**5. Cellular response to frequent milking**

248 Milk Production – An Up-to-Date Overview of Animal Nutrition, Management and Health

**4. Endocrine response to frequent milking or suckling** 

stimulus during this time.

negative feedback on the gland.

Wall & McFadden, 2007a). It was unknown whether a shorter duration or altered timing of frequent milking during early lactation would still elicit a persistent effect on milk production; however, since any costs associated with extra labor are increased only during frequent milking, it was of great interest to shorten the duration of frequent milking if a persistent increase in milk yield could still be observed. To answer this question, we assigned cows to UFM (4X of the right udder half, 2X of the left udder half) for d 1 to 14 or d 7 to 21 of lactation (Wall & McFadden, 2007b). We observed an acute milk yield response in both treatments; and a significant carry-over effect in the d 7 to 21 group. There was a numerical carry-over for the d 1 to 14 group; however it was not significant. Our results indicate that within the first 21 DIM, an interval of frequent milking as short as 2 wk can elicit a persistent increase in milk production. As mentioned previously, similar observations have been made on the response of cows and heifers to suckling during early lactation (Fulkerson et al., 1978; Peel et al., 1979). Further narrowing of this "window" within the first 21 of lactation, as well as characterization of the cellular response could provide insight into the mechanisms underlying the receptiveness of the mammary gland to

It has long been thought that the hormones released at milking may be involved in regulating the galactopoietic effects of frequent milking on milk production. Indeed, multiple hormones are released during milking including glucocorticoids, oxytocin, and prolactin (Tucker et al., 1975; Carruthers & Hafs, 1980; Akers & Lefcourt, 1982). Oxytocin is responsible for milk ejection. Cows suckling calves are thought to have more efficient milk ejection due to increased secretion of oxytocin elicited by the presence of the calf. In fact, on dairies using cross breeds and cows not bred for high milk production, the calf is often used as a facilitator of milk letdown during milkings (Little et al., 1991; Krohn, 2001; Bruckmaier & Wellnitz, 2008). In addition, treatment with exogenous oxytocin was associated with increased milk production of both dairy cows (Nostrand et al., 1991; Ballou et al., 1993; Lollivier & Marnet, 2005) and sheep (Zamiri et al., 2001). Therefore, it is possible that oxytocin is involved in regulating the increase in milk production elicited by frequent milking or suckling, perhaps by allowing for more complete milk removal and a decrease in

Along with enhanced milk production, Bar-Peled et al. (1995) observed increased concentrations of growth hormone, insulin-like growth factor-1, oxytocin and prolactin in circulation of cows that were frequently milked or suckled. In addition, the magnitude of milking-induced PRL release declines concomitantly with the decrease in milk production as lactation progresses (Koprowski & Tucker, 1973). Consequently, PRL has been hypothesized as a candidate regulator of the effects of frequent milking on milk production (Dahl et al., 2004a). In an attempt to determine whether milking-induced PRL release indeed mediates the effects of frequent milking on milk production, we assigned cows to 2X, 4X, or 2X + twice daily injections of PRL (Crawford et al., 2004; Wall et al., 2006). Four times daily milking or PRL injections increased milk production relative to 2X (Crawford et al., 2004); Several authors have speculated that frequent milking increases milk yield via an increase in mammary cell number and (or) activity (Bar-Peled et al., 1995; Stelwagen & Knight, 1997; Sanders et al., 2000; Hale et al., 2003), both of which are critical to improved lactation performance (Capuco et al., 2003). Hillerton et al. (1990) observed an increase in activity of mammary enzymes, protein and lactose synthesis (in heifers only), DNA synthesis, and alveolar area in response to increased milking frequency, and concluded that cellular differentiation and proliferation were optimized with frequent milking. Hale et al. (2003) reported an increase in mammary cell proliferation at 7 DIM in cows that were milked 4X for the first 3 wk of lactation compared to cows milked 2X; however, differences in proliferation were only observed in one of the two frequently milked cow groups. In contrast to those experiments, Norgaard et al. (2005) reported that despite an increase in milk yield (+18%), there was no effect of frequent milking on cell death, proliferation, or enzyme activities in the mammary gland. In agreement with that report, we have observed across multiple experiments that relative to 2X, 4X did not affect mammary cell proliferation or apoptosis (Wall et al., 2006; Wall & McFadden, 2010; Wall et al., 2011b), indicating that changes in milking frequency influence milk yield through an alternative mechanism.

Using a unilateral frequent milking model and a functional genomics approach, we determined that the increase in milk yield associated with frequent milking is regulated by changes in gene expression elicited by removal of milk from the gland (Wall et al., 2011a). We then used a sequential biopsy approach and obtained mammary tissue at various times during and after exposure to unilateral frequent milking and determined that the temporal expression of 64 genes was co-regulated by unilateral frequent milking (Wall et al., 2011b). Importantly, the pattern of differential expression of the 64 genes was negatively correlated with differential milk yield (Figure 4); therefore, we hypothesize that we have identified a pathway for the autocrine regulation of milk production. Furthermore, this transcriptional signature appears to be malleable and adaptable to the needs of the offspring (mimicked by changes in milking frequency), since expression of some of the genes was still different between udder halves nearly three weeks after cessation of treatment (Figure 4). Future experiments will clarify the role of these genes in the mammary gland and their involvement in the autocrine regulation of milk production.

What is unique to early lactation, when the stimulus of frequent milking for a short duration can elicit a persistent increase in milk production? This question remains

unanswered, but work by Stelwagen and Knight (1997) has provided some clues. Using a half-udder model, they compared 1X to 2X of cows in early or late lactation and reported a more dramatic increase in milk secretion efficiency in response to 2X during early lactation compared to late lactation (Stelwagen & Knight, 1997). In agreement, Walsh (1974) observed different effects of suckling during early vs. late lactation on mammary health. During early lactation, suckling of a calf was associated with a 27% decrease in clinical mastitis, whereas suckling during late lactation had no effect (Walsh, 1974). Taken together, the observations of Walsh (1974) and Stelwagen and Knight (1997) indicate that there are distinct differences in the cell population during early vs. late lactation. It is possible that during early lactation, there are more secretory cells present in the mammary gland, and these cells may have more potential to respond to stimulus than cells present in late lactation. Frequent milking may prevent otherwise unused cells from undergoing apoptosis, or may provide the stimulus to push the cells to reach higher levels of differentiation and secretory capacity. These scenarios could result in an increase in the number of cells in the gland throughout lactation, an increase in the activity of cells throughout the lactation, or both. Shorten et al. (2002) proposed a hypothetical model by which frequent milking for the entire lactation increases the number of active alveoli by reducing the rates of quiescence and senescence in the mammary gland. If such an event occurs with frequent milking during early lactation, this could permanently increase the number of actively secreting alveoli and enhance milk production potential for the remainder of lactation. Many of the biopsy studies that have been previously conducted could have captured changes in mammary cell activity, but would not have captured changes in total cell number or in rates of quiescence and senescence within the gland.

**Figure 4.** Unilateral four-times daily milking for days 1 to 21 of lactation is associated with coordinated changes in mammary expression of 64 genes, and this is negatively correlated with differential milk yield. Solid vertical line represents cessation of unilateral frequent milking.

## **6. Conclusions**

250 Milk Production – An Up-to-Date Overview of Animal Nutrition, Management and Health

unanswered, but work by Stelwagen and Knight (1997) has provided some clues. Using a half-udder model, they compared 1X to 2X of cows in early or late lactation and reported a more dramatic increase in milk secretion efficiency in response to 2X during early lactation compared to late lactation (Stelwagen & Knight, 1997). In agreement, Walsh (1974) observed different effects of suckling during early vs. late lactation on mammary health. During early lactation, suckling of a calf was associated with a 27% decrease in clinical mastitis, whereas suckling during late lactation had no effect (Walsh, 1974). Taken together, the observations of Walsh (1974) and Stelwagen and Knight (1997) indicate that there are distinct differences in the cell population during early vs. late lactation. It is possible that during early lactation, there are more secretory cells present in the mammary gland, and these cells may have more potential to respond to stimulus than cells present in late lactation. Frequent milking may prevent otherwise unused cells from undergoing apoptosis, or may provide the stimulus to push the cells to reach higher levels of differentiation and secretory capacity. These scenarios could result in an increase in the number of cells in the gland throughout lactation, an increase in the activity of cells throughout the lactation, or both. Shorten et al. (2002) proposed a hypothetical model by which frequent milking for the entire lactation increases the number of active alveoli by reducing the rates of quiescence and senescence in the mammary gland. If such an event occurs with frequent milking during early lactation, this could permanently increase the number of actively secreting alveoli and enhance milk production potential for the remainder of lactation. Many of the biopsy studies that have been previously conducted could have captured changes in mammary cell activity, but would not have captured changes in total cell number or in rates of quiescence and senescence within the gland.

**Figure 4.** Unilateral four-times daily milking for days 1 to 21 of lactation is associated with coordinated changes in mammary expression of 64 genes, and this is negatively correlated with differential milk

yield. Solid vertical line represents cessation of unilateral frequent milking.

Research in the area of frequent milking of dairy cows has established a robust milk yield response to increased milking frequency or suckling, and has identified a window of time during early lactation wherein the mammary gland is especially responsive to the stimulus of frequent milk removal. In addition, there is now evidence that this response is regulated within the mammary gland. Consequently, the concept of 'use it or lose it' is becoming more clearly established, that is, the stimulus of frequent milking or suckling during early lactation permanently increases the milk production capacity of the mammary gland. Exciting research opportunities now present themselves, and ongoing experiments seek to identify the local factor(s) that are involved in the regulation of milk production efficiency of dairy cows. The opportunity now exists for dairy scientists to identify the mechanisms involved in local regulation of milk production potential, and for dairy producers to further refine milking management practices to maximize milk production efficiency of their operations.
