I.M. Thompson

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

Heat stress during the dry period affects the cows 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 heat stress during the dry period on mammary gland development

Heat stress during the dry period negatively affects hepatic metabolism and cellular immune function during the transition period, and milk production in the subsequent lactation. However, the cellular mechanisms involved in the depressed mammary gland function remain unknown. The objective of the present study was to determine the effect of heat stress during the dry period on various indices of mammary gland development of multiparous cows. Cows were dried off approximately 46 d before expected calving and randomly assigned to 2 treatments, heat stress (HT, n=15) or cooling (CL, n=14), based on mature equivalent milk production. Cows in the CL treatment were provided with sprinklers and fans that came on when ambient temperatures reached 21.1°C, whereas HT cows were housed in the same barn without fans and sprinklers. After parturition, all cows were housed in a freestall barn with cooling. Rectal temperatures were measured twice daily (0730 and 1430 h) and respiration rates recorded at 1500 h on a Monday-Wednesday-Friday schedule from dry off to calving. Milk yield and composition were recorded daily up to 280 d in milk. Daily dry matter intake was measured from dry off to 42 d relative to calving. Mammary biopsies were collected at dry off, -20, 2, and 20 d relative to calving from a subset of cows (HT, n=7; CL, n=7). Labeling with Ki67 antigen and terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick-end labeling were used to evaluate mammary cell proliferation and apoptosis, respectively. The average temperature-humidity index during the dry period was 76.6 and not different between treatments. Heat-stressed cows had higher rectal temperatures in the morning (38.8 vs. 38.6°C) and afternoon (39.4 vs. 39.0°C), greater respiration rates (78.4 vs. 45.6 breath/min), and decreased dry matter intake (8.9 vs. 10.6 kg/d) when dry compared with CL cows. Relative to HT cows, CL cows had greater milk production (28.9 vs. 33.9 kg/d), lower milk protein concentration (3.01 vs. 2.87%), and tended to have lower somatic cell score (3.35 vs. 2.94) through 280 d in milk. Heat stress during the dry period decreased mammary cell proliferation rate (1.0 vs. 3.3%) at -20 d relative to calving compared with CL cows. Mammary cell apoptosis was not affected by prepartum heat stress. We conclude that heat stress during the dry period compromises mammary gland development before parturition, which decreases milk yield in the next lactation.

Effects of lactation and pregnancy on gene expression of endometrium of Holstein cows at day 17 of the estrous cycle or pregnancy

Abstract Objectives were to determine effects of lactation and pregnancy on endometrial gene expression on d 17 of the estrous cycle and pregnancy. Heifers (n=33) were assigned randomly after parturition to lactating (L, n=17) or nonlactating (NL, n=16) groups. Cows were subjected to an ovulation synchronization program for a timed artificial insemination (TAI); 10 cows in L and 12 in NL were inseminated. Slaughter occurred 17 d after the day equivalent to TAI, and intercaruncular endometrial tissues were collected. Gene expression was determined by DNA microarray analysis for pregnant (L, n=8; NL, n=6) and noninseminated cyclic (L, n=7; NL, n=4) cows. Differentially expressed genes were selected with a P-value <0.01 and absolute expression >40. In addition, a fold effect >1.5 was used as a criterion for genes affected by pregnancy. In total, 210 genes were differentially regulated by lactation (136 downregulated and 74 upregulated), and 702 genes were differentially regulated by pregnancy (407 downregulated and 295 upregulated). The interaction effect of pregnancy and lactation affected 61 genes. Genes up- and downregulated in pregnant cows were associated with several gene ontology terms, such as defense response and interferon regulatory factor, cell adhesion, and extracellular matrix. The gene ontology analyses of up- and downregulated genes of lactating cows revealed terms related to immunoglobulin-like fold, immune response, COMM domain, and non-membrane-bounded organelle. Several genes upregulated by lactation, such as IGHG1, IGLL1, IGK, and TRD, were related to immune function, particularly for B cells and γδ T cells. Developmental genes related to limb and neural development and glucose homeostasis (e.g., DKK1, RELN, PDK4) were downregulated by lactation, whereas an interaction was also detected for RELN. The stated genes associated with immune function and developmental genes expressed in the endometrium affected by lactational state are possible candidate genes for interventions to improve fertility of lactating dairy cows.

Effects of resynchronization programs on pregnancy per artificial insemination, progesterone, and pregnancy-associated glycoproteins in plasma of lactating dairy cows

Objectives were to develop a timed artificial insemination (TAI) resynchronization program to improve pregnancy per AI and to evaluate responses of circulating progesterone and pregnancy-associated glycoproteins in lactating cows. Cows (n=1,578) were presynchronized with 2 injections of PGF2alpha, given 14 d apart starting on d 45+/-3 postpartum, followed by Ovsynch [2 injections of GnRH 7 d before and 56 h after injection of PGF2alpha, TAI 16 h after second injection (d 0)]. The Resynch-treated cows received an intravaginal progesterone insert from d 18 to 25, GnRH on d 25, and pregnancy diagnosis on d 32, and nonpregnant cows received PGF2alpha., GnRH 56 h later, and TAI 16 h later (d 35). The control cows were diagnosed for pregnancy on d 32 and nonpregnant cows received GnRH, PGF2alpha 39 d after TAI, GnRH 56 h later, and TAI 16 h later (d 42). Pregnancy was reconfirmed on d 60 after AI. Ovarian structures were examined in a subset of cows at the time of GnRH and PGF2alpha injections. Blood samples for analyses of progesterone and pregnancy-associated glycoproteins were collected every 2 d from d 18 to 30 in 100 cows, and collection continued weekly to d 60 for pregnant cows (n=43). Preenrollment pregnancies per AI on d 32 did not differ for cows subsequently treated as Resynch (45.8%, n=814) and control (45.9%, n=764), and pregnancy losses on d 60 were 6.7 and 4.0%, respectively. Resynchronized service pregnancy per AI (36%, n=441; 39.5%, n=412) and pregnancy losses (6.3 and 6.7%) did not differ for Resynch and control treatments, respectively. Days open for pregnant cows after 2 TAI were less for the Resynch treatment than for the control treatment (96.2+/-0.82 vs. 99.5+/-0.83 d). Cows in the Resynch treatment had more large follicles at the time of GnRH. The number of corpora lutea did not differ between treatments at the time of PGF2alpha. Plasma progesterone for pregnant cows was greater for Resynch cows than for control cows (18-60 d; 6.6 vs. 5.3 ng/mL), and plasma concentrations of progesterone on d 18 were greater for pregnant cows than for nonpregnant cows (5.3 vs. 4.3 ng/mL). Plasma pregnancy-associated glycoproteins during pregnancy were lower for cows in the Resynch treatment compared with control cows on d 39 (2.8 vs. 4.1 ng/mL) and 46 (1.3 vs. 3.0 ng/mL). Cows pregnant on d 32 that lost pregnancy by d 60 (n=7) had lower plasma concentrations of pregnancy-associated glycoproteins on d 30 than cows that maintained pregnancy (n=36; 2.9 vs. 5.0 ng/mL). Pregnancy-associated glycoproteins on d 30 (>0.33 ng/mL) were predictive of a positive d 32 pregnancy diagnosis (sensitivity=100%; specificity=90.6%). In conclusion, Resynch and control protocols had comparable pregnancy per AI for first and second TAI services, but pregnancy occurred 3.2 d earlier in the Resynch group because inseminations in the Resynch treatment began 7 d before those in the control treatment. Administration of an intravaginal progesterone insert, or GnRH, or both increased progesterone during pregnancy. Dynamics of pregnancy-associated glycoproteins were indicative of pregnancy status and pregnancy loss.

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 lactation and pregnancy on metabolic and hormonal responses and expression of selected conceptus and endometrial genes of Holstein dairy cattle

Objectives were to characterize postpartum metabolic and hormonal differences between nonlactating and lactating dairy cows, evaluate lactation and pregnancy effects on endometrium and conceptus expression of selected genes, and characterize associations between conceptus and endometrial expression of genes in early pregnancy (d 17). Pregnant heifers were assigned randomly after calving to a lactating group (L, n=17) and a nonlactating group (NL, n=16). The L cows were fed a total mixed ration [1.65 Mcal of net energy for lactation (NE(L))/kg, 16.5% crude protein (CP)] ad libitum, and the NL cows were fed a maintenance ration (1.45 Mcal of NE(L)/kg, 12.2% CP) once per day. All cows were presynchronized and enrolled in a timed artificial insemination (TAI) protocol; 10 cows in the L and 12 in the NL received TAI. On d 17 after GnRH and TAI, cows were slaughtered and endometrial and conceptus tissues collected. The Affymetrix Bovine Genome DNA Microarray (Affymetrix Inc., Santa Clara, CA) was used to assess conceptus and endometrial gene expression. The L cows had higher body temperature than the NL cows (38.4 vs. 38.2°C), and the NL cows cycled earlier than the L cows (26.3 vs. 34.7 d in milk). Cows in the L group had greater plasma concentrations of β-hydroxybutyrate (4.90 vs. 2.97 mg/dL) and blood urea N (11.6 vs. 6.5mg/dL) and lower concentrations of glucose (74.0 vs. 79.9 mg/dL) compared with NL cows. Insulin-like growth factor-1 was lower for L compared with NL (140.5 vs. 198.2 ng/mL) and was greater for cows subsequently classified pregnant compared with cyclic (191.0 vs. 147.6 ng/mL). The concentration of progesterone from GnRH or TAI (d 0) until d 17 was lower for L cows than for NL cows. Gene expression analyses indicated that all conceptuses (n=13) expressed pregnancy-associated glycoprotein (PAG) genes PAG2, PAG8, PAG11, and PAG12. The same PAG family genes were observed in the endometrium of some pregnant cows. Simple and standard partial correlation analyses detected associations of conceptus PAG11 with prostaglandin regulatory genes. In conclusion, lactation altered metabolic status, delayed initiation of cyclicity, and decreased concentrations of progesterone in pregnant cows. Early expression of PAG genes in the conceptus may contribute to successful development of early pregnancy and possibly alter mechanisms related with embryo survival such as prostaglandin synthesis.

LACTATION BIOLOGY SYMPOSIUM: Effects of photoperiod on mammary gland development and lactation

Photoperiod, or the daily sequence of light and dark, has dramatic effects on many physiological systems across animal species. Light patterns alter melatonin secretion profiles and, subsequently, the release profiles and circulating concentrations of several hormones that influence a variety of physiological responses. Although the impact of photoperiod on reproductive processes is perhaps the most common example, it is often the seasonal aspects of ovulation and anestrus that are considered. However, in cattle, the final phase of reproduction, that is, lactation, is significantly influenced by photoperiod. In contrast to short days (SDPP; 8 h light:16 h dark), exposure to long days (LDPP) of 16 to 18 h of light and 6 to 8 h of darkness increases milk yield 2 to 3 kg/d, regardless of the stage of lactation. There is evidence that this LDPP effect is due to increased circulating IGF-I, independent of any effect on GH concentrations. Cows that are housed under SDPP during the dry period have increased mammary growth and produce 3 to 4 kg/d more milk in the subsequent lactation compared with cows on LDPP when dry. While cows are on SDPP, circulating prolactin (PRL) diminishes but expression of PRL receptor increases in mammary, liver, and immune cells. Moreover, PRL signaling pathways within those tissues are affected by photoperiod. Further, replacement of PRL to cows on SDPP partially reverses the effects of SDPP on production in the next lactation. Thus, effects on dry cows are mediated through a PRL-dependent pathway. Before maturity, LDPP improve mammary parenchymal accumulation and lean body growth, which lead to greater yields in the first lactation. The accumulated evidence supports the concept that photoperiod manipulation can be harnessed to improve the efficiency of production across the life cycle of the dairy cow.

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 heat stress during the dry period on gene expression in mammary tissue and peripheral blood mononuclear cells

Heat stress (HT) during the dry period compromises mammary gland development, decreases future milk production, and impairs the immune status of dairy cows. Our objective was to evaluate the effect of cooling HT cows during the dry period on gene expression of the mammary gland and peripheral blood mononuclear cells (PBMC). Cows were dried off 46 d before their expected calving and assigned to 2 treatments, HT or cooling (CL). Cows in the CL group were cooled with sprinklers and fans whereas HT cows were not. After parturition, all cows were housed in a freestall barn with cooling. The PBMC were isolated at dry-off and at -20, 2, and 20 d relative to calving from a subset of cows (HT, n=9; CL, n=10), and mammary biopsies were taken at the same intervals (HT, n=7; CL, n=6) for RNA extraction. Gene expression was assessed using a custom multiplex gene expression assay based on traditional reverse transcription-PCR. Genes involved in prolactin (PRL) signaling [PRL receptor long form, PRL receptor short form, suppressor of cytokine signaling (SOCS)2, SOCS3, IGF2, IGF binding protein 5, and cyclin D1], fatty acid metabolism (acetyl-CoA carboxylase α (ACACA) and lipoprotein lipase (LPL)], and IGF1 were evaluated in mammary tissue, and genes related to fatty acid metabolism [ACACA, fatty acid synthase (FASN), and LPL], cytokine production [IL6, IL8, and tumor necrosis factor (TNF)], and IGF1 were evaluated in PBMC. No differences were observed in PRL signaling or fatty acid metabolism gene expression in the mammary gland. In PBMC, HT cows had greater mRNA expression of IGF1 and TNF during the transition period relative to CL and upregulated IL8 and downregulated FASN mRNA expression at 2 d relative to calving. We conclude that cooling HT cows during the dry period alters expression of genes involved in cytokine production and lipid metabolism in PBMC.

Environmental regulation of pregnancy-specific protein B concentrations during late pregnancy in dairy cattle.

Environmental factors, such as photoperiod and heat stress, can be manipulated during the dry period to influence health, productivity, and reproductive performance of dairy cows in their subsequent lactation. The impacts of photoperiod and heat stress on subsequent lactation are related to alterations in prolactin (PRL) signaling and may affect the expression of pregnancy-specific protein B (PSPB). Additionally, exposure of cows to heat stress during the dry period decreases gestation length; however, the mechanism involved in this process is unknown. The objective of these experiments was to evaluate the influence of environmental factors (i.e., heat stress and photoperiod) during late gestation (i.e., dry period) on PSPB concentrations in plasma of dairy cows. In Exp. 1, cows were dried off in the summer months approximately 46 d before expected calving and assigned randomly to heat stress (HT; n=30) or cooling (CL; n=30) treatment. Cooling cows were housed with sprinklers, fans, and shade, whereas HT cows were provided only shade. In Exp. 2, cows were dried off at approximately 60 d before expected calving in summer/fall months and randomly assigned to 3 treatments: long day photoperiod (LDPP: 16L:8D; n=15), short day photoperiod (SDPP: 8L:16D; n=14) and SDPP+PRL implant (12 mg/d of PRL at 28 d or 16 mg/d of PRL at 39 d; n=11). In both experiments, plasma samples were collected at dry off and at -32, -18, -7, -3 and 0 d relative to calving. In Exp. 1, greater concentrations of PSPB were detected in plasma of CL versus HT cows (388.3±24.7 vs. 287.4±23.8 ng/mL; P<0.01). Concentrations of PSPB did not differ between -46 to -18 d before calving (66.0 ng/mL). However, PSPB concentrations were greater (P<0.01) for CL cows at d -7 (534.7>357.2 ng/mL), -3 (807.2>572.2 ng/mL) and 0 (800.8>563.5 ng/mL) relative to calving. Additionally, HT cows in Exp. 1 had increased PRL plasma concentrations compared with CL cows (21.01±1.6 vs. 13.78±1.6 ng/mL). In Exp. 2, no differences were detected in plasma concentrations of PSPB (ng/mL) among LDPP, SDPP, or SDPP+PRL groups on d -60 (41.5), -32 (51.7), -18 (58.5), -7 (532.9), -3 (838.2), and 0 (729.4) relative to parturition. Photoperiodic PRL concentrations were 10.81, 7.84, and 4.22 ng/mL for LDPP, SDPP+ PRL, and SDPP, respectively. Results indicate that HT alters PSPB concentrations in late pregnancy, suggesting that placental activity is altered in cows exposed to excessive elevated temperatures around the time of calving. However, the mechanism involved likely is not associated with changes in PRL secretion.