M. Van Eetvelde

Insulin response of the glucose and fatty acid metabolism in dry dairy cows across a range of body condition scores

The objective of the present research was to determine the insulin response of the glucose and fatty acid metabolism in dry dairy cows with a variable body condition score (BCS). Ten pregnant Holstein Friesian dairy cows (upcoming parity 2 to 5) were selected based on BCS at the beginning of the study (2mo before expected parturition date). During the study, animals were monitored weekly for BCS and backfat thickness and in the last 2wk, blood samples were taken for determination of serum nonesterified fatty acid (NEFA) concentration. Animals underwent a hyperinsulinemic euglycemic clamp test in the third week before the expected parturition date. The hyperinsulinemic euglycemic clamp test consisted of 4 consecutive insulin infusions with increasing insulin doses: 0.1, 0.5, 2, and 5mIU/kg per minute. For each insulin infusion period, a steady state was defined as a period of 30min where no or minor changes of the glucose infusion were necessary to keep the blood glucose concentration constant and near basal levels. During the steady state, the glucose infusion rate [steady state glucose infusion rate (SSGIR) in µmol/kg per minute] and NEFA concentration [steady state NEFA concentration (SSNEFA) in mmol/L] were determined and reflect the insulin response of the glucose and fatty acid metabolism. Dose response curves were created based on the insulin concentrations during the steady state and the SSGIR or SSNEFA. The shape of the dose response curves is determined by the concentration of insulin needed to elicit the half maximal effect (EC50) and the maximal SSGIR or the minimal SSNEFA for the glucose or fatty acid metabolism, respectively. The maximal SSGIR was negatively associated with variables reflecting adiposity of the cows (BCS, backfat thickness, NEFA concentration during the dry period, and absolute weight of the different adipose depots determined after euthanasia and dissection of the different depots), whereas the EC50 of the glucose metabolism was positively associated with these variables. These results reflect a decreased insulin sensitivity and a decreased insulin responsiveness of the glucose metabolism in overconditioned dry dairy cows. The minimal SSNEFA and the EC50 of the fatty acid metabolism were not associated with variables reflecting adiposity of the cows, meaning that the insulin response of the fatty acid metabolism was not associated with the level of fat accumulation in dry dairy cows. Additionally, within individual cows, the EC50 of the glucose metabolism was higher than the EC50 of the fatty acid metabolism, meaning that the response of the fatty acid metabolism occurs at lower insulin concentrations compared with the response of the glucose metabolism. It can be concluded that a negative association exists between the level of fat accumulation in pregnant dairy cows at the end of the dry period and the insulin response of the glucose metabolism.

Age at calving in heifers and level of milk production during gestation in cows are associated with the birth size of Holstein calves

The objective of the present study was to evaluate environmental and dam factors associated with birth size of Holstein calves. Data of 1,594 births from dairy herds in Belgium and Germany were analyzed in a retrospective cross-sectional study. Immediately after birth, the birth weight of the calves was measured. On the next day, the heart girth (HG), withers height, and diagonal length of the dams and calves were measured. Parity, body condition score, gestation length (GL), and age at calving were recorded for all dams. For the cows, days open, lactation length, length of the dry period, and calving interval were also calculated. The magnitude and shape of the lactation that took place during gestation was quantified using the MilkBot model based on monthly milk weights. Using the same procedure, cumulative milk production from conception to drying off (MGEST) was calculated. After descriptive analyses, mixed models were used to identify factors that are significantly associated with the birth weight (most consistent measure of size at birth) of the calves born to both heifers and cows. Of the variables offered to the offspring birth weight model in heifers (n=540), calf sex, season of calving, GL, HG, withers height, diagonal length, and age at calving were significant. The mean birth weight of the calves born to heifers was estimated to be 41.3±1.01kg. In comparison to calves born to old (25.5 to 37.3mo; n=99) heifers, the birth weight was estimated to be 2.75, 3.29, and 2.35kg heavier when the calves were born to very young (20.3 to <22mo; n=98), young (22 to <23.5mo; n=145), and standard aged (23.5 to <25.5mo; n=198) heifers, respectively. Of the variables offered to the offspring birth weight model in cows (n=1,054), calf sex, season of calving, GL, parity, dry period, and MGEST were significant. The mean birth weight of the calves born to cows was estimated to be 44.1±0.99kg. For cows having an identical HG, the birth weight of the calves was estimated to be 0.97 and 1.11kg higher in cows with low (1,400 to <5,400kg) and high (6,500 to <7,200) MGEST, respectively, compared with cows with very high (7,200 to 11,600kg) MGEST. The decisive effects of age at calving in heifers and of high milk production levels during gestation in cows on the birth weight of their calves may provide a basis for developing managerial interventions to improve long-term health and productivity of the offspring.

Influence of adipocyte size and adipose depot on the in vitro lipolytic activity and insulin sensitivity of adipose tissue in dairy cows at the end of the dry period

The aim of the present research was to describe characteristics of adipose tissue lipolysis in dairy cows with a variable body condition score (BCS). Ten clinically healthy Holstein Friesian cows were selected based on BCS and euthanized 10 to 13 d before the expected parturition date. Immediately after euthanasia, adipose tissue samples were collected from subcutaneous and omental fat depots. In both depots, we observed an increase in adipocyte size with increasing BCS. Using an in vitro explant culture of subcutaneous and omental adipose tissue, we aimed to determine the influence of adipocyte size and localization of adipose depot on the lipolytic activity in basal conditions and after addition of isoproterenol (nonselective β-agonist) and insulin in different concentrations. Glycerol release in the medium was used as a measure for lipolytic activity. We observed that the basal lipolytic activity of subcutaneous and omental adipose tissue increased with adipocyte volume, meaning that larger fat cells have higher basal lipolytic activity independent of the location of the adipose depot. Dose-response curves were created between the concentration of isoproterenol or insulin and the amount of glycerol released. The shape of the dose-response curves is determined by the concentration of isoproterenol and insulin needed to elicit the half-maximal effect and the maximal amount of stimulated glycerol release or the maximal inhibitory effect of insulin. We observed that larger fat cells released more glycerol upon maximal stimulation with isoproterenol and this was more pronounced in subcutaneous adipose tissue. Additionally, larger fat cells had a higher sensitivity toward lipolytic signals. We observed a trend for larger adipocytes to be more resistant to the maximal antilipolytic effect of insulin. The insulin concentration needed to elicit the half-maximal inhibitory effect of insulin was within the physiological range of insulin and was not influenced by adipocyte size or adipose depot. We conclude that overconditioned cows have larger adipocytes and are predisposed to excessive mobilization of body fat due to a higher basal and stimulated lipolytic activity of large adipocytes while the antilipolytic effect of insulin is preserved.

Effect of uterine lavage on neutrophil counts in postpartum dairy cows

Subclinical endometritis affects approximately 30% of lactating dairy cows, causing significant economic losses to the dairy industry. Yet, there is no efficient treatment available for this condition. The present study examines the effect of uterine lavage in clinically normal cows with sterile saline solution at 30 days in milk (DIM) on the percentage of polymorphonuclear cells (PMNs) detected with endometrial cytology as an indicator of subclinical endometritis. It was hypothesized uterine lavage would be a technique to reduce the number of PMNs in the uterus, and hence be beneficial for cows affected by subclinical endometritis. Cytology samples were taken by low-volume flushing from 50 Holstein Friesian cows on 30 and 40 DIM. On Day 30, cows were clinically examined and randomly assigned into a treatment and control group. In the treatment group, the cytology sampling on Day 30 was immediately followed by uterine lavage with 500-600 mL of sterile physiological saline (35-40°C). Cytology sampling was repeated in all cows at 40 DIM. Lactation numbers >2, peripheral progesterone concentrations >1 ng/mL and uterine lavage at 30 DIM all were significantly associated with lesser PMN percentages at 40 DIM (P=0.0041; 0.0187 and 0.0043, respectively). Uterine lavage might, therefore, be a useful and practical method to decrease the number of PMNs in the uterus of cattle. Results from the current study can be used as preliminary data for designing in depth therapeutic protocols for treatment of subclinical endometritis in cattle.

Prevalence of cytological endometritis and effect on pregnancy outcomes at the time of insemination in nulliparous dairy heifers

The objectives of the present study were to assess the prevalence of cytological endometritis (CYTO) at the time of artificial insemination (AI) and its effect on pregnancy outcomes in nulliparous dairy heifers. In total, 512 endometrial cytology samples were taken during AI from 351 nulliparous Holstein-Friesian heifers using cytotape (a 1.5-cm piece of paper tape rolled on the top of an AI catheter covered with a double guard sheet). After sampling, the top of the AI catheter was gently rolled onto a glass slide, air-dried, and stained using Diff-Quick (Fisher Diagnostics, Newark, DE). For each slide, 300 nucleated cells were counted, and the polymorphonuclear cell ratio (% PMN) was assessed at 400× magnification. We constructed a receiver operating characteristic curve to find the cutoff point at which sensitivity and specificity (% PMN) affected pregnancy outcomes. The receiver operating characteristic curve revealed that the threshold level for diagnosing CYTO in nulliparous dairy heifers was 1% PMN. An insemination was considered successful when pregnancy was confirmed by rectal palpation at least 45d post-AI. Heifers were considered not pregnant when they received a subsequent insemination or were diagnosed empty by rectal palpation. We built multilevel generalized mixed-effect models to test factors affecting pregnancy outcomes and the occurrence of CYTO at AI. We excluded 16 samples harvested from 12 heifers due to poor sample quality or unavailability of reproductive data. Of the 496 AI samples, the prevalence of CYTO at AI was 7.86% (n=39). The conception rate was 62.8% (n=287) in CYTO-negative samples (n=457) and 38.46% (n=15) in CYTO-positive samples. Risk factors for non-pregnancy were a previous AI (odds ratio 2.96; 95% confidence interval: 1.21-7.26) and the interaction between CYTO and previous AI. The only risk factor identified as being associated with the occurrence of CYTO was a previous AI (odds ratio 4.7; 95% confidence interval: 2.15-10.34). The performance of unsuccessful inseminations significantly affects reproductive outcomes in subsequent AI and may lead to CYTO in nulliparous dairy heifers.

Evidence for placental compensation in cattle.

Prenatal development is known to be extremely sensitive to maternal and environmental challenges. In this study, we hypothesize that body growth and lactation during gestation in cattle reduce nutrient availability for the pregnant uterus, with consequences for placental development. Fetal membranes of 16 growing heifers and 27 fully grown cows of the Belgian Blue (BB) breed were compared to determine the effect of body growth on placental development. Furthermore, the fetal membranes of 49 lactating Holstein Friesian (HF) cows and 27 HF heifers were compared to study the impact of dam lactation compared to dam body growth. After parturition, calf birth weight and body measurements of dam and calf were recorded, as well as weight of total fetal membranes, cotyledons and intercotyledonary membranes. All cotyledons were individually measured to calculate both the surface of each individual cotyledon and the total cotyledonary surface per placenta. Total cotyledonary surface was unaffected by breed or the breed×parity interaction. Besides a 0.3 kg lower cotyledonary weight (P=0.007), heifer placentas had a smaller total cotyledonary surface compared with placentas of cows (0.48±0.017 v. 0.54±0.014 m2, respectively, P<0.001). Within the BB breed, fetal membranes of heifers had a 1.5 kg lower total weight and 1.0 kg lower intercotyledonary membrane weight (P<0.005) compared with cows. A cotyledon number of only 91±5.4 was found in multiparous BB dams, while growing BB heifers had a higher cotyledon number (126±6.7, P<0.001), but a greater proportion of smaller cotyledons (<40 cm2). Within the HF breed, no parity effect on intercotyledonary membrane weight, cotyledon number and individual cotyledonary surface was found. Placental efficiency (calf weight/total cotyledonary surface) was similar in HF and BB heifers but significantly higher in multiparous BB compared with multiparous HF dams (106.0±20.45 v. 74.3±12.27 kg/m2, respectively, P<0.001). Furthermore, a seasonal effect on placental development was found, with winter and spring placentas having smaller cotyledons than summer and fall placentas (P<0.001). Main findings of the present study are that lactation and maternal growth during gestation entail a comparable nutrient diverting constraint, which might alter placental development. However, results suggest that the placenta is able to manage this situation through two potential compensation mechanisms. In early pregnancy the placenta might cope by establishing a higher number of cotyledons, while in late gestation a compensatory expansion of the cotyledonary surface is suggested to meet the nutrient demand of the fetus.

Short communication: Limitations of glucose tolerance tests in the assessment of peripheral tissue insulin sensitivity during pregnancy and lactation in dairy heifers

The aim of the present study was to point at the limitations of glucose tolerance tests (GTT) to assess peripheral tissue insulin sensitivity in dairy heifers in different physiological states (pregnancy and lactation). Intravenous GTT were performed in 5 nonpregnant, nonlactating heifers, 5 heifers at the end of pregnancy (12-7 d before calving), and 5 lactating primiparous cows (11-14 d after calving). Glucose and insulin concentrations were determined and area under the curve (AUC) and clearance rate of glucose and insulin were calculated. Additionally, data were analyzed using the minimal model to derive the insulin sensitivity parameter (Si). Basal glucose and insulin concentrations were greater in the nonpregnant, nonlactating heifers. Clearance rate of glucose and Si were lowest, whereas the AUC for glucose was greatest in the nonpregnant, nonlactating heifers. Insulin concentrations during the GTT were greater for the nonpregnant, nonlactating heifers. Results from the GTT in pregnant heifers and lactating primiparous cows are biased by the fact that a large part of the glucose disappearance during an intravenous GTT occurs independently of insulin by the pregnant uterus or the lactating mammary gland. As such, greater AUC of glucose, lower clearance rate of glucose, or lower Si derived from GTT performed in nonpregnant, nonlactating dairy heifers in the present study might indicate decreased peripheral tissue insulin sensitivity of the glucose metabolism or decreased insulin-independent glucose disappearance. Based on the results from a GTT, it is impossible to discriminate between both metabolic pathways. It can be concluded that parameters derived from GTT are not suited to compare peripheral tissue insulin sensitivity of the glucose metabolism between dairy heifers in different physiological states due to the large variation in insulin secretion and the substantial difference in insulin-independent glucose disposal associated with these physiological states.

Effects of overconditioning on pancreatic insulin secretory capacity, fat infiltration, and the number and size of islets in dairy cows at the end of the dry period

In the present study, we tested the hypothesis that overconditioning in dairy cows at the end of the dry period leads to infiltration of fat and alterations of the insulin secretory capacity of the pancreas. Pregnant Holstein Friesian dairy cows were selected based on body condition score (BCS) at the start of the dry period. Body condition score varied between cows to have optimal conditioned (2.5 < BCS ≤3.5, n = 5) and overconditioned (3.5 < BCS ≤5, n = 5) cows. All animals underwent an intravenous glucose tolerance test (IVGTT) at an average of 260 d of gestation to measure the pancreatic insulin secretory capacity and assess peripheral insulin sensitivity regarding glucose metabolism. Eight days after the IVGTT, animals were slaughtered. The pancreas was dissected and weighed and tissue samples were taken for histological analysis. Results revealed that overconditioning in dairy cows led to fat infiltration in the pancreas and an increase in size of pancreatic islets expressed relative to the total area of pancreatic tissue. In addition, results revealed a positive correlation between serum fatty acid concentration and peak insulin concentration and area and number of pancreatic islets expressed relative to the total area of pancreatic tissue. The IVGTT revealed that overconditioned animals have a higher insulin secretory capacity of the pancreas, as demonstrated by higher peak insulin concentration, higher acute insulin response to glucose, and higher area under the curve (AUC) for insulin compared with optimal conditioned cows. A higher AUC for glucose during the first 60 min following administration of the glucose bolus in overconditioned cows indicates an insulin-resistant state regarding glucose metabolism. Our results suggest that the pancreas of overconditioned dairy cows at the end of gestation compensates for the concomitantly elevated level of peripheral insulin resistance by greater secretion of insulin.

Novel approaches to assess the quality of fertility data stored in dairy herd management software

Scientific journals and popular press magazines are littered with articles in which the authors use data from dairy herd management software. Almost none of such papers include data cleaning and data quality assessment in their study design despite this being a very critical step during data mining. This paper presents 2 novel data cleaning methods that permit identification of animals with good and bad data quality. The first method is a deterministic or rule-based data cleaning method. Reproduction and mutation or life-changing events such as birth and death were converted to a symbolic (alphabetical letter) representation and split into triplets (3-letter code). The triplets were manually labeled as physiologically correct, suspicious, or impossible. The deterministic data cleaning method was applied to assess the quality of data stored in dairy herd management from 26 farms enrolled in the herd health management program from the Faculty of Veterinary Medicine Ghent University, Belgium. In total, 150,443 triplets were created, 65.4% were labeled as correct, 17.4% as suspicious, and 17.2% as impossible. The second method, a probabilistic method, uses a machine learning algorithm (random forests) to predict the correctness of fertility and mutation events in an early stage of data cleaning. The prediction accuracy of the random forests algorithm was compared with a classical linear statistical method (penalized logistic regression), outperforming the latter substantially, with a superior receiver operating characteristic curve and a higher accuracy (89 vs. 72%). From those results, we conclude that the triplet method can be used to assess the quality of reproduction data stored in dairy herd management software and that a machine learning technique such as random forests is capable of predicting the correctness of fertility data.

Effect of β-hydroxybutyric acid, parity, and body condition score on phenotype and proliferative capacity of colostral mononuclear leukocytes of high-yielding dairy cows

In neonatal calves, the ingestion of colostrum is imperative for preventing infectious diseases. Investigations into the transfer of passive immunity of cattle have primarily focused on the importance of colostral immunoglobulins, with a recent increase in focus on understanding the role of colostral leukocytes. The main objective of the present study was to measure the influence of parity, body condition score, serum nonesterified fatty acids, and serum β-hydroxybutyrate concentrations of periparturient cows on phenotype and mitogen- and antigen-induced proliferative capacity of bovine colostral leukocytes. Holstein-Friesian cows (n=141) were intramuscularly vaccinated at 60 and 30 d before the expected parturition date with a tetanus toxoid vaccine. Of these 141 animals, 28 primiparous and 72 multiparous cows were sampled immediately after parturition. Colostrum mononuclear cell populations were identified by flow cytometry using bovine cluster of differentiation markers, and the proliferative capacity of these cells was determined using a (3)H-thymidine proliferation assay. Under-conditioned cows had a significantly higher percentage of colostral macrophages than normal-conditioned animals, whereas over-conditioned cows had significantly more colostral B-lymphocytes. Serum β-hydroxybutyrate was significantly associated with higher numbers of colostral T-lymphocytes and macrophages. Heifers had significantly higher mitogen- and antigen-induced proliferation of their colostral leukocytes than third parity or older cows. In conclusion, body condition score, parity, and serum β-hydroxybutyrate concentration of periparturient high-yielding dairy cows were shown to influence the number of colostral macrophages or the mitogen- and antigen-induced proliferation of colostral leukocytes, possibly influencing the cellular immunity of the newborn calf.