation (i.e., the dry period) have lower birth weight and weaning weight and compromised passive immune transfer compared with those born to dams that are cooled. However, it is unknown if heat stress in utero has carryover effects after weaning. The objective was to evaluate the effect of heat stress (HT) or cooling (CL) in late gestation dairy cows on the survival, growth, fertility, and milk production in the first lactation of their calves. Data of animals obtained from previous experiments conducted during 5 consecutive summers in Florida were pooled and analyzed. Cows were dried off 46d before expected calving and randomly assigned to 1 of 2 treatments, HT or CL. Cooled cows were housed with sprinklers, fans, and shade, whereas only shade was provided to HT cows. Within 4h of birth, 3.8 L of colostrum was fed to calves from both groups of cows. All calves were managed in the same manner and weaned at 49d of age. Birth weight and survival of 146 calves (HT=74; CL=72) were analyzed. Additionally, body weight, growth rate, fertility, and milk production in the first lactation from 72 heifers (HT=34; CL=38) were analyzed. As expected, HT calves were lighter (means ± SEM; 39.1±0.7 vs. 44.8±0.7kg) at birth than CL calves. Cooled heifers were heavier up to 1yr of age, but had similar total weight gain (means ± SEM; 305.8±6.3 vs. 299.1±6.3kg, respectively) compared with HT heifers. No effect of treatment was observed on age at first insemination (AI) and age at first parturition. Compared with CL heifers, HT heifers had a greater number of services per pregnancy confirmed at d 30 after AI, but no treatment effect was observed on number of services per pregnancy confirmed at d 50 after AI. A greater percentage of CL heifers reached first lactation compared with HT heifers (85.4 vs. 65.9%). Moreover, HT heifers produced less milk up to 35wk of the first lactation compared with CL heifers (means ± SEM; 26.8±1.7 vs. 31.9±1.7kg/d), and no difference in body weight during lactation was observed (means ± SEM; HT: 568.4±14.3kg; CL: 566.5±14.3kg). These data suggest that heat stress during the last 6wk of gestation induces a phenotype that negatively affects survival and milk production up to and through the first lactation of offspring.

Short communication: Effect of maternal heat stress in late gestation on blood hormones and metabolites of newborn calves.

Maternal heat stress alters immune function of the offspring, as well as metabolism and future lactational performance, but its effect on the hormonal and metabolic responses of the neonate immediately after birth is still not clear. The objective of this study was to investigate the blood profiles of hormones and metabolites of calves born to cows that were cooled (CL) or heat-stressed (HS) during the dry period. Within 2 h after birth, but before colostrum feeding, blood samples were collected from calves [18 bulls (HS: n=10; CL: n=8) and 20 heifers (HS: n=10; CL: n=10)] born to CL or HS dry cows, and hematocrit and plasma concentrations of total protein, prolactin, insulin-like growth factor-I, insulin, glucose, nonesterified fatty acid, and ß-hydroxybutyrate were measured. Compared with CL, HS calves had lower hematocrit and tended to have lower plasma concentrations of insulin, prolactin, and insulin-like growth factor-I. However, maternal heat stress had no effect on plasma levels of total protein, glucose, fatty acid, and ß-hydroxybutyrate immediately after birth. These results suggest that maternal heat stress desensitizes a calf's stress response and alters the fetal development by reducing the secretion of insulin-like growth factor-I, prolactin, and insulin.

Short communication: Effect of heat stress on markers of autophagy in the mammary gland during the dry period.

Heat stress (HT) during the dry period compromises mammary gland (MG) growth, thus negatively affecting subsequent milk yield. Cooling during the late dry period, when mammary tissue proliferates, is a common management practice. However, it neglects MG involution during the early dry period, a process that is accomplished by both apoptosis and autophagy. Our objective was to evaluate the effect of HT on MG autophagy during the early dry period. Holstein cows were dried off ~45d before expected calving and randomly assigned to 1 of 2 treatments: HT or cooling (CL). All cows were housed in the same free stall barn during the dry period, but only the stall area for CL cows was equipped with soakers and fans. Rectal temperature and respiration rate were measured daily during the dry period. Mammary gland biopsies were collected from each cow 3d before dry-off and on d 3, 7, 14, and 22±2 after dry-off. Autophagy in the MG was determined by measuring protein expression of 2 autophagic markers, autophagy-related protein 7 and microtubule-associated protein light chain 3 (LC3). The average temperature-humidity index during the dry period was 77.7, which indicated that HT and CL cows were exposed to significant heat stress. However, the cooling system effectively alleviated heat strain in CL cows by decreasing the rectal temperature (39.0 vs. 39.4°C) and respiration rate (47.3 vs. 71.2 breaths per minute) relative to HT cows. Protein expression of autophagy-related protein 7, a marker for early autophagosome formation, did not change within or between groups. In contrast, protein expression of LC3-II, a marker of autophagosomes, and its precursor LC3-I showed a dynamic expression pattern in MG from CL cows during the early dry period. Relative to HT cows, MG from CL cows displayed higher expression of LC3-I and LC3-II on d 7 and lower expression of LC3-II on d 14 and 22 after dry-off. Collectively, our data provide a possible mechanistic explanation for the impairment of MG capacity in HT dairy cows. Heat stress-related perturbations of autophagic activity may compromise the regenerative MG involution that is necessary for optimal cell proliferation.

Conceptus development and transcriptome at preimplantation stages in lactating dairy cows of distinct genetic groups and estrous cyclic statuses.

The objectives were to compare development and transcriptome of preimplantation conceptuses 15 d after synchronized ovulation and artificial insemination (AI) according to the genetic background of the cow and estrous cyclicity at the initiation of the synchronization program. On d 39±3 postpartum, Holstein cows that were anovular (HA; n=10), Holstein cows that were estrous cyclic (HC; n=25), and Jersey/Holstein crossbred cows that were estrous cyclic (CC; n=25) were randomly selected in a grazing herd and subjected to the Ovsynch protocol. All cows were inseminated on d 49±3 postpartum, which was considered study d 0. Blood was sampled and analyzed for concentrations of progesterone, estradiol, insulin, and insulin-like growth factor 1 (IGF-1) on study d -10, -3, -1, 7, and 15 relative to AI. On study d 15, uteri were flushed and recovered fluid had IFN-τ concentrations measured and subjected to metabolomic analysis. Morphology of the recovered conceptuses was evaluated, and mRNA was extracted and subjected to transcriptome microarray analysis. Compared with HC, CC presented greater concentrations of progesterone and estradiol in plasma, with corpora lutea and preovulatory follicles of similar size. Conceptuses from CC were larger, tended to secrete greater amounts of IFN-τ, and had greater transcript expression of peroxisome proliferator-activated receptor gamma (PPARγ), an important transcription factor that coordinates lipid metabolism and elongation at preimplantation development. In addition, pregnant CC had greater concentrations of anandamide in the uterine flush, which might be important for elongation of the conceptus and early implantation. Conceptuses from HA were also longer and secreted greater amounts of IFN-τ than conceptuses from HC, likely because of the distinct progesterone profiles before and after AI. Nonetheless, anovular cows had reduced concentrations of IGF-1 in plasma, and their conceptuses presented remarkable transcriptomic differences. Some of the altered transcripts suggest that conceptus cells from anovular cows might be under greater cellular stress and presented markers suggesting increased apoptosis and autophagy, which could lead to increased mortality after d 15 of development. Estrous cyclicity had more impact on transcriptome of bovine conceptus than genetic background, and the developmental changes observed during the preimplantation period might be linked to differences in fertility among groups.

Effect of maternal heat stress during the dry period on growth and metabolism of calves.

Preliminary studies suggest that maternal heat stress (HS) during late gestation exerts carryover effects on a calf's insulin response after weaning, but a comprehensive evaluation of how maternal HS affects calf intake, growth, and metabolic response from birth to weaning is lacking. Our objective was to evaluate the effects of maternal HS during the dry period on dry matter intake, growth, and metabolism from birth to weaning. After birth, 20 heifers born to either HS (n=10) or cooled (CL, n=10) dry cows were immediately separated from their dams and fed 3.8 L of colostrum from a common pool within 4h of birth. All heifers were managed identically and weaned at 49 d of age (DOA). Calf starter intake was recorded daily, and body weight was assessed at birth and every 2 wk from birth to 56 DOA. Blood samples were collected twice a week until 56 DOA to assess hematocrit and concentrations of insulin and metabolites. To evaluate metabolic responses to maternal HS, a glucose tolerance test, insulin, and epinephrine challenge were performed on 3 consecutive days for all heifers at 8, 29, and 57 DOA. Maternal HS during the dry period did not affect heifer birth weight. Compared with HS, CL calves consumed more starter (0.53 vs. 0.34kg/d) from birth to 56 DOA and were heavier (71.7 vs. 61.4kg) at 56 DOA. Relative to HS calves, CL calves tended to have higher hematocrit (27.4 vs. 24.7%). No differences were found between treatments in plasma concentrations of insulin and glucose, but HS calves had higher nonesterified fatty acids and ß-hydroxybutyrate concentrations after 32 DOA. Compared with CL, HS calves had a faster glucose clearance after a glucose tolerance test and a slower insulin clearance after an insulin challenge. In conclusion, maternal HS during late gestation reduces calf starter intake and growth, alters blood metabolite profile, and increases noninsulin-dependent glucose uptake.

Effects of late-gestation heat stress on immunity and performance of calves.

Lactating cows that experience heat stress will have reduced dry matter intake and milk yield and shift metabolism, which ultimately reduces the efficiency of milk production. Dry cows that are heat stressed similarly experience lower intake, reduced mammary growth, and compromised immune function that ultimately results in a poorer transition into lactation and lower milk yield in the next lactation. A recent focus in our laboratory is on the effects of late gestation, in utero heat stress on calf survival and performance. We have completed a series of studies to examine preweaning growth and health, and later reproductive and productive responses, in an attempt to quantify acute and persistent effects of in utero heat strain. Late gestation heat stress results in calves with lower body weight at birth, shorter stature at weaning, and failure to achieve the same weight or height at 12 mo of age observed in calves from dams that are cooled when dry. A portion of the reduced growth may result from the lower immune status observed in calves heat stressed in utero, which begins with poorer apparent efficiency of immunoglobulin absorption and extends to lower survival rates through puberty. Heat-stressed calves, however, have permanent shifts in metabolism that are consistent with greater peripheral accumulation of energy and less lean growth relative to those from cooled dams. Comparing reproductive performance in calves heat stressed versus those cooled in utero, we observe that the cooled heifers require fewer services to attain pregnancy and become pregnant at an earlier age. Tracking the milk production in calves that were heat stressed in utero versus those cooled in late gestation revealed a significant reduction of yield in the first lactation, approximately 5 kg/d through 35 wk of lactation, despite similar body weight and condition score at calving. These observations indicate that a relatively brief period of heat stress in late gestation dramatically alters the health, growth, and ultimate performance of dairy calves. Thus, it is critical to effectively manage heat stress of dry cows to avoid negative effects on the calf.

Effect of cooling during the dry period on immune response after Streptococcus uberis intramammary infection challenge of dairy cows.

Heat stress in the dry period affects the immune status of dairy cows in the subsequent lactation. We hypothesized that cooling during the dry period improves immune response to postpartum intramammary infection (IMI) by environmental pathogens such as Streptococcus uberis. Cows were dried off 46 d before expected calving and assigned to cooling (CL, n = 15) or heat stress (HT, n = 15). Cooled cows were housed with sprinklers, fans, and shade, whereas the HT group had only shade. All cows were cooled postpartum. Rectal temperature (RT) and respiration rate (RR) were recorded thrice weekly during the dry period. From −46 to 42 d relative to calving, dry matter intake was recorded daily, and both body weight (BW) and body condition score (BCS) weekly. Milk yield and composition were recorded daily after calving. Streptococcus uberis IMI was induced at 5 d postpartum in a subset of cows (CL, n = 5; HT, n = 5). Blood was collected at 0, 12, 18, 24, and 36 h after IMI. Hematological analysis was performed, and neutrophils isolated for RNA extraction. Immune response genes (TLR2, IL1-ß, IL6, IL8, IL10, and TNFα) were assessed by real-time, reverse transcription-PCR. Relative to HT cows, CL cows had lower RT and RR during the dry period. The CL cows also consumed more feed prepartum but not postpartum, gained more BW prepartum but lost more BW in lactation, and had higher BCS score prepartum and a lower BCS postpartum. During 40 wk of lactation, CL cows produced more milk (33.8 vs. 30.0 kg/d) than HT cows but milk composition was not affected. Cows in the CL group had greater white blood cell counts and more neutrophils than HT cows during IMI. From 0 to 36 h post-IMI, TNFα mRNA expression decreased, whereas that of IL6 and IL8 increased in both treatments. Additionally, CL cows had lower IL10 mRNA expression at 18 h post-IMI. Expression of TLR2 mRNA decreased over time in both treatments. However, CL cows had greater overall TLR2 mRNA expression than HT. No differences were detected for mRNA expression of IL1-ß, IL6, IL8, or TNFα. Cooling cows during the dry period alters immune function and neutrophil response to IMI in early lactation.

Effect of heat stress during late gestation on immune function and growth performance of calves: isolation of altered colostral and calf factors.

Calves born to cows exposed to heat stress during the dry period and fed their dams' colostrum have compromised passive and cell-mediated immunity compared with calves born to cows cooled during heat stress. However, it is unknown if this compromised immune response is caused by calf or colostrum intrinsic factors. Two studies were designed to elucidate the effects of colostrum from those innate to the calf. The objective of the first study was to evaluate the effect of maternal heat stress during the dry period on calf-specific factors related to immune response and growth performance. Cows were dried off 46 d before expected calving and randomly assigned to 1 of 2 treatments: heat stress (HT; n=18) or cooling (CL; n=18). Cows of the CL group were housed with sprinklers, fans and shade, whereas cows of HT group had only shade. After calving, the cows were milked and their colostrum was frozen for the subsequent study. Colostrum from cows exposed to a thermoneutral environment during the dry period was pooled and stored frozen (-20 °C). Within 4h of birth, 3.8L of the pooled colostrum from thermoneutral cows was fed to calves born to both HT and CL cows. Day of birth was considered study d 0. All calves were exposed to the same management and weaned at d 49. Blood samples were collected before colostrum feeding, 24h after birth and twice weekly up to d 28. Total serum IgG concentrations were determined. Body weight was recorded at birth and at d 15, 30, 45, and 60. Relative to CL calves, HT calves were lighter at birth (38.3 vs. 43.1 kg), but no difference in weight gain was observed at d 60. Additionally, HT calves had lower apparent efficiency of IgG absorption (26.0 vs. 30.2%), but no differences were observed for total IgG concentration. The objective of the second study was to evaluate the isolated effect of the colostrum from HT cows on calf immune response and growth performance. The experimental design was identical to the first study, but all calves were born to cows under thermoneutral conditions during the dry period. At birth, calves were blocked by sex and birth weight and then randomly assigned to 1 of 2 treatments, which meant they received pooled colostrum from HT cows or CL cows. No treatment effect was observed on passive immune transfer or on postnatal growth. Thus, heat stress during the last 6 wk of gestation negatively affects the ability of the calf to acquire passive immunity, regardless of colostrum source.

Short communication: Maternal heat stress during the dry period alters postnatal whole-body insulin response of calves.

Heat stress during the dry period not only negatively affects a cow's performance but also affects her offspring. Previous studies indicate that calves born to cows heat-stressed during late gestation have lower birth weight but similar overall weight gain during the prepubertal period compared with those cooled in utero. However, it is unclear if whole-body insulin response, and thus metabolism, of calves is altered in their postnatal life after in utero heat stress. The aim of the present study was to examine the effects of maternal heat stress during the dry period on whole-body insulin response of calves after weaning. Calves (10/treatment) were born to cows exposed to heat stress (HT) or cooling (CL) when dry. Calves were immediately separated from their dams and fed 3.8L of high-quality colostrum within 1h after birth and then 1.9L 12h later. All calves were fed 1.9 to 3.8L of pasteurized milk in the morning and afternoon from 2 to 42 d of age and then only in the morning until weaning at 49 d. Calf starter and water were offered ad libitum starting at 2 d of age. All calves were managed in the same manner throughout the study. All calves were subjected to a glucose tolerance test (GTT) and an insulin challenge (IC) at 55 d of age. Calves heat-stressed in utero were born lighter (40 ± 1.4 vs. 45 ± 1.4 kg) compared with CL calves. Both groups of calves had similar weaning weights (HT: 68 ± 3.2 kg; CL: 71 ± 3.3 kg) and body weight gain from birth to weaning (HT: 28 ± 2.2 kg; CL: 26 ± 2.3 kg). Compared with those cooled in utero, HT calves had a similar insulin response to GTT and insulin clearance during IC but faster glucose clearance during GTT and IC. In conclusion, in addition to impaired fetal growth, maternal heat stress during the dry period enhances the whole-body insulin response of calves after weaning, which suggests the possibility of accelerated lipogenesis and fat deposition in early life.

Prevalence of periparturient diseases and effects on fertility of seasonally calving grazing dairy cows supplemented with concentrates.

The objectives were to characterize the prevalence of periparturient diseases and their effects on reproductive performance of dairy cows in seasonal grazing farms. A total of 957 multiparous cows in 2 farms (555 in farm A and 402 in farm B) were evaluated and diseases characterized. At calving, dystocia, twin birth, stillbirth, and retained fetal membranes were recorded and grouped as calving problems. On d 7±3 and 14±3 postpartum, cows were evaluated for metritis and on d 28±3 for clinical endometritis based on scoring of the vaginal discharge. From parturition to 30 d after artificial insemination (AI), prevalence of mastitis, lameness, and digestive and respiratory problems were recorded. For subclinical diseases, diagnosis was based on blood samples collected from 771 cows and analyzed for concentrations of Ca, nonesterified fatty acids (NEFA), and ß-hydroxybutyrate. Cows were considered as having elevated NEFA concentration if the concentration was ≥0.70 mM, subclinical ketosis if the ß-hydroxybutyrate concentration was ≥0.96 mM, and subclinical hypocalcemia if the Ca concentration was ≤2.14 mM. Ovaries were scanned on d 35±3 and 49±3 postpartum for determination of estrous cyclicity. All cows were enrolled in a timed AI program and inseminated on the first day of the breeding season: on average, 86 d postpartum. Overall, 37.5% (359/957) of the cows presented at least 1 clinical disease and 59.0% (455/771) had at least 1 subclinical health problem. Prevalence of individual diseases was 8.5% for calving problems, 5.3% for metritis, 15.0% for clinical endometritis, 13.4% for subclinical endometritis, 15.3% for mastitis, 2.5% for respiratory problems, 4.0% for digestive problems, 3.2% for lameness, 20.0% for elevated NEFA concentration, 35.4% for subclinical ketosis, and 43.3% for subclinical hypocalcemia. Clinical and subclinical diseases had additive negative effects on reproduction, delaying resumption of estrous cyclicity and reducing pregnancy per AI (P/AI). Occurrence of multiple diseases further reduced reproductive efficiency compared with a single disease. Individually, subclinical hypocalcemia, elevated NEFA concentration, metritis, and respiratory and digestive problems reduced estrous cyclicity by d 49 postpartum. Elevated NEFA concentration, calving problem, metritis, clinical and subclinical endometritis, and digestive problems reduced P/AI on d 65 after AI. Moreover, calving problems and clinical endometritis increased the risk of pregnancy loss between gestation d 30 and 65. Serum concentrations of Ca and NEFA were negatively correlated, and both were associated with prevalence of uterine diseases. In conclusion, periparturient diseases were highly prevalent in seasonally calving grazing dairies and affected cows had delayed resumption of estrous cyclicity, reduced P/AI, and increased risk of pregnancy loss.

Effect of late-gestation maternal heat stress on growth and immune function of dairy calves.

Heat stress during the dry period affects the cow's mammary gland development, metabolism, and immunity during the transition period. However, the effect of late-gestation heat stress on calf performance and immune status is unknown. Our objective was to evaluate the effect of heat stress during the final ~45 d of gestation on growth and immune function of calves. Calves (17/treatment) were born to cows that were exposed to cooling (CL) or heat stress (HT) during the dry period. Only heifer calves (CL, n=12; HT, n=9) were used in measurements of growth and immune status after birth. Heifer calves were managed under identical conditions. All were fed 3.78 L of colostrum from their respective dams within 4 h of birth and were weaned at 2 mo of age (MOA). Body weight (BW) was obtained at weaning and then monthly until 7 MOA. Withers height (WH) was measured monthly from 3 to 7 MOA. Hematocrit and plasma total protein were assessed at birth, 1, 4, 7, 11, 14, 18, 21, 25, and 28 d of age. Total serum IgG was evaluated at 1, 4, 7, 11, 14, 18, 21, 25, and 28 d of age, and apparent efficiency of absorption was calculated. Peripheral blood mononuclear cells were isolated at 7, 28, 42, and 56 d of age, and proliferation rate was measured by (3)H-thymidine incorporation in vitro. Blood cortisol concentration was measured in the dams during the dry period and in calves in the preweaning period. Gestation length was 4d shorter for HT cows compared with CL cows. Calves from CL cows had greater BW than calves from HT cows at birth (42.5 vs. 36.5 kg). Compared with CL heifers, HT heifers had decreased weaning BW (78.5 vs. 65.9 kg) but similar BW (154.6 vs. 146.4 kg) and WH (104.8 vs. 103.4 cm) from 3 to 7 MOA. Compared with CL, heifers from HT cows had less total plasma protein (6.3 vs. 5.9 g/dL), total serum IgG (1,577.3 vs. 1,057.8 mg/dL), and apparent efficiency of absorption (33.6 vs. 19.2%), and tended to have decreased hematocrit (33 vs. 30%). Additionally, CL heifers had greater peripheral blood mononuclear cell proliferation relative to HT heifers (23.8 vs. 14.1 fold). Compared with CL, late-gestation HT did not affect the blood cortisol concentration of dams during the dry period or that of the calves in the preweaning period, but CL calves tended to have increased circulating cortisol at birth (7.6 vs. 5.7 µg/dL). We conclude that heat stress of the dam during the dry period compromises the fetal growth and immune function of offspring from birth through weaning.

Effect of cooling heat-stressed dairy cows during the dry period on insulin response.

Heat stress (HT) during the dry period affects hepatic gene expression and adipose tissue mobilization during the transition period. In addition, it is postulated that HT may alter insulin action on peripheral tissues. Our objective was to evaluate the effect of cooling heat-stressed cows during the dry period on insulin effects on peripheral tissues during the transition period. Cows were dried off 46 d before expected calving and assigned to 1 of 2 treatments: HT (n = 16) or cooling (CL, n = 16). During the dry period, the average temperature-humidity index was 78, but CL cows were cooled with sprinklers and fans, whereas HT cows were not. After calving, all cows were housed and managed under the same conditions. Rectal temperatures were measured twice daily (0730 and 1430 h) and respiration rate recorded 3 times weekly during the dry period. Dry matter intake was recorded daily from dry-off to 42 d relative to calving (DRC). Body weight and body condition score were measured weekly from dry-off to 42 DRC. Milk yield and composition were recorded daily to 42 wk postpartum. Glucose tolerance tests (GTT) and insulin challenges (IC) were performed at dry-off, -14, 7, and 28 DRC in a subset of cows (HT, n = 8; CL, n = 8). Relative to HT, CL cows had lower rectal temperatures (39.3 vs. 39.0°C) in the afternoon and respiration rate (69 vs. 48 breath/min). Cows from the cooling treatment tended to consume more feed than HT cows prepartum and postpartum. Compared with HT, CL cows gained more weight before calving but lost more weight and body condition in early lactation. Cows from the cooling treatment produced more milk than HT cows (34.0 vs. 27.7 kg/d), but treatments did not affect milk composition. Treatments did not affect circulating insulin and metabolites prepartum, but CL cows had decreased glucose, increased nonesterified fatty acid, and tended to have lower insulin concentrations in plasma postpartum compared with HT cows. Cooling prepartum HT cows did not affect the insulin responses to GTT and IC during the transition period and glucose responses to GTT and IC at -14 and 28 DRC were not affected by treatments. At 7 DRC, CL cows tended to have slower glucose clearance to GTT and weaker glucose response to IC relative to HT cows. Cows from the cooling treatment had stronger nonesterified fatty acid responses to IC postpartum but not prepartum compared with HT. In conclusion, cooling heat-stressed dairy cows in the dry period reduced insulin effects on peripheral tissues in early lactation but not in the dry period.

Effects of presynchronization and length of proestrus on fertility of grazing dairy cows subjected to a 5-day timed artificial insemination protocol.

The objectives were to compare the effects of 2 methods of presynchronization and 2 lengths of proestrus on fertility of grazing dairy cows subjected to a 5-d timed artificial insemination (AI) protocol at initiation of breeding season. Lactating dairy cows (n=1,754) from 3 seasonal grazing farms were blocked within farm by breed, parity, and days in milk (DIM). Study d 0 was considered the day of AI of cows in COS72 (72h of proestrus). Within each block, cows were randomly assigned to 1 of 2 presynchronization treatments: a PGF(2α)-based program, Presynch, consisting of 2 injections of PGF(2α) administered on d -32 and -18, or a PGF(2α)-GnRH-based program, Double-Ovsynch (DO), consisting of GnRH on d -25, PGF(2α) on d -18, and GnRH on d -15. Within each of the 2 presynchronization treatments, cows were randomly assigned to 1 of 2 lengths of proestrus within the 5-d timed AI protocol, consisting of GnRH on d -8, PGF(2α) on d -3 and -2, and GnRH+AI at either 58 h (COS58) or 72 h (COS72) after the d -3 PGF(2α) injection. Ovaries were scanned by ultrasonography twice, on d -42 and -32, to determine estrous cyclicity before enrollment in the study. Blood was sampled and analyzed for concentrations of estradiol on the day of AI. Pregnancies per AI (P/AI) were determined 30 and 65 d after AI. Presynchronization did not affect the concentration of estradiol at AI (DO=6.4 vs. Presynch=5.8 pg/mL), detection of estrus at AI (20.8 vs. 25.9%), or P/AI on d 30 (56.8 vs. 59.1%) and 65 (52.5 vs. 52.4%) after the first AI. Cows receiving COS72 had increased concentration of estradiol (6.6 vs. 5.5 pg/mL) and detection of estrus at AI (28.5 vs. 10.8%) compared with cows receiving COS58. Length of proestrus did not affect P/AI on d 30 (COS72=58.7 vs. COS58=56.1%) but, in Presynch cows, COS58 was detrimental to fertility on d 65 after AI (54.9 vs. 46.5%). Pregnancy loss between gestational d 30 and 65 was greater for Presynch than for DO (7.6 vs. 11.3%), but it was not affected by length of proestrus. Estrous cyclic cows had greater P/AI than anovular cows on d 30 (61.7 vs. 35.1%) and 65 (56.1 vs. 30.7%), but no interaction between estrous cyclic status and treatments was detected. Crossbred Holstein/Jersey cows had superior fertility than their purebred counterparts during the breeding season. The Presynch and DO protocols resulted in similar fertility with no overall difference between the presynchronization methods; however, limiting the length of proestrus to 58 h reduced P/AI in the 5-d timed AI protocol when cows had their estrous cycle presynchronized with Presynch but not with DO.

Antianxiety and antidepressant effects of riparin III from Aniba riparia (Nees) Mez (Lauraceae) in mice.

This work presents behavioral effects of methyl ethers of N-(2,6-dihydroxybenzoyl) tyramine (riparin III) isolated from the unripe fruit of Aniba riparia on the open field, elevated plus maze (EPM), rotarod, hole board, barbiturate-induced sleeping time, tail suspension and forced swimming tests in mice. Riparin III was administered intraperitoneally to male mice at single doses of 25 and 50 mg/kg. The results showed that riparin III with both doses had no effects on spontaneous motor activity in mice or in the rotarod test, but decreased the number of grooming and rearing. At the dose of 50 mg/kg, riparin III increased the number of entries in the open arms of the EPM test as compared with control. Similarly, in the hole-board test, both doses increased the number of head dips. There was a reduction on the sleeping latency with both doses and a prolongation of the pentobarbital-induced sleeping time with the dose of 25 mg/kg. In the tail suspension test, similar to imipramine (30 mg/kg), riparin III at the dose of 50 mg/kg presented a reduction in the immobility time. In the forced swimming test, both doses of riparin III decreased the immobility time. These results showed that riparin III potentiated the barbiturate-induced sleeping time and presented antidepressant- and anxiolytic-like effects.