Increased milking frequency (IMF) during early lactation increases milk yield not only during the IMF period, but also after the cow is returned to twice daily milking (2X). The increase in yield is locally regulated within the gland; however the mechanism for the increase in yield is unknown. The objective of this study was to demonstrate a difference in milk and component yield, both during the IMF period and throughout the remainder of lactation, and examine potential local mechanisms driving the increase in production. Eight multiparous dairy cows were assigned to unilateral frequent milking [UFM ; 2X left udder half and 4-times-daily milking (4X) right udder half] for the first 21 days of lactation. Both udder halves were milked 2X for the remainder of lactation. Early lactation IMF significantly increased milk, fat, and protein yields in the right udder halves by 2.27 kg/d, 73.5 g/d, and 68 g/d respectively through the first 210 DIM (P < 0.001). At d 21, the right udder halves had a significant increase in activated signal transducer and activator of transcription 5 (STAT5), as well as a reduction in activated Akt (P ≤ 0.05). There was no difference in STAT3 expression at d 21. There was no significant difference in gene expression of prolactin, insulin-like growth factor 1 (IGF-1), insulin-like growth factor binding protein 5 (IGFPB5), or chitinase 3-like-1 (CHI3L1) in mammary tissue at d 21 or 60; and no difference in protein expression of STAT5, Akt, or STAT3 in mammary tissue at d 60. / Master of Science

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Six primiparous Holstein cows were assigned to a half udder design (n=6) 40 days prior to parturition. Beginning at parturition, one udder half was milked once daily (24hr interval) and the other four times daily (6hr interval). Udder halves were biopsied at days 15, 60, 120, and 230 of lactation for mammary tissue to perform mitochondrial staining and apoptosis studies. Increasing the milking frequency from 1x to 4x elevated the 4x udder half milk yield at early (d1-45) (P<0.0001), mid (d46-150) (P<0.0001) and late (d151-230) (P<0.0001) lactation. Milk protein percent (P= 0.013), lactose percent (P=0.004) and SNF percent (P=0.006), were elevated in milk from 4x udder halves over milk from 1x udder halves. We did not detect an effect of increased milking frequency on milk fat percent (P=0.25); however, yield of all components was increased. Increased milking frequency also increased mitochondrial numbers in mammary cells from 4x udder half (P=0.002) compared to 1x. We did not detect an effect of increased milking frequency on mammary apoptosis percentage. We also did not detect a difference in the abundance of gene transcripts for SOCS1, SOCS2, SOCS3 and CIS in milk; but could find an increase in alpha-lactalbumin (P=0.04) and beta-casein (P=0.001) 4x udder half gene transcripts.

The objective of this thesis was to determine the effects of frequency of milking and feed delivery on the behavioural patterns and productivity of lactating dairy cows. In two independent experiments, twelve free-stall housed, lactating Holstein dairy cows were exposed to either varying milking frequency or varying feed delivery frequency over 21-d periods. In the first study, cows milked three times per day and multiparous cows produced more milk than those milked twice per day and primiparous cows. Milking three times per day altered the distribution of feeding activity throughout the day. Multiparous cows had longer, and larger meals, while primiparous cows had smaller, more frequent meals throughout the day when milked three times per day. In the second study, cows delivered feed three times per day consumed more DM than those fed once per day and twice per day. Feed delivery frequency had little effect on feeding behaviour and the distribution of feeding activity, but altered the magnitude of DMI following feed delivery. Cows delivered feed more frequently achieve greater daily DMI by consuming more feed following the return from milking and the delivery of feed. / This project was financially supported by a Dairy Farmers of Ontario (Mississauga, Ontario, Canada) research grant and an Ontario Ministry of Agriculture, Food and Rural Affairs (OMAFRA; Guelph, Ontario, Canada)/University of Guelph Production Systems research grant. Additional project support was received from the Canadian Foundation for Innovation (CFI; Ottawa, Ontario, Canada) and the Ontario Research Fund (Toronto, Ontario, Canada).

Increased milking frequency (IMF) in early lactation is a time and cost-effective farm management practice to enhance profit in the dairy industry. The process involves milking cows more often in early lactation alone. On farms that milk twice daily (2X), early lactation cows are milked four times daily (4X) for 21 d postpartum. Cows produce significantly more milk during this timeframe and continue to have increased milk yield (MY) when returned to 2X milking for the remainder of lactation. The objective of this dissertation was to discover the physiological processes of early lactation IMF that cause increased MY throughout lactation. All studies involved unilateral frequent milking (UFM) with 2X and 4X udder halves for 21 d in early lactation. The first study manipulated milking interval (MI), or the time between milkings, during early lactation IMF. Cows were either milked on an even MI every 6 h or unevenly on a 9:3:9:3 h MI. Unevenly milked cows produced more milk on the final day of 4X treatment, but there was no significant difference in the increased MY carry-over effect between MI groups. Therefore, farmers can utilize any MI that fits their schedule and still achieve significantly enhanced profits. The second study aimed to infer the metabolic mechanisms of early lactation IMF that increase MY by comparing it to bovine somatotropin (bST). Cows that underwent early lactation IMF received bST at 80 DIM. Both IMF and bST treatments significantly enhanced MY, but there was no interaction or synergistic effect between treatments. We surmised that IMF and bST cause increased MY through different metabolic mechanisms since IMF functions locally and bST operates systemically. The final study analyzed mammary tissue from 2X and 4X udder halves on the final day of UFM treatment. The mechanism in which IMF enhanced MY involved increased protein levels of signal transducer and activator of transcription (STAT5), activated and total protein kinase B (Akt), and total extracellular signal-regulated kinase 1 and 2 (ERK1/2) and reduced protein levels of total mammalian target of rapamycin (mTOR) and total mitogen activated protein kinase (MAPK) in 4X udder halves compared to 2X. / Doctor of Philosophy / Increased milking frequency in early lactation is the process of milking cows more often for the first 3 weeks after calving. Cows not only produce more milk during this timeframe but continue to have elevated milk yield throughout lactation. This phenomenon is called the increased milk yield carry-over effect. This dissertation aimed to further enhance the increased milk yield carry-over effect of early lactation increased milking frequency. All studies utilized unilateral frequent milking with left udder halves milked twice daily and right udder halves milked four-times daily. The first study compared even and uneven milking intervals alongside early lactation increased milking frequency. The right udder halves of the even groups were milked every 6 hours. Cows in the uneven milking interval group were milked on a 9:3:9:3 hour interval. The uneven milking interval group produced more milk than even milking interval group on the final day of milking frequency treatment. However, there was no difference in milk yield between groups at any other time point. All cows had an increased milk yield carry-over effect throughout 300 days of lactation. In the second study, cows received bovine somatotropin in mid-lactation to observe possible synergistic effects in enhancing milk yield after early lactation increased milking frequency. Both the bovine somatotropin and increased milking frequency increased milk yield, but there was no synergistic effect when the two treatments were combined. The final study analyzed proteins within mammary tissue after 3 weeks of unilateral frequent milking. Udder halves milked four-times daily had significantly elevated activated and total STAT5, activated and total Akt, and total ERK1/2. Udder halves milked twice daily had elevated total MAPK and total mTOR. These findings helped to understand the metabolic functioning of increased milking frequency in early lactation that causes a persistent increase in milk yield throughout lactation.

Milk production is one of the most important parts of agriculture. Task of this paper was to examinate relation between milking frequency and total dairy production in Automatic Milking Systems. The thesis evaluates impact of selected factors, such as lactation stage, parity and season of calving, on dairy production and milking frequency. Total of 44,000 records of milk yield from 3 farms was analysed. Data was collected from 2009 to 2011. Relation between milk yield and milking frequency was prooved. The milk production from a cow rises with parity and is highest in second stage of lactation (cca 60th day after calving). Environment is one of very important factors affecting milk yield. Automatic milking system does not deteriorate milking parameters comparing to traditional way of milking.

Increasing the milking frequency of early lactation dairy cows increases their milk yield, both during increased milking and after the cows are returned to a normal twice-daily milking schedule. When milked four times a day (4X) for only the first twenty-one days of lactation, the right udder half produces 3 kg/d more milk than the left half, which is milked only two times a day (2X) over the course of lactation (Hale et al., 2003). Alterations to this increased milking frequency (IMF) approach have been investigated in order to maximize production of the animals and determine the most efficient practice for producers. The aim of this study was to determine the appropriate duration of early lactation IMF treatment by increasing milking frequency of early lactation cows for various lengths of time, and subsequently increasing the use of this management practice on Virginia dairy farms. The right udder half of twenty-three primiparous and multiparous Holstein cows were milked 4X for 10, 20, or 40 days at the beginning of lactation, and the left udder half 2X for the entire lactation. Udder-half milk yields were measured at various time points throughout lactation and used to calculate the difference between right (4X) and left (2X) udder halves. Overall, treatment did not have a significant effect on milk yield difference throughout the entire lactation; the udder half differences for each group were -0.45 kg, 1.92 kg and 4.62 kg for the 10 d, 20 d and 40 d treatments (P > 0.05). In addition to the IMF portion of the experiment, two different methodologies were used to investigate the possible mechanism of local regulation of milk yield in response to IMF treatment. Milk fatty acid analysis was performed on milk samples obtained from the above experiment. Three different groups of fatty acids were analyzed to detect potential changes in the right udder half (4X) when compared to the left (2X); the three groups were denovo, C16, and preformed fatty acids. There was no significant effect of treatment on fatty acid composition of right and left udder halves for any of the three groups (P > 0.05). The second methodology used to explore a possible mechanism behind increased milk yield following IMF treatment was immunohistochemistry of mammary gland tissue samples obtained after IMF treatment in a previous experiment. The key target investigated was a component of the Hippo signaling pathway, Yes-associated protein (YAP). Intensity of YAP staining in the cytoplasmic area of mammary epithelial cells (MEC) and number of YAP-positive stained nuclei located in the MEC were quantified for each of the images obtained. There was no effect of treatment or day on intensity of staining (P > 0.05) with no difference in the intensity of staining between 4X and 2X samples or d 21 and d 60 samples. However, the interaction for treatment x day tended to be significant (P < 0.06), with the d 60 samples tending to have higher intensity of staining than d 21 samples. For YAP-positive nuclei, there was a significant effect of day (P < 0.05), with d 60 samples having significantly more YAP positive nuclei. There was not a significant effect of treatment or treatment x day interaction (P > 0.05) with 2X and 4X samples having the same number of YAP positive nuclei. Results from the first experiment reveal that 40 d of IMF during early lactation is sufficient to produce an increase in milk and component yields throughout lactation. This practice could be implemented on Virginia dairy farms as a way to increase efficiency and milk yield per cow. Results from the second half of this research indicates that further research is needed to investigate the fatty acid content of milk from cows subject to IMF treatment during early lactation. In addition, YAP potentially plays a role in the changes occurring in the mammary gland, with increased intensity of YAP staining and increased number of YAP positive nuclei observed at 60 DIM. Understanding of this protein and its involvement in the mammary gland could lead to identifying a mechanism for which this increase in milk yield and components following IMF is occurring. Further research needs to be done to provide results supporting the current experiment. / MSLFS

The aim of this thesis was to analyze the effect of milking frequency per day on milk efficiency and fertility during the course of lactation. The observation of dairy Holstein cattle herd took place in agricultural company Brloh (Blanský les Region) from January 2010 to February 2011. There were observed 55 dairy cows: Holstein cattle (75 %), share cross cows with race Czech Pied cattle (22 %) and Fleckvieh cattle (3%). Microsoft Excel has been used for processing the statistic data. For results evaluation, some basic statistic characteristics have been counted. Differences between the groups have been verified by the simple factor analysis of variance. By evaluation of milk efficiency including milk constituents for standardized lactation on individual lactation phases, reached breeding cows on first lactation 9460.00 kg M (3.22 % B and 3.88 % T), on second lactation 10632.15 kg M (3.23 % B and 3.96 % T) and breeding cows on third and next lactation 9738.60 kg M (3.18 % B a 4.08 % T). In the assessment of milking frequency visited breeding cows the AMS on the first lactation at 1/3 lactation phases 2.51 per a day, breeding cows at 2/3 lactation phases 2.72 per a day and 2.70 per a day at 3/3 lactation phases. This trend continued in next lactation, but breeding cows on the second lactation at 3/3 lactation phases visited most often robotic milking machine 2.80 per a day. Relationship between the number of milking and milk efficiency was found statistical significance. With the increasing number of milking performance also increases. By evaluation reproductive parameters the following values were observed ? the average first calving age 774.9 days (25 months), the average meantime 382.3 days, the average servis period 110.4 days and the average insemination period 77.8 days. The relationship between the number milking and service period has been established by the positive correlation, i.e. to the growing number of milking is to the extension service period.

La lipolyse est une réaction enzymatique qui influence négativement les qualités organoleptiques et technologiques du lait. La lipolyse spontanée (LS) correspond à la part de la lipolyse qui dépend de l’animal et du système d’élevage. La LS résulte de l’action de la lipoprotéine lipase (LPL) et de ses cofacteurs sur les globules gras (GG). L’objectif de la thèse a été de comprendre les variations de LS à l’échelle zootechnique et à l’échelle du lait. Les vaches (VL) ont pu être classées en 2 groupes selon leur phénotype : « susceptible » et « non susceptible » à la LS, confirmant l’importance de l’effet individu. Chez les VL susceptibles, nous avons confirmé un effet de la race/génétique, de la parité, du stade physiologique, du moment de la traite, de la fréquence de traite et de l’alimentation.La LS était plus élevée dans les laits issus de la traite du soir, chez les VL Holstein, génotypées KK au locus de DGAT-1. Elle l’était également en fin de lactation et en début de lactation uniquement chez les multipares hautes productrices. Un bilan énergétique négatif pourrait expliquer ces variations. L’augmentation de la fréquence de traite, la restriction alimentaire, l’alimentation à base d’ensilage de maïs comparé à l’herbe conservée/fraîche et la supplémentation lipidique ont également augmenté la LS. Le mécanisme d’action implique probablement une inhibition de la LS par la protéose peptone 5. La membrane des GG semblerait avoir un rôle crucial dans le maintien de l’intégrité du GG, l’interaction avec la LPL et l’équilibre des cofacteurs. L’impact des facteurs zootechniq / Lipolysis is an enzymatic reaction which leads to off-flavor in milk and impairs technological properties of milk. Spontaneous lipolysis (SL) is the fraction of lipolysis which depends on cows and breeding systems. SP corresponds to the hydrolysis of milk fat in milk fat globules (MFG) by the lipoprotein lipase (LPL) and its cofactors. The aim of the PhD was to understand SL variations at both zootechnical and biochemical levels. Cows were sorted in two groups according their phenotype: “susceptible” and “non-susceptible” to SL, confirming the strong impact of the individual effect. Among cows “susceptible” to SL, we confirmed the effects of breed/genetics, parity, physiological stage, milking moment, milking frequency and feeding systems. SL was higher in evening milks of Holstein cows and of cows having the KK genotype at the DGAT-1 locus. SL was higher in late lactation and, in early lactation only for high merit multiparous cows, probably in relation to negative energy balanceAn increase in milking frequency, feeding restriction, maize silage based diets compared to fresh grass and conserved grass based diets and lipid supplementation enhanced SL. At a biochemical level, LS might be inhibited by proteose peptone 5. The MFG membrane might play an important role on MFG integrity, LPL and MFG interactions, and cofactors balance. The impact of zootechnical and biochemical factors on SL is still difficult to hierarchize