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.     

共轭亚油酸对乳腺脂类代谢和乳腺发育的作用及其机理

共轭亚油酸(Conjugated Linoleic Acid,CLA)是亚油酸具有共轭双键的一组同分异构体,由瘤胃细菌在生物氢化过程中合成,具有抗癌、抗炎症、抗动脉粥样硬化等多种生物学功能。CLA在代谢方面也有调节作用,能影响全身代谢,影响肝脏、乳腺以及富含脂肪的相关组织代谢。在此介绍了CLA的来源、结构、对动物乳腺脂类代谢和乳腺发育的作用及其机理。     

Performance strategies affect mammary gland development in prepubertal heifers

In Brazil, the majority of dairy cattle are Holstein × Gyr (H×G). It is unknown whether excessive energy intake negatively affects their mammary development to the same extent as in purebred Holsteins. We hypothesized that mammary development of H×G heifers can be affected by dietary energy supply. We evaluated the effect of different average daily gains (ADG) achieved by feeding different amounts of a standard diet during the growing period on biometric measurements, development of mammary parenchyma (PAR) and mammary fat pad (MFP), and blood hormones. At the outset of this 84-d experiment, H×G heifers (n = 18) weighed 102.2 ± 3.4 kg and were 3 to 4 mo of age. Heifers were randomly assigned to 1 of 3 ADG programs using a completely randomized design. Treatments were high gain (HG; n = 6), where heifers were fed to gain 1 kg/d; low gain (LG; n = 6), where heifers were fed to gain 0.5 kg/d; and maintenance (MA; n = 6), where heifers were fed to gain a minimal amount of weight per day. Heifers were fed varying amounts of a single TMR to support desired BW gains. Over the 84 d, periodic biometric and blood hormone measurements were obtained. On d 84, all heifers were slaughtered and carcass and mammary samples were collected. At the end, HG heifers weighed the most (181 ± 7.5 kg), followed by LG (146 ± 7.5 kg) and MA (107 ± 7.5 kg) heifers. The ADG were near expected values and averaged 0.907, 0.500, and 0.105 ± 0.03 kg/d for HG, LG, and MA, respectively. In addition, body lengths, heart girths, and withers heights were affected by dietary treatment, with MA heifers generally being the smallest and HG heifers generally being the largest. Body condition scores differed by treatment and were highest in HG and lowest in MA heifers; in vivo subcutaneous fat thickness measurement and direct analysis of carcass composition supported this. The HG heifers had the heaviest MFP, followed by LG and then MA heifers. Amount of PAR was highest in LG heifers and was the same for HG and MA heifers. The percentage of udder mass occupied by PAR was lowest in HG heifers, differing from LG and MA heifers. Composition of MFP was not evaluated. Regarding PAR composition, no differences in ash or DM were found. On the other hand, CP concentration of PAR for HG heifers was lower than that for LG heifers, which was lower than that for MA heifers. Regarding the fat content, HG treatment was higher than LG and MA treatment, which did not differ from each other. In PAR, differences in relative abundance of genes related to both stimulation and inhibition of mammary growth were observed to depend on dietary treatment, sampling day, or both. The same can be said for most of the blood hormones that were measured in this experiment. In this experiment, high ADG achieved by feeding different amounts of a standard diet during the growing period negatively affected mammary development.     

Distribution of lysosomal enzymes within the non-secretory epithelium of the bovine mammary gland

The levels of N-acetyl-beta-glucosaminidase (NAGase) and acid phosphatase were analysed in tissue from the teat and lactiferous sinus epithelia of dairy cows at different stages of lactation. Enzyme levels were lower in the epithelia of the teat sinus than of the lactiferous sinus. For both enzymes, levels were highest in tissue from post-partum glands, lowest in those from dry and mid-lactation glands. In involuting glands the levels were high, but not as high as the post-partum glands.     

Plant inhibitors of proteolytic enzymes

The presence of inhibitors of proteinases was stated in many species of plants. There are macropeptides of the molecular weight ranging from 3700 to 8000, often bound to carbohydrates. Potential sources of inhibitors of proteinases are legumes, cereals, potatoes and also some fruits. They are characterized by different activity. "Single-headed" inhibitors inhibit one type of proteolytic enzyme, when "double-headed" inhibitors, possessing two independent active sites, can inhibit several types of proteolytic enzymes at the same time. They also differ in the resistance to temperature and change of pH. The role and importance of inhibitors of proteinases is not exactly explained. They are used in the pharmaceutical, baking and beer-industry as well as in the therapy of numerous diseases.     

Lactoferrin concentration during involution of the bovine mammary gland.

Electroimmunodiffusion assay was used to quantitate changes in lactoferrin concentration in mammary secretions during involution of the bovine mammary gland. Concentration of lactoferrin began to increase 2 to 4 days after cessation of regular milking and continued to increase linearly at a rate of 1.15 mg/ml per day as a result of increased net synthesis of lactoferrin during the first 14 to 21 days of involution. Maximum lactoferrin concentration (approximately 20 mg/ml) was attained after 3 to 4 wk of involution. These changes represent a 100-fold increase in lactoferrin concentration over that in normal milk. Maximum lactoferrin concentration was variable between cows. In some cows, the concentration of lactoferrin plateaued at less than 10 mg/ml after 10 days of involution. In others, much higher lactoferrin concentrations of 75 to 100 mg/ml were measured. Lactoferrin concentration decreased markedly prior to parturition and onset of lactation. The increase in lactoferrin concentration during mammary gland involution appeared to be related closely to the process of involution.     

The influence of proteolytic and cytolytic enzymes on starch degradation during mashing

The objective of this research was to investigate the impact of proteolysis and cytolysis on starch degradation over the course of the mashing process. A proteolytic enzyme (Neutrase 0.8L, Novozymes) and a number of cytolytic enzymes (barley β‐glucanase from Megazyme, Shearzyme 500L and Ultraflo Max from Novozymes) were used to test their efficiency on starch degradation during mashing. The proteolytic and cytolytic enzymes had positive effects on the levels of starch‐degrading enzymes and starch solubilization during mashing, resulting in higher levels of wort sugar compared with the control, indicating that proteins and residual non‐starchy polysaccharides limited the digestibility of starch during mashing. Moreover, the proteolytic enzyme showed a significantly greater improvement than the cytolytic enzymes, yielding a 57% increase in β‐amylase, a 173% increase in limit dextrinase, rapid starch solubilization during mashing and a higher percentage of fermentable sugars in resultant wort. The increases in limit dextrinase and β‐amylase promoted by the protease suggest that sufficient proteinaceous inhibitor existed in the wort during mashing to inhibit their activities, leading to the unavailability of β‐amylase and especially limiting dextrinase. Furthermore, Ultraflo Max, which contained a β‐glucanase–xylanase mixture, showed a greater improvement than either the individual β‐glucanase or the xylanase on starch hydrolysis. These findings suggest that β‐glucanase is the major enzyme responsible for the degradation of cell walls, and that the complete hydrolysis of the residual cell walls depends on the synergistic effect of β‐glucanase and xylanase. The results suggest that brewers should adjust the degradation of the cell walls and correct the degree of protein modification in order to obtain the desired wort composition. Copyright © 2014 The Institute of Brewing & Distilling     

Growth of Corynebacterium bovis in mammary secretions during physiological transitions of the bovine mammary gland

An in vitro microassay was used to evaluate growth of five strains of Corynebacterium bovis in mammary secretions collected from quarters of five Holstein cows at 0, 14, and 28 d of involution, at parturition, and 14 d after parturition. Variation in growth among different strains of Corynebacterium bovis was observed. Corynebacterium bovis grew well in mammary secretions obtained at the last milking of lactation, at parturition, and 14 d after parturition. However, growth of four strains of Corynebacterium bovis in mammary secretions obtained at 14 and 28 d of involution was reduced significantly. In contrast, a streptomycin-resistant strain of Corynebacterium bovis grew well in mammary secretions obtained during involution. These data suggest that mammary secretions support growth of Corynebacterium bovis during lactation but inhibit growth during the nonlactating period. Inhibition of growth in secretions collected during the nonlactating period may be associated with the high rate of spontaneous elimination of Corynebacterium bovis intramammary infection from cessation of milking to parturition. Conversely, enhanced growth in milk may be related to persistent Corynebacterium bovis intramammary infections during lactation.     

奶牛不同发育时期乳腺细胞凋亡规律的研究

为系统地研究奶牛乳腺不同发育时期细胞凋亡的规律,应用TUNEL法对奶牛不同发育时期正常乳腺组织(冰冻切片)的乳腺细胞凋亡进行了系统检测。结果表明,青春期乳腺发育缓慢,结构变化较小,乳腺细胞凋亡量相对较少;妊娠期乳腺导管持续发育,凋亡量上升,其中妊娠初期2月,乳腺腺泡大量出现,脂肪细胞凋亡随之增加,出现了一个高峰;泌乳期乳腺的结构和功能最为完善,乳腺结构变化很小,细胞凋亡量维持在很低水平;退化期腺泡瓦解,大量细胞发生凋亡,其中退化初期乳腺细胞凋亡持续增加,退化3 d达到最大值,之后凋亡量逐渐降低,退化30 d后乳腺已经基本恢复到青春期状态,凋亡细胞量也随之减少。     

Role of the isocitrate dehydrogenases and other Krebs cycle enzymes in lactating bovine mammary gland

The role of the isocitrate dehydrogenases and other Krebs cycle enzymes in bovine mammary metabolism was studied by investigation of their distribution between cytosol and mitochondria. Citrate synthase was used as a marker for mitochondrial disruption, and distributions were normalized to this enzyme. Aconitase, fumarase, and NAD+:malate dehydrogenase were distributed between the mitochondria and the cytosol; evidence for the possible involvement of an aspartate:malate shuttle was also found. The NADP+:isocitrate dehydrogenase is predominantly cytosolic with a small but significant amount of mitochondrial component. Using the dye dichlorophenol-indophenol, a low level of NAD+:isocitrate dehydrogenase activity was observed in bovine mammary tissues. This assay also allows for detection of the enzyme in fresh mitochondria from a variety of other bovine tissues (heart, liver, kidney, and brain). Activities of the isocitrate dehydrogenases were also examined as a function of gestation and lactation. The NAD+:isocitrate dehydrogenase is apparently depressed during gestation with the NADP+ form of the enzyme (cytosolic) elevated postpartum. These results indicate that a substantial portion of Krebs cycle activity may become extramitochondrial in bovine mammary gland at the onset of lactation.     

IGFs对小鼠乳腺发育及细胞凋亡的影响

为揭示IGFs在乳腺发育和乳腺上皮细胞凋亡中的作用，运用荧光显微镜及光镜技术，采用小鼠乳腺组织培养及小鼠乳腺上皮细胞培养方法，对IGFs的作用进行了研究。结果表明，IGFs可促进小鼠乳腺导管的生长发育，并且IGF—I促小鼠乳腺导管生长发育的功能强于IGF—II；IGFs抑制泌乳期小鼠乳腺上皮细胞凋亡，并且IGF—I抑制凋亡的作用强于IGF—II。     

Secretion and mammary gland uptake of prolactin in dairy cows during lactogenesis

Mammary arteriovenous differences of prolactin concentration and net mammary uptake of prolactin from blood were quantified near parturition in 9 dairy cows. Six cows were milked once daily for at least 6 d before parturition, and prepartum lactogenesis occurred in 3 of 6 cows. Prepartum milking 2 or more d before parturition abruptly increased secretion of prolactin into blood, but milkings within 1 d before or after parturition did not increase prolactin secretion. Concentrations of prolactin in whole milk sampled over 8 d before parturition (64.5 ng/ml) were substantially greater than those occurring several days after parturition (19 ng/ml). Milk prolactin concentrations were unaffected by the successful induction of prepartum lactogenesis, which greatly increased prepartum yields of milk (2 to 8 kg/milking). Therefore, the alveolar lumenal content of prolactin was greatest in pregnant cows with prepartum lactogenesis. This enhanced content of intraalveolar prolactin before parturition was associated with an absence of mammary uptake of prolactin immediately prior to ejection of the prolactin-containing milk from the alveoli. However, prolactin uptake was quickly restored to about 2 micrograms/min per half udder shortly after milk ejection. During the prepartum period, an enhanced intraalveolar reservoir of 200 to 400 micrograms prolactin, due to induction of prepartum lactogenesis, appears to saturate temporarily all putative sites for uptake of prolactin from blood.     

Genetic manipulation of the IGF-I axis to regulate mammary gland development and function

Insulin-like growth factor I (IGF-I) is known to regulate mammary gland development. This regulation occurs through effects on both cell cycle progression and apoptosis. Our laboratory has studied the IGF-I-dependent regulation of these processes by using transgenic and knockout mouse models that exhibit alterations in the IGF-I axis. Our studies of transgenic mice that overexpress IGF-I during pregnancy and lactation have demonstrated that this growth factor slows the apoptotic loss of mammary epithelial cells during the declining phase of lactation but has minimal effects during early lactation on milk composition or lactational capacity. In contrast, our analysis of early developmental processes in mammary tissue from mice carrying a targeted mutation in the IGF-I receptor gene suggests that IGF-dependent stimulation of cell cycle progression is more important to early mammary gland development than potential anti-apoptotic effects. With both models, the effects of perturbing the IGF-I axis are dependent on the physiological state of the animal. The diminished ductal development that occurs in response to loss of the IGF-I receptor is dramatically restored during pregnancy, whereas the ability of overexpressed IGF-I to protect mammary cells from apoptosis does not occur if the mammary gland is induced to undergo forced involution. Data from our laboratory on the expression of IGF-signaling molecules in the mammary gland suggest that this effect of physiological context may be related to the expression of members of the insulin receptor substrate family.