Effect of inulin polymerization degree on various properties of synbiotic fermented milk including Lactobacillus acidophilus La-5 and Bifidobacterium animalis Bb-12, Publication date: August 2019 Source: Journal of Dairy Science, Volume 102, Issue 8 Author(s): Sebnem Ozturkoglu-Budak, H. Ceren Akal, İlkay Buran, Atila Yetişemiyen ABSTRACT The effects of inulin degree of polymerization (DP) on the viabilities of Lactobacillus acidophilus La-5 and Bifidobacterium animalis Bb-12 and on some parameters of fermented milk, such as microbiological, rheological, biochemical, and sensory properties, were investigated during 30 d of storage. Samples were produced using L. acidophilus La-5 and B. animalis Bb-12, along with inulin having different DP as prebiotic, and the effects of high-DP (DP ≥ 23) and low-DP (DP ≤ 10) inulin on fermented milk, were determined. The viability of both strains increased when they were used with inulin having any DP. The addition of inulin increased the consistency index of all samples. During storage, we observed an increase in lactic and acetic acid contents of samples in which high-DP inulin was used, for both strains of bacteria. Of the combinations we tested, the sample produced with L. acidophilus La-5 and high-DP inulin demonstrated the highest rheological and sensory performance as well as the best viability of probiotics.

Graduate Student Literature Review: The milk behind the mustache: A review of milk and bone biology Publication date: August 2019 Source: Journal of Dairy Science, Volume 102, Issue 8 Author(s): Brandon S. Batty, Massimo Bionaz ABSTRACT Exploring the relationship between nutrition, skeletal development, and aging is important in maintaining bone health. Even further, understanding the complexity of skeletal homeostasis may assist in reducing the prevalence of skeletal disease, especially osteoporosis. The skeleton is unique in that it can adapt to various physical pressures, maintain shape, and remodel itself to increase integrity and strength. For decades, it was thought that increasing skeletal health was as simple as drinking three 8-oz. glasses of milk per day due to high levels of bioavailable calcium. New research into the bioactive components of milk have revealed other roles in promoting skeletal health. Milk contains various bioactive peptides, houses genetic information in milk-derived exosomes, and supplies relevant amounts of nutrients important for bone health. In this review, we discuss the basics of skeletal formation and homeostasis, dive into the potential effects of milk on the growing skeleton, and present contrasting findings.

Short communication: Effect of manipulating fatty acid profile on gluconeogenic gene expression in bovine primary hepatocytes Publication date: August 2019 Source: Journal of Dairy Science, Volume 102, Issue 8 Author(s): K.A. Weld, S.J. Erb, H.M. White ABSTRACT During the peripartum period, dairy cows experience both an increase in circulating fatty acid (FA) profile and a change in circulating FA profile, which have been shown to alter regulation of gluconeogenic genes. The objective was to quantify gene expression of key enzymes involved in gluconeogenesis and FA transport into the mitochondria in primary hepatocytes in response to exposure to an FA mixture mimicking what is circulating in a transition dairy cow with or without enrichment of C16:0, C18:0, and C18:1. Primary hepatocytes were isolated from 4 Holstein bull calves 3 d of age (± standard deviation 2 d) and cultured. Twenty-four hours after plating, treatments were applied to the cells for 24-h incubation. Treatments consisted of (1) control (1% BSA), (2) 0.75 mM FA cocktail (3% C14:0, 27% C16:0, 23% C18:0, 31% C18:1, 8% C18:2, and 8% C18:3 to mimic the FA profile of dairy cattle at calving), (3) 0.90 mM FA cocktail, (4) 0.75 mM FA cocktail + 0.15 mM C16:0, (5) 0.75 mM FA cocktail + 0.15 mM C18:0, and (6) 0.75 mM FA cocktail + 0.15 mM C18:1. After harvest in Trizol (Life Technologies, Carlsbad, CA), samples were stored at −80°C until RNA extraction, purification, and reverse transcription. Abundance of mRNA was measured using quantitative real-time PCR. Expression of genes of interest [carnitine palmitoyltransferase 1A, pyruvate carboxylase, cytosolic phosphoenolpyruvate carboxykinase (PCK1), mitochondrial phosphoenolpyruvate carboxykinase, and glucose-6-phosphatase] was calculated relative to the average abundance of 2 reference genes (ribosomal protein L32 and glyceraldehyde 3-phosphate dehydrogenase), which were the most stable out of 3 tested. Data were analyzed using PROC MIXED (SAS version 9.4; SAS Institute, Cary, NC) with the fixed effect of treatment and calf in the random statement. The addition of FA compared with the 1% BSA treatment increased the expression of carnitine palmitoyltransferase 1A and cytosolic PCK1. Enrichment with individual FA did not further regulate pyruvate carboxylase or PCK1 beyond that achieved by the basal profile. These results suggest that shifts in circulating FA profile within a biological range, without a difference in the total FA concentration, have minimal effects on transcriptional regulation of hepatic gluconeogenic genes in primary bovine hepatocytes.

Short communication: Blood samples before and after embryonic attachment accurately determine non-pregnant lactating dairy cows at 24 d post-artificial insemination using a commercially available assay for pregnancy-specific protein B Publication date: August 2019 Source: Journal of Dairy Science, Volume 102, Issue 8 Author(s): E.L. Middleton, J.R. Pursley ABSTRACT Early pregnancy diagnosis is critical to reproductive success on dairy farms. Reproductive success depends on cows becoming pregnant before 130 d in milk and then maintaining that pregnancy. The earlier non-pregnant cows are identified, the sooner they can be reinseminated, thus reducing days to pregnancy. Assays for pregnancy-specific protein B (PSPB) and pregnancy-associated glycoproteins can be used to diagnose pregnancy >28 d post-artificial insemination (AI) in lactating cows. The objective of this study was to determine whether percentage change in serum levels of PSPB within cow from d 17 to 24 can be used to identify non-pregnant cows using a commercially available assay. This study was performed on a large commercial dairy. Blood samples were taken at d 17 and 24 post-AI. The d 17 sample served as a baseline based on previous data. Cows with a 10% increase in serum PSPB levels from d 17 to 24 were considered pregnant. Lactating dairy cows (n = 206; 39% primiparous and 61% multiparous) were synchronized using G6G-Ovsynch. The PSPB diagnosis was compared with the herd veterinarian's diagnosis via ultrasound on d 34. The sensitivity for a 10% cutoff as a non-pregnant diagnosis was 100%, and the specificity was 93.58%. The positive predictive value was 93.27%, and the negative predictive value was 100%. Low PSPB levels at d 24 were predictive of early pregnancy loss by 60 d post-AI. To our knowledge no other method can diagnose non-pregnancy with 100% accuracy and predict pregnancy loss earlier than 24 d post-AI. Using comparative PSPB samples at d 17 and 24 post-AI provides an accurate non-pregnancy diagnosis earlier than any other pregnancy diagnosing method.

Potential for a localized immune response by the ruminal epithelium in nonpregnant heifers following a short-term subacute ruminal acidosis challenge Publication date: August 2019 Source: Journal of Dairy Science, Volume 102, Issue 8 Author(s): C. Kent-Dennis, A. Pasternak, J.C. Plaizier, G.B. Penner ABSTRACT The aim of this study was to investigate whether the ruminal epithelium activates a local inflammatory response following a short-term subacute ruminal acidosis (SARA) challenge. Seven ruminally cannulated, nonpregnant, nonlactating beef heifers, fed a baseline total mixed ration (TMR) with 50:50 forage-to-concentrate ratio, were used in a crossover design with 2 periods and 2 treatments: SARA and control (CON). Induction of SARA included feed restriction (25% of dry matter intake [DMI] for 24 h) followed by a grain overload (30% of baseline DMI) and provision of the full TMR; whereas, the CON group received the TMR ad libitum. Ruminal pH was recorded using indwelling probes, and ruminal lipopolysaccharide (LPS) concentration was measured daily following the challenge until d 6. Biopsies of ruminal papillae from the ventral sac were collected on d 2 and 6 after the grain overload. Transcript abundance of genes associated with acute inflammation was measured by quantitative real-time PCR, normalized to the geometric mean of 3 stable housekeeping genes. Target genes included toll-like receptor-2 (TLR2), TLR4, TLR9, tumor necrosis factor-α (TNFA), prostaglandin endoperoxide synthase-1 (PTGS1), PTGS2 transforming growth factor β-1 (TGFB1), and 4 intermediate enzymes of leukotriene synthesis (ALOX5, ALOX5AP, LTA4H, and LTC4S). Protein localization and expression of TLR4 were quantified by image analysis of fluorescence intensity. Statistical analysis was performed using as a crossover design with fixed effects of treatment, day, and the treatment × day interaction with the random effect of day within period. Ruminal pH was below 5.6 for 4.5 h/d and below 5.8 for 6.9 h/d in the SARA group compared with 22 and 72 min/d, respectively, for CON. Ruminal LPS concentration peaked on d 2 in SARA heifers at 51,481 endotoxin units (EU)/mL compared with 13,331 EU/mL in CON. Following grain overload, small but statistically significant decreases in the transcriptional abundance of TLR2, TLR4, TNF, PTGS2, ALOX5, and ALOX5AP were seen in SARA versus CON heifers. A functionally relevant decrease in TLR4 expression in SARA heifers compared with CON was confirmed by a decrease in fluorescence intensity of the corresponding protein following immunohistofluorescent staining of papillae. The study results indicate a suppression of the inflammatory response in the ruminal epithelium and suggest that the response is tightly regulated, allowing for tissue recovery and return to homeostasis following SARA.

Intramammary pressure and udder firmness during a 72-h interruption of milking to simulate dry-off, with and without feed restriction Publication date: August 2019 Source: Journal of Dairy Science, Volume 102, Issue 8 Author(s): Ulrich Blau, Lisa Zanini, Rupert M. Bruckmaier ABSTRACT The goal of the present study was to quantify the increase of intramammary pressure (IMP) in dry-off during an extended milking interval of 72 h. In particular, we tested the hypothesis that feed restriction (no concentrate and roughage with reduced energy) causes earlier cessation of milk secretion and a lower IMP than continued feeding of the lactational diet. In addition to repeated IMP measurements, we tested a noninvasive method that records udder firmness (UF) via external application of pressure on the udder. Two experimental groups consisted of 10 Holstein cows each, with a daily milk yield of 20 to 25 kg. The restricted group (RG) was changed to restricted feeding on the afternoon of the final milking (0 h), whereas late-lactation feeding was continued in the control group (CG). Both IMP and UF were measured before and after the final milking immediately before milking was stopped for 72 h. These measurements represented IMP and UF levels at 10 h and 0 h milking intervals, respectively. Further measurements were performed at 18, 24, 30, 36, 42, 48, and 72 h after final milking. Milk samples (2 mL) were taken through the IMP catheter at each sampling event, for analysis of somatic cell count (SCC) and serum albumin (SA). Both IMP and UF increased with time, and both parameters peaked at 30 h in CG and at 24 h in RG. The mean IMP from 18 to 72 h, compared with the 10-h IMP (normal milking interval) was higher in CG than in RG. The duration of elevated IMP and UF was prolonged in CG compared with RG (>36 h vs. 12 h). The Pearson correlation between IMP and UF was r = 0.67. Thus, the noninvasive measurement of UF is suitable to replace invasive IMP measurements. However, due to individual differences in udder shape, the correlation between UF and IMP was too low to predict exact IMP levels using UF. Both SCC (presented as logSCC) and SA increased after the final milking until the end of the experiment. The mean increase from 18 to 72 h, compared with levels immediately after final milking, was higher in CG than in RG for SCC but did not differ between treatments for SA. In conclusion, feed restriction causes a faster cessation of milk secretion and therefore limits the increase of IMP at dry-off.

Low abundance of mitofusin 2 in dairy cows with moderate fatty liver is associated with alterations in hepatic lipid metabolism Publication date: August 2019 Source: Journal of Dairy Science, Volume 102, Issue 8 Author(s): Jihong Dong, Juan J. Loor, Rankun Zuo, Xiying Chen, Yusheng Liang, Yazhe Wang, Xin Shu, Xudong Sun, Hongdou Jia, Guowen Liu, Zhe Wang, Xiaobing Li, Xinwei Li ABSTRACT High blood concentrations of nonesterified fatty acids (NEFA) and altered lipid metabolism are key characteristics of fatty liver in dairy cows. In nonruminants, the mitochondrial membrane protein mitofusin 2 (MFN2) plays important roles in regulating mitochondrial function and intrahepatic lipid metabolism. Whether MFN2 is associated with hepatic lipid metabolism in dairy cows with moderate fatty liver is unknown. Therefore, to investigate changes in MFN2 expression and lipid metabolic status in dairy cows with moderate fatty liver, blood and liver samples were collected from healthy dairy cows (n = 10) and cows with moderate fatty liver (n = 10). To determine the effects of MFN2 on lipid metabolism in vitro, hepatocytes isolated from healthy calves were used for small interfering RNA–mediated silencing of MFN2 or adenovirus-mediated overexpression of MFN2 for 48 h, or treated with 0, 0.6, 1.2, or 2.4 mM NEFA for 12 h. Milk production and plasma glucose concentrations in dairy cows with moderate fatty liver were lower, but concentrations of NEFA and β-hydroxybutyrate (BHB) were greater in dairy cows with moderate fatty liver. Dairy cows with moderate fatty liver displayed hepatic lipid accumulation and lower abundance of hepatic MFN2, peroxisome proliferator-activated receptor-α (PPARα), and carnitine palmitoyltransferase 1A (CPT1A). However, sterol regulatory element-binding protein 1c (SREBP-1c), acetyl CoA carboxylase 1 (ACACA), fatty acid synthase (FASN), and diacylglycerol acyltransferase 1 (DGAT1) were more abundant in the livers of dairy cows with moderate fatty liver. In vitro, exogenous NEFA treatment upregulated abundance of SREBP-1c, ACACA, FASN, and DGAT1, and downregulated the abundance of PPARα and CPT1A. These changes were associated with greater lipid accumulation in calf hepatocytes, and MFN2 silencing aggravated this effect. In contrast, overexpression of MFN2-ameliorated exogenous NEFA-induced lipid accumulation by downregulating the abundance of SREBP-1c, ACACA, FASN, and DGAT1, and upregulating the abundance of PPARα and CPT1A in calf hepatocytes. Overall, these data suggest that one cause for the negative effect of excessive NEFA on hepatic lipid accumulation is the inhibition of MFN2. As such, these mechanisms partly explain the development of hepatic steatosis in dairy cows.

Establishing a model of primary bovine hepatocytes with responsive growth hormone receptor expression Publication date: August 2019 Source: Journal of Dairy Science, Volume 102, Issue 8 Author(s): S. Witte, Y. Brockelmann, J.-D. Haeger, M. Schmicke ABSTRACT The liver becomes resistant to growth hormone before parturition in dairy cows (uncoupling of the somatotropic axis). However, the mechanism of growth hormone insensitivity has not been fully described. The aim of the present study was to improve a previous model of adult bovine hepatocytes in a sandwich culture system to ensure growth hormone receptor (GHR) expression. First, we modified the protocol for hepatocyte retrieval and tested the effect of short (18 min) and long (up to 30 min) warm ischemia on hepatocyte viability. Second, we used medium additives that affect GHR expression in vivo—insulin (INS), dexamethasone (DEX), both (INS+DEX), or no hormone additives (CTRL)—to ensure the functionality of hepatocytes, as measured by lactate dehydrogenase activity and urea concentration in the medium. We also used reverse transcriptase PCR of hepatocytes to evaluate expression of albumin (ALB), hepatocyte nuclear factor 4α (HNF4A), nuclear factor-κ-B-inhibitor α (NFKBIA), cytosolic phosphoenolpyruvate carboxykinase (PCK1), and vimentin (VIM) mRNA. Moreover, we analyzed the expression of GHRtot (GHR), GHR1A, insulin-like growth factor-1 (IGF1), and IGF binding protein-2 (IGFBP2) in response to exposure to media with the different compositions. Modification of the protocol (changes in rinsing and perfusion times, buffer composition, and the volume and standardization of collagenase) led to increased cell counts and cell viability. Short warm ischemia with the modified protocol significantly increased cell count (4.7 × 107 ± 1.9 × 107 vs. 3.5 × 106 ± 1.5 × 106 vital cells/g of liver) and viability (79.1 ± 8.4 vs. 37.1 ± 8.9%). Therefore, we gathered hepatocytes from the liver after short warm ischemia with the modified protocol. For these hepatocytes, lactate dehydrogenase activity was lower in media with INS and with DEX than in media with INS+DEX or CTRL; urea concentrations were highest at d 4 for INS+DEX. As well, HNF4A and ALB were more highly expressed in hepatocytes cultured with INS and INS+DEX than in those cultured with DEX or CTRL, and the substitution of DEX suppressed VIM and NFKBIA expression but increased PCK1 expression. The expression of GHR, GHR1A, and IGF1 was suppressed by dexamethasone (DEX and INS+DEX), whereas INS distinctly increased GHR, GHR1A, and IGF1 mRNA expression. Hepatocytes in a sandwich culture showed influenceable GHR expression; this study provides a model that can be used in studies examining factors that influence the expression and signal transduction of GHR in dairy cows.

Effect of manipulating progesterone before timed artificial insemination on reproductive and endocrine outcomes in high-producing multiparous Holstein cows Publication date: August 2019 Source: Journal of Dairy Science, Volume 102, Issue 8 Author(s): P.D. Carvalho, V.G. Santos, H.P. Fricke, L.L. Hernandez, P.M. Fricke ABSTRACT 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.

Short communication: Summer on-farm environmental condition assessments in Québec tiestall farms and adaptation of temperature-humidity index calculated with local meteorological data Publication date: August 2019 Source: Journal of Dairy Science, Volume 102, Issue 8 Author(s): V. Ouellet, A.L. Bellavance, S. Fournel, É. Charbonneau ABSTRACT Temperature-humidity index (THI) calculation following the equation developed by the National Research Council (A Guide to Environmental Research on Animals, 1971) requires ambient temperature (AT) and relative humidity (RH). Those data are widely and readily available at local meteorological stations. However, studies showed that using average AT and RH retrieved from the closest stations is not appropriate for estimating on-farm conditions. The present objectives were (1) to study summer on-farm environmental conditions, (2) to explore the relationship between summer THI calculated with on-farm data and summer THI calculated with local weather station data, and (3) to verify whether THI calculated with summer meteorological station data could be adapted to better represent summer on-farm conditions. Six tiestall dairy farms located in 2 regions of the province of Québec [Eastern Québec (EQ) and Southwestern Québec (SWQ)] were enrolled in this study. Within-barn conditions were monitored using 3 remote data loggers from August 2016 through August 2017. Two loggers were installed inside at varying distances relative to the ventilation inlet (L1: closest to inlet; L2: farthest from inlet) and a third was installed just outside of the barn (L3). Values retrieved from each logger and the closest local meteorological station were used to calculate daily THI according to the National Research Council formula and were ultimately compared. Our results showed that THI varied within the barn depending on the proximity relative to the inlet because THI measured by L1 was lower than THI measured by L2 in both regions. Moreover, our results showed that in both regions AT measured on-farm was consistently higher than AT measured at the weather station. The opposite was observed with RH, as it was significantly lower on-farm in EQ and numerically lower in SWQ compared with RH extracted from weather stations. Overall, this led to THI being lower by 4.6 and 3.7 units at the weather stations compared with within-barn conditions for EQ and SWQ farms, respectively. Hence, using local meteorological station data to estimate on-farm conditions would lead to an underestimation of heat stress level in dairy cows. Adapting THI calculations by including daily maximum AT and minimum RH retrieved from the local weather station instead of their average counterparts led to a better estimation of within-barn conditions. However, the difference between THI measured on-farm and the adapted THI calculated with weather station data remained significant. Although the adaption made to THI allowed for a closer relation to on-farm conditions, THI calculated with weather station data should only be used to assess heat stress level in dairy cows when heat stress thresholds are adapted for such data.