Mechanisms by which feeding synthetic zeolite A and dietary cation-anion difference diets affect mineral metabolism in multiparous Holstein cows: Part I.

The periparturient period is characterized by the increased demand for calcium (Ca) in dairy cows. This has resulted in the use of several different prepartal nutritional strategies to prevent hypocalcemia postpartum. The objective of our study was to determine the effects of feeding synthetic zeolite A (XZ), a diet with negative dietary cation-anion difference (-DCAD), or a positive-DCAD diet (CON) during the close-up period on peripartal mineral dynamics and hormones involved in calcium metabolism. To this end, 121 multiparous Holstein cows, blocked by lactation number and expected due date, were enrolled at 254 d of gestation and randomly assigned to 1 of 3 prepartum diets: CON (+190 mEq/kg; n = 40), -DCAD (-65 mEq/kg; n = 41), or a diet supplemented with sodium aluminum silicate (XZ; +278 mEq/kg, fed at 3.3% DM, targeting 500 g/d; n = 40; Protekta Inc.). Blood, urine, and saliva samples were collected from enrollment until parturition, with data analyzed and presented beginning 14 d before parturition (d -14) until parturition (d 0), and on d 1, 2, 3, 6, 9, 12, 15, 18, 21, 35, and 49 postpartum, to assess mineral and hormone dynamics. Total fecal collections were performed in a subset of 8 cows per treatment group to assess fecal mineral loss. Data were analyzed as a randomized complete block design in SAS. Cows fed XZ and -DCAD had higher blood Ca concentrations compared with CON-fed cows, with XZ-fed cows exhibiting the highest blood Ca concentrations pre- and postpartum. Cows fed XZ had decreased blood and salivary phosphorus (P), increased fecal water-extractable phosphate, and the highest blood calcium concentrations pre- and postpartum. Parathyroid hormone was unaffected by diet but was increased at parturition in all treatments. Serotonin concentrations were increased in -DCAD and XZ cows compared with CON during the prepartum period. Our data indicate that the XZ group's improvement in blood Ca concentrations pre- and postpartum is most likely regulated by a dietary P restriction. Taken together, these data suggest that XZ and -DCAD diets improve postpartum calcium metabolism; however, they appear to work through different mechanisms.

Mechanisms by which feeding synthetic zeolite A and dietary cation-anion difference diets affect feed intake, energy metabolism, and milk performance: Part II.

The objectives of this study were to assess the effects of feeding 2 different diets, a diet with low dietary cation-anion difference (DCAD) or a diet with synthetic zeolite A, to multiparous Holstein cows during the close-up period on dry matter intake (DMI) and energy metabolism, as well as to evaluate colostrum and milk production. A total of 121 multiparous Holstein cows, blocked by lactation number and expected parturition date were enrolled at 254 d of gestation and randomly assigned to 1 of 3 dietary treatments: control (CON; +190 mEq/kg; n = 40), negative DCAD (-DCAD, -65 mEq/kg; n = 41; Ultra Chlor; Vita Plus, Lake Mills, WI), or a diet containing sodium aluminum silicate zeolite (XZ; +278 mEq/kg, fed at 3.3% dry matter, targeting 500 g/d; n = 40; X-Zelit, Protekta Inc., Lucknow, ON, Canada/Vilofoss, Graasten, Denmark). Prepartum DMI was measured daily using Insentec roughage intake control (RIC) gates (RIC System, Holofarm Group, the Netherlands). All cows received the same postpartum diet. Blood and urine samples were collected daily beginning 14 d before parturition (d -14) until parturition (d 0), and on 1, 2, 3, 6, 9, 12, 15, 18, 21, 35, and 49 d postpartum. Colostrum collected within 6 h of parturition, weighed, and based on samples' Brix value, IgG concentrations, and nutrient composition were analyzed. Prepartum, cows fed the XZ diet had decreased DMI (11.70 ± 0.26, 13.88 ± 0.26, and 13.45 ± 0.25 kg/d for XZ, CON, and -DCAD, respectively) and lower rumination (487 ± 8.1, 531 ± 8.3, and 527 ± 8.5 min for XZ, CON, and -DCAD, respectively) compared with CON and -DCAD. However, rumination was not different postpartum due to treatment. No prepartum or postpartum differences were observed for glucose or BHB concentrations in blood between dietary treatments. Colostrum collected from cows fed XZ had the highest IgG concentrations (91.10 ± 2.63, 78.00 ± 2.63, and 78.90 ± 2.63 mg/mL for XZ, CON, and -DCAD, respectively), but yield did not differ between dietary treatments. Additionally, cows in their third lactation or greater fed XZ had the highest milk production (51.0 ± 1.1 kg) during the first 49 d in milk. This study demonstrates that despite a decrease in DMI and rumination in cows fed XZ prepartum, blood BHB concentrations were not altered. Additionally, cows fed XZ had higher colostral IgG concentrations and cows in their third lactation or greater fed XZ produced the most milk. These data suggest that feeding XZ prepartum may improve colostrum quality and milk yield in mature cows, and does not affect energy metabolism.

Influence of prepartum dietary cation-anion difference and the magnitude of calcium decline at the onset of lactation on mineral metabolism and physiological responses.

The onset of lactation is characterized by substantially altered calcium (Ca) metabolism; recently, emphasis has been placed on understanding the dynamics of blood Ca in the peripartal cow in response to this change. Thus, the aim of our study was to delineate how prepartum dietary cation-anion difference (DCAD) diets and the magnitude of Ca decline at the onset of lactation altered blood Ca dynamics in the periparturient cow. Thirty-two multiparous Holstein cows were blocked by parity, previous 305-d milk yield and expected parturition date, and randomly allocated to either a positive (+120 mEq/kg) or negative (-120 mEq/kg) DCAD diet from 251 d of gestation until parturition (n = 16/diet). Immediately after parturition cows were continuously infused for 24 h with (1) an intravenous solution of 10% dextrose or (2) Ca gluconate (CaGlc) to maintain blood ionized (iCa) concentrations at ∼1.2 mM (normocalcemia) to form 4 treatment groups (n = 8/treatment). Blood was sampled every 6 h from 102 h before parturition until 96 h after parturition and every 30 min during 24 h continuous infusion. Cows fed a negative DCAD diet prepartum exhibited a less pronounced decline in blood iCa approaching parturition with lesser magnitude of decline relative to positive DCAD-fed cows. Cows fed a negative DCAD diet prepartum required lower rates of CaGlc infusion to maintain normocalcemia in the 24 h postpartum relative to positive DCAD-fed cows. Infusion of CaGlc disrupted blood Ca and P dynamics in the immediate 24 h after parturition and in the days following infusion. Collectively, these data demonstrate that prepartum negative DCAD diets facilitate a more transient hypocalcemia and improve blood Ca profiles at the onset of lactation whereas CaGlc infusion disrupts mineral metabolism.

Eucalcemia during lipopolysaccharide challenge in postpartum dairy cows: II. Calcium dynamics.

Hypocalcemia induced by immune activation is a conserved response among mammals. Early postpartum cows will experience decreased circulating Ca concentrations following acute immune activation; however, the cause for decreased Ca concentration is unknown. Our objectives were to (1) describe Ca dynamics following an intravenous (IV) LPS challenge in early postpartum cows, and (2) compare inflammatory-induced changes in Ca dynamics between IV Ca-treated cows and control cows. Cows (n = 14, 8 ± 1 d in milk) were enrolled in a matched-pair randomized controlled design to receive IV Ca (IVCa) in a eucalcemic clamp for 12 h, or 0.9% NaCl (CTRL) following an IV LPS infusion (0.040 or 0.045 µg of LPS/kg of body weight over 1 h). During the 24 h following LPS infusion, circulating concentrations of parathyroid hormone and serotonin were measured, serum and urine samples were collected to calculate urinary fractional excretion of Ca (FECa), and fecal samples were collected to calculate Ca apparent digestibility (ADCa) using amylase-treated and ash-corrected undigested neutral detergent fiber after 240 h (uNDFom240) as an internal marker. Changes in Ca intake and milk Ca secretion were also quantified and compared with baseline values. Cows were fasted during challenge and dry matter intake was 20 ± 5% less than baseline values on the day of challenge and did not differ between groups. On the day of challenge, milk Ca concentration increased, but milk yield decreased such that total Ca secreted in milk did not change from baseline. Urine FECa was low overall, but an interaction of treatment and time was identified such that FECa increased in IVCa but decreased in CTRL. Concentrations of parathyroid hormone increased and serotonin decreased following challenge. Fecal dry matter decreased from baseline, but did not differ between 6, 12, and 24 h, and did not differ between groups. An interaction of treatment and time was identified for ADCa and apparent digestibility of dry matter such that digestibility was decreased in CTRL but not IVCa at 6 h. Acute immune activation induced hypocalcemia in CTRL, and although urinary Ca excretion was not a primary cause, it is unclear to what degree hypocalcemia was due to altered ADCa. Eucalcemia appeared to alter adaptations in Ca homeostasis during immune activation as FECa was increased in IVCa animals.

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.

Physiological adaptations in early-lactation cows result in differential responses to calcium perturbation relative to nonlactating, nonpregnant cows.

The peripartal cow experiences a rapid change in calcium metabolism at the onset of lactation. Research has focused on understanding how mammary-derived factors, such as serotonin (5HT) and parathyroid hormone like hormone (PTHLH), aid in coordinating these calcemic adaptations to lactation. Therefore, the aim of our study was to determine how induced subclinical hypocalcemia influences physiological responses, specifically the 5HT-PTHLH-Ca axis, in lactating and nonlactating dairy cows to elucidate the potential contribution of the mammary gland. Twelve nonlactating, nonpregnant (NL) multiparous Holstein cows and 12 early-lactation (EL) multiparous Holstein cows received either (1) a continuous 24-h intravenous solution of 0.9% NaCl or (2) 5% ethylene glycol tetraacetic acid (EGTA) solution in 0.9% NaCl (n = 6 EL, n = 6 NL per treatment) with the aim of maintaining blood ionized calcium (iCa) less than 1.0 mM. Mammary gland biopsies were taken immediately after and 48 h after termination of infusion. Blood was sampled hourly during infusion and 4, 8, 12, 24, 48, and 72 h after termination of infusion. Infusion of EGTA successfully decreased blood iCa concentrations. However, EL EGTA-infused cows required increased rates of EGTA infusion to maintain iCa below 1.0 mM. Circulating and mammary serotonin concentrations were increased in EL relative to NL cows, with no difference as a result of EGTA infusion. Mammary PTHLH expression was increased in EL cows, with highest expression observed in EL EGTA-infused cows. Collectively, these data demonstrate the robust adaptations EL cows have to maintain Ca homeostasis and the supporting roles 5HT and PTHLH may play.

Graduate Student Literature Review: Serotonin and calcium metabolism: A story unfolding.

The peripartum period is characterized by dynamic shifts in metabolic, mineral, and immune metabolism as the dairy cow adapts to the demands of lactation. Emphasis over the past decade has been placed on understanding the biology of the large shift in calcium metabolism in particular. Moreover, research has also focused on exploring the role of serotonin during the transition period and lactation and further unraveling its relationship with calcium. This review aimed to demonstrate the integration of calcium physiology during the peripartal period and throughout lactation. More specifically, we sought to discuss the knowledge gained in recent years on calcium metabolism, mammary calcium transport, serotonin metabolism, and the serotonin-calcium axis. Herein we also discuss the challenges and limitations of current research and where that leaves the present understanding of the serotonin-calcium axis as we seek to move forward and continue exploring this interesting relationship.

Elevated serotonin alters whole-blood expression of serotonin receptor and metabolism genes in the lactating dairy cow.

Serotonin's role as a hormone has been demonstrated through modulating calcium metabolism, energy homeostasis, and immune function in the preweaned and lactating bovine. Recently, manipulation of the serotonergic axis in calves via administration of 5-hydroxy-l-tryptophan (5-HTP), the serotonin precursor, elicited immunomodulatory effects and regulated serotonin transport and metabolism genes in leukocytes. However, serotonin's ability to modulate whole-blood gene expression in lactating dairy cows is unknown. Our objective was to explore the effect of infusing 5-HTP on whole-blood expression of genes regulating serotonin transport (reuptake transporter), signaling (receptors), metabolism (synthesis and degradation), and cytokines in dairy cows. Twelve lactating multiparous Holstein dairy cows were blocked by parity, milk production, and days in milk in a randomized complete block design and randomly assigned to intravenous infusion of either 1 L of 1.5 mg/kg 5-HTP dissolved in saline (n = 6) or 1 L of saline solution (n = 6) for 3 consecutive days. Whole blood was collected at 48, 56, and 72 h relative to termination of first infusion to analyze whole-blood gene expression. Infusion of 5-HTP increased whole-blood mRNA expression of monoamine oxidase-A and serotonin receptor 7 across the experimental period. Forty-eight hours from termination of first infusion, the mRNA of monoamine oxidase-A, serotonin reuptake transporter, and serotonin receptor 7 were increased in blood relative to the control. Interleukin-8 concentrations and mRNA were unchanged in response to 5-HTP infusion. Collectively, these data suggest that infusion of 5-HTP may alter mRNA of serotonin metabolism, transport, and signaling genes in whole blood of lactating dairy cows.

Short communication: The effect of ruminal administration of 5-hydroxy-l-tryptophan on circulating serotonin concentrations.

The monoamine serotonin has been shown to regulate peripartal calcium homeostasis in multiparous cows and be a possible mitigation tool for hypocalcemia. Increasing circulating serotonin concentrations via prepartum intravenous (IV) administration of the serotonin precursor 5-hydroxy-L-tryptophan (5-HTP) increases postpartum calcium concentrations. However, the ability of 5-HTP to be used orally or ruminally to alter circulating serotonin concentrations has not been established. Hence, our objective was to determine if ruminal administration of 5-HTP altered circulating serotonin concentrations. Four ruminally cannulated, nonlactating, nonpregnant multiparous Holstein dairy cows were randomly assigned to 1 of 4 treatments in a 4 × 4 replicated Latin square with 4-d periods separated by a 7-d washout. On d 1 and 2 of each period, cows were dosed with 1 of 4 experimental treatments as follows: (1) 0 mg/kg of body weight (BW) of 5-HTP, (2) 1 mg/kg of BW of intraruminal 5-HTP, (3) 2 mg/kg of BW of intraruminal 5-HTP, or (4) 1 mg/kg of BW of IV 5-HTP. Infusions were administered over a 1-h period, and all groups not receiving 5-HTP IV were infused with an equal volume of IV saline to that of IV 1 mg/kg of BW of 5-HTP treatment. Continuous serial blood samples were collected beginning after d 2 of treatment administration. Whole blood serotonin concentrations were higher in cows dosed with 2 mg/kg of BW of intraruminal 5-HTP immediately after dosing when compared with cows dosed with 0 mg/kg of BW of 5-HTP on d 2, but were similar on d 3 and 4 of the experimental period. Cows receiving IV 5-HTP had the highest circulating serotonin concentrations relative to all other treatments. These findings demonstrated that 2 intraruminal dosings of 5-HTP at 2 mg/kg of BW resulted in elevated circulating serotonin concentrations relative to the control immediately after dosing. This supports the potential for 5-HTP to be used orally to manipulate circulating serotonin concentrations.

Review: Endocrine pathways to regulate calcium homeostasis around parturition and the prevention of hypocalcemia in periparturient dairy cows.

Calcium homeostasis is crucial for the normal function of the organism. Parathyroid hormone, calcitriol and calcitonin play critical roles in the homeostatic regulation of calcium. Serotonin and prolactin have also been shown to be involved in the regulation of calcium homeostasis. In modern dairy cows, the endocrine pathways controlling calcium homeostasis during non-lactating and non-pregnant physiological states are unable to fully support the increased demand of calcium required for milk synthesis at the onset of lactation. This review describes different endocrine systems associated with the regulation of calcium homeostasis in mammalian species around parturition with special focus on dairy cows. Additionally, classic and novel strategies to reduce the incidence of hypocalcemia in parturient dairy cows are discussed.

Transcriptomic analysis investigating the interaction between peripheral serotonin and high-fat diet feeding on mammary gene expression in midlactation mice.

To understand the role of peripheral serotonin and its interaction with diet in midlactation mammary gene expression, our study uses tryptophan hydroxylase 1 knockout (Tph1-KO) mice fed a high-fat diet (HFD). It has previously been demonstrated that HFD feeding increases inflammatory and immune pathways in peak lactation mammary glands of mice and increases pup mortality in wild-type (WT) mice compared with dams fed a low-fat diet (LFD). Peripheral serotonin inhibition has been associated with resistance to obesity in male mice fed an HFD. Little is known about the function of Tph1 and how peripheral serotonin affects mammary gland function during pregnancy and lactation. In this study, WT and Tph1-KO models were used to investigate global transcriptomic changes in peak lactation mammary glands when dams were fed either an HFD or LFD. WT and Tph1-KO female mice were assigned to either an LFD or HFD beginning at 3 wk of age (n = 4/group). Dams were euthanized on lactation day 11. Differentially expressed genes (DEGs) were first filtered by adjusted P value (cutoff ≤ 0.05) and fold-change (FC, cutoff ≥2). Genes were further filtered by mean normalized read count with a cutoff 310. We did not observe many differentially expressed genes in WT and Tph1-KO dams fed LFD. However, 3,529 DEGs were observed between WT-HFD and Tph1-KO-HFD mice, including cell cycle regulation and MAPK pathways being significantly enriched. Further research is required to completely understand the physiological significance of our results on peak lactation mammary physiology and the contribution of serotonin.

Effect of manipulating progesterone before timed artificial insemination on reproductive and endocrine outcomes in high-producing multiparous Holstein cows.

Our objective was to evaluate the effect of manipulating progesterone (P4) concentrations before timed artificial insemination (TAI) on reproductive and endocrine outcomes in high-producing Holstein cows. Multiparous lactating Holstein cows (n = 80) were synchronized for first TAI using a Double-Ovsynch protocol and were randomly assigned to receive 25 mg of PGF2α 1 d after the first GnRH treatment of the Breeding-Ovsynch protocol that included a once-used P4 insert (low-P4 group) or to receive 2 new P4 inserts during the Breeding-Ovsynch protocol (high-P4 group). Blood samples were collected thrice weekly from -10 to 32 d relative to TAI for all cows and from 32 to 67 d after TAI for pregnant cows and were analyzed for P4 and pregnancy-specific protein B (PSPB) concentrations. Expression of IFNτ-stimulated gene 15 (ISG15) was assessed in blood leukocytes 18 and 20 d after TAI. As expected, P4 concentrations were greater for high-P4 cows than for low-P4 cows from 3 to 8 d before TAI. Incidence of double ovulation was 3-fold greater for low-P4 cows than for high-P4 cows (33 vs. 10%), which resulted in more twin pregnancies 32 d after TAI for low-P4 cows than for high-P4 cows (29 vs. 0%). Low-P4 cows had larger preovulatory follicles at the last GnRH treatment of the Double-Ovsynch protocol and greater P4 concentrations than high-P4 cows after TAI. Relative expression of ISG15 mRNA 18 and 20 d after TAI was greater for low-P4 cows than for high-P4 cows and for pregnant cows than for nonpregnant cows. Overall, PSPB concentrations tended to be greater for low-P4 cows than for high-P4 cows, and pregnant cows had greater P4 concentrations than nonpregnant cows. In summary, cows with low P4 before TAI had increased preovulatory follicle diameter, PSPB concentrations, relative expression of ISG15 mRNA 18 and 20 d after TAI, double ovulations, and twinning compared with cows with high P4 before TAI. Increasing P4 before TAI may effectively decrease double ovulation and twinning in high-producing multiparous Holstein cows.

Effect of treatment with human chorionic gonadotropin 7 days after artificial insemination or at the time of embryo transfer on reproductive outcomes in nulliparous Holstein heifers.

Our objective was to assess the effect of treatment with human chorionic gonadotropin (hCG) 7 d after artificial insemination (AI) or at the time of in vitro-fertilized (IVF) embryo transfer on reproductive outcomes, including progesterone (P4), interferon-tau stimulated gene 15 (ISG15), pregnancy-specific protein B (PSPB), and pregnancies per AI (P/AI) or pregnancies per embryo transfer (P/ET), in nulliparous Holstein heifers. Heifers in experiment 1 were randomly assigned to receive no treatment (control; n = 129) or 2,000 IU of hCG 7 d after AI to a detected estrus (estrus = experimental d 0; hCG; n = 132). Heifers in experiment 2 were randomly assigned to receive no treatment (control; n = 143) or 2,000 IU of hCG (hCG; n = 148) at transfer of an IVF embryo 7 d after the last GnRH treatment of a 5-d controlled internal drug release-synch protocol (last GnRH = experimental d 0). Blood samples were collected from a subgroup of heifers (experiment 1, n = 82; experiment 2, n = 104) at d 7, 11, 18, 20, 25, 28, and 32, and blood samples from heifers diagnosed pregnant were collected on d 35, 39, 46, 53, 60, and 67. Blood samples were assayed for P4 by RIA and for PSPB by ELISA, and expression of ISG15 was assessed in mRNA isolated from blood leukocytes on d 18 and 20. Data were analyzed by ANOVA and logistic regression using the MIXED and GLIMMIX procedures. In both experiments, treatment with hCG increased P4 concentrations from d 11 to 32; however, treatment did not affect P/AI or P/ET at d 32 or 67, PSPB concentrations from d 11 to 67 of pregnancy, or relative ISG15 mRNA concentrations on d 18 or 20. Heifers diagnosed not pregnant at d 32 in experiment 2 with an extended luteal phase (>20 d) and treated with hCG had greater relative ISG15 mRNA concentrations on d 20 than control heifers. Treatment with hCG did not affect pregnancy loss in experiment 1, whereas heifers treated with hCG at the time of IVF embryo transfer had fewer pregnancy losses from d 32 to 67 than control heifers. We concluded that treatment with 2,000 IU of hCG 7 d after AI or at the time of embryo transfer increased P4 concentrations without affecting P/AI or P/ET in nulliparous Holstein heifers.

Short communication: Mammary gland tight junction permeability after parturition is greater in dairy cows with elevated circulating serotonin concentrations.

After parturition, the start of copious milk production in dairy cows requires the closure of tight junctions (TJ) to form the blood-milk barrier and prevent paracellular transfer of blood constituents into milk [e.g., lactate dehydrogenase (LDH) and serum albumin (SA)] and vice versa [e.g., appearance of α-lactalbumin (α-LA) in blood]. Serotonin (5-HT) has been demonstrated to alter tight junction permeability in the mammary gland. The present study investigated individual differences of TJ permeability of mammary epithelium at the beginning of lactation in relation to circulating 5-HT in dairy cows. Blood and milk samples were obtained from 11 multiparous Holstein dairy cows for the first time at 4 h after parturition, at the following 5 milkings, and at the evening milkings on d 5, 8, 10, and 14 of lactation. Retrospectively, cows were split into 2 groups according to their calculated areas under the curve of serum 5-HT during the entire experimental period: a high-serum 5-HT (HSS) group (5 cows) and a low-serum 5-HT (LSS) group (6 cows). The areas under the curve of serum 5-HT concentrations over the 324-h experimental period were 62 ± 2 × 103 ng/mL in HSS and 25 ± 5 × 103 ng/mL in LSS. Plasma α-LA concentration was greater in LSS than in HSS cows at the first milking, but no difference between groups was found from the second to sixth milking. Yield of α-LA in milk was lower in HSS than in LSS during the first 6 milkings postpartum, especially in colostrum. Concentrations of α-LA, IgG1, and IgG2 in milk did not differ between groups during the entire experiment except for higher IgG observed in LSS than in HSS at the second milking and for higher IgG2 found in HSS compared with LSS on d 5. In contrast, SA concentrations and LDH activity in milk were lower in LSS compared with HSS cows during the first 6 milkings postpartum, particularly in colostrum. Milk somatic cell count was higher in HSS than in LSS throughout the study. Higher circulating 5-HT concentrations were associated with an increased transfer of the paracellularly transported SA, LDH, and somatic cell count, especially at the first milking, suggesting that 5-HT affects TJ permeability during closure of the blood-milk barrier at the onset of lactation. Furthermore, higher serum 5-HT concentrations were associated with a lower α-LA yield in milk. A consistent relationship with serum 5-HT concentrations was neither observed for the transfer of IgG2 nor the primarily transcellular transport of IgG1 during the first milkings after parturition.

Effect of high-fat diet feeding and associated transcriptome changes in the peak lactation mammary gland in C57BL/6 dams.

Maternal consumption of a high-fat diet (HFD) during pregnancy has established adverse effects on the developing neonate. In this study, we aimed to investigate the effect of an HFD on the murine mammary gland during midlactation. Female C57BL/6J mice were placed on either a low-fat diet (LFD/10% fat) or HFD (60% fat) from 3 wk of age through peak lactation (lactation day 11/L11). After 4 wk of consuming either the LFD or HFD, female mice were bred. There were no significant differences in milk yield between treatment groups, which was measured from L1 to L9. On L10, mice were subjected to an overnight fast and then euthanized on the morning of L11. Total RNA was isolated from inguinal mammary glands for whole transcriptome sequencing. We found 628 genes that were differentially expressed between the treatment groups. Notably, HFD feeding resulted in expression alterations of genes involved in collagen and cytoplasmic components. Additionally, genes related to inflammatory and immune responses were also impacted. Differential expression in gene transcript isoforms between the treatment groups was detected in three genes related to mammary duct development. This study sheds light as to how an HFD may affect the mammary gland transcriptome during midlactation.

Short communication: Circulating serotonin is related to the metabolic status and lactational performance at the onset of lactation in dairy cows.

Serotonin (5-hydroxytryptamine, 5-HT) affects many physiological functions because it is involved in glucose and lipid metabolism, calcium homeostasis, and regulation of lactation in dairy cows. This study aimed to examine physiological differences in serum 5-HT concentrations (high vs. low) and their association with metabolic status and milk production at the onset of lactation. Twelve multiparous Holstein dairy cows were milked within 4 h of calving, and blood and milk samples were collected at the first 6 subsequent milkings after parturition and at the evening milkings on d 5, 8, 10, and 14. Cows were retrospectively divided into 2 groups (6 cows/group): low serum 5-HT (LSS) and high serum 5-HT (HSS) according to their calculated areas under the curve (AUC) for serum 5-HT for the entire experimental period (cut-off: 46,000 ng/mL × 324 h). Concentrations of 5-HT, free fatty acids (FFA), ß-hydroxybutyrate (BHB), glucose, calcium, and IGF-1 were measured in blood. Milk was analyzed for fat, protein, lactose, and 5-HT concentrations. Milk yield was recorded at each milking and energy-corrected milk yield was calculated. Serum 5-HT concentrations were higher in HSS than in LSS [AUC (ng/mL × 324 h): 57,830 ± 4,810 vs. 25,005 ± 5,930]. The amount of energy-corrected milk was lower in HSS than in LSS. The HSS group produced less colostrum and had decreased milk yield, specifically during the first 6 milkings. Concentrations of FFA, BHB, and glucose in plasma did not differ between groups. Concentrations of IGF-1 in serum were elevated in HSS compared with LSS throughout the experiment. Total circulating calcium concentrations in serum tended to be higher in HSS than in LSS. Milk fat and protein yields were decreased in HSS compared with LSS. Milk 5-HT decreased overall during the experimental period, with LSS maintaining higher 5-HT concentrations than HSS until d 14 of lactation. In conclusion, cows with high serum 5-HT concentrations showed a reduced metabolic load at the onset of lactation, concomitantly lower milk yield, and a reduced energy output via milk.

Association of quarter milking measurements and cow-level factors in an automatic milking system.

The primary aim of this observational study, in a single herd milked using multiple automatic milking system units, was to describe associations of quarter milk yield variability and quarter peak milk flow rate with cow-level factors. Information from the current lactation of 1,549 primiparous and multiparous cows was collected from January to December 2015. Data from each individual milking used in the analysis included quarter milk yield (QMY), udder milk yield, quarter peak milk flow rate (QPMF), quarter average milk flow rate (QAMF), quarter milking time, and milking interval. Milking interval and milk yield were used to calculate milk production rate (kg/h) at the quarter and udder levels. We investigated associations between QPMF and milking interval, QPMF and days in milk, and QMY and QAMF. A strong association between QPMF and both QAMF and milking interval was observed. A moderate association was found between QPMF and stage of lactation. However, QMY was not a useful indicator of QPMF because of the weak association observed between these variables. In this study, rear quarter QPMF was significantly increased by 3% compared with front quarter QPMF (1.45 vs 1.41 kg/min). Quarter milk yield was calculated as a percentage contribution of total udder milk yield per 10-d in milk window and ranked from lowest to highest contribution. Quarter contribution to udder milk yield showed a high level of variability, with 39% of animals having all 4 quarters change contribution rank at least once during part of or the whole lactation. Only 14% of cows were observed to have no change in quarter rank. When quarter contribution was assessed, irrespective of physical position of quarter within the udder, the percent of highest to lowest contribution across the lactation was relatively stable. The standard deviation of quarter milk production rate for each cow was regressed against the same cow's peak udder milk production rate, within a lactation, to ascertain whether quarter milk production rate variance could be used to predict peak udder milk production rate. Knowledge of the intra-udder quarter milk production rate standard deviation for an individual cow is not useful in predicting peak udder milk production rate. Quarter milking time appears to be a useful indicator to predict the optimal order of teatcup attachment. Analysis from this large, single-herd population indicates that QPMF is associated with the cow-level factors milking interval and days in milk, and that intra-udder QMY is highly variable.

Influence of sampling technique and bedding type on the milk microbiota: Results of a pilot study.

The objective of this pilot study was to evaluate the influence of sampling technique and exposure to different bedding types on the milk microbiome of healthy primiparous cows. Primiparous Holstein cows (n = 20) with no history of clinical mastitis or monthly somatic cell counts >150,000 cells/mL were selected for this study. From each enrolled cow, a composite milk sample was aseptically collected from all 4 mammary quarters (individual quarter somatic cell counts <100,000 cells/mL), 1 individual quarter milk sample was collected using conventional aseptic technique, and 2 individual quarter milk samples were collected directly from the gland cistern using a needle and vacuum tube. All milk samples were cultured using standard milk microbiological techniques and DNA was extracted. Extracted DNA was subjected to PCR and next-generation sequencing for microbiota determination. All samples yielded relatively little total DNA. Amplification of PCR was successful in 45, 40, and 83% of composite, conventional, and cisternal samples, respectively. Bacteria were successfully cultured from 35% of composite milk samples but from none of the quarter milk samples collected using conventional or cisternal sampling techniques. Bacterial DNA sequences were assigned to operational taxonomic units (OTU) based on 97% sequence similarity, and bacterial richness and diversity were determined. Most samples were dominated by low-prevalence OTU and of the 4,051 identified OTU, only 14 were prevalent at more than 1% each. These included bacteria typically recovered from environmental sources. Chao richness was greatest in composite samples and was 636, 347, and 356 for composite, conventional quarter, and cisternal milk samples, respectively. Shannon diversity was similar among sample types and ranged from 3.88 (quarter) to 4.17 (composite). Richness and diversity did not differ by bedding type among cisternal samples, but the power of this pilot study was limited due to small sample size. Despite the small sample size, for milk samples collected from the gland cistern, overall bacterial community composition differed among bedding types. These results demonstrate that sampling technique and bedding type may be associated with the microbiota detected in bovine milk, and we suggest that these variables should be considered in designing and reporting studies about the milk microbiota.

ADSA Foundation Scholar Award: A role for serotonin in lactation physiology-Where do we go from here?

Lactation is a physiological event that is exclusive to mammals. Lactation evolved as a strategy to improve the survival of the young by providing them with the complete nutrition that is required for survival upon birth as well as maternal-offspring bonding. Typically, milk production by the dam matches the demand of the young. The dairy cow is a unique exception in which the discoveries and genetic selection related to lactation physiology have been applied and resulted in a dramatic increase in milk yield of dairy cows. Studies on the role of mammary-derived serotonin and the coordination of various aspects of milk production and maternal metabolism have revealed novel mechanisms by which milk production and maternal metabolism can be improved. Furthermore, the investigation into molecular and cellular mechanisms regulating mammary gland function has revealed the importance of epigenetics on mammary gland function. Understanding mammary gland function at the cellular and physiological levels will be important for improving mammary gland control of maternal metabolism during early lactation. The early lactation period is a critical time for a dairy cow as that is when she is most susceptible to disease and metabolic disorders that can lead to negative effects on her productive capacity and overall health. Our research in the area of serotonin physiology has illustrated the importance of serotonin on the regulation of lactation and maternal homeostasis. Future research in the area of lactation physiology should be targeted at improving maternal health and longevity in the herd through manipulation of the signals the mammary gland sends to coordinate maternal metabolism and synthesize milk. Specifically, we believe that serotonin will play a central role in understanding the communication between the mammary gland and the maternal physiology during lactation.

Interaction of 5-hydroxy-l-tryptophan and negative dietary cation-anion difference on calcium homeostasis in multiparous peripartum dairy cows.

Hypocalcemia affects almost 50% of all dairy cows. Our laboratory has previously demonstrated that infusions of the serotonin precursor 5-hydroxy-l-tryptophan (5-HTP) increase circulating calcium concentrations in the Holstein transition cow. It is unknown whether feeding a negative dietary cation-anion difference (DCAD) diet alters the relationship between 5-HTP and hypocalcemia. The main objective of this study was to determine whether feeding a negative DCAD (-DCAD) diet before calving in conjunction with 5-HTP treatment could further diminish the magnitude of hypocalcemia at the time of calving. We used a randomized complete block design with a 2 × 2 factorial arrangement. Thirty-one multiparous Holstein cows were fed either a positive (+13 mEq/100 g) or negative (-13 mEq/100 g) DCAD diet 21 d before parturition and were intravenously infused daily with saline or 5-HTP (1 mg/kg) starting 7 d before the estimated date of parturition. Cows were blocked by parity and were randomly assigned to 1 of 4 treatment groups: positive DCAD plus saline, positive DCAD plus 5-HTP, negative DCAD plus saline, and negative DCAD plus 5-HTP, resulting in n = 8 per group. Total calcium (tCa), ionized calcium (iCa), and feed intake were recorded. The iCa was elevated prepartum in the -DCAD/5-HTP group compared with the other treatment groups as well as on d 0 and 1 postpartum. Although differences in tCa were not significant across the pre- or postpartum periods, tCa was numerically higher on d 0 and significantly higher on d 1 in -DCAD/5-HTP cows compared with all other groups. Prepartum the -DCAD/5-HTP treatment group ate less than the other treatment groups; however, postpartum dry matter intake differences were not significant. These findings demonstrate that feeding a -DCAD diet in conjunction with 5-HTP prepartum can increase postpartum circulating iCa concentrations and therefore diminish the magnitude of hypocalcemia at the time of parturition.