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

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 focus of this study is increasing milk yield by increasing milking frequency of early lactation cows. We will use a technique where each half of the cow's udder is milked with different milking frequencies. Previous studies have shown an increase in milk yield due to increased milking frequency. When milked four times a day for 21 days, the right udder half produced 2,032 lb. more milk than the left over the course of a lactation. This project will investigate modifications to the 21-day increase in milking frequency that might increase the use of this management practice on Virginia dairy farms. This experiment will determine the increase in milk yield when cows are milked four times a day for 10, 20, or 40 days. Farmers are concerned about return on investment - including time invested engaging extra milkings. This experiment will determine the effective duration of increased milking frequency needed to gain an increase in milk production with the minimum investment of time necessary to apply increased milking frequency - can the increase achieved with 20 days be achieved in only 10 or does increasing to 40 days provide an even greater benefit than 20-day application?