Published December 5, 2014

Induction of milk ejection and milk removal in different production systems1 R. M. Bruckmaier2 and O. Wellnitz Veterinary Physiology, Vetsuisse Faculty, University of Bern, Switzerland

ABSTRACT: Milk ejection is important during milking or suckling to obtain the alveolar milk fraction, which can represent more than 80% of the milk stored in the udder of dairy cows. In response to tactile teat stimulation, either manually or by the milking machine, milk ejection is induced by the release of oxytocin and resultant myoepithelial contraction. The time from the start of tactile stimulation until the occurrence of milk ejection spans 40 s to >2 min and increases with a decreasing degree of udder filling. Therefore, cows need a longer prestimulation in the late stages of lactation or if the milking is performed shortly after the previous milking, whereas in full udders prestimulation is less important. Milk ejection is disturbed under several conditions, such as during milking in unfamiliar surroundings (i.e., a novel milking environment) or for several weeks immediately after parturition in primiparous cows. Disturbed milk ejection is due to a reduction of or absence of oxytocin release from the pituitary. The severity of disturbed milk ejection and the coping capacity toward a novel milking environment is related

to cortisol release in response to ACTH (i.e., adrenal cortex activity). Therefore, susceptibility of individual cows to the inhibition of oxytocin release and milk ejection can be predicted by an ACTH challenge test. Comfortable surroundings, such as feeding in and lighting of the milking parlor, can increase the secretion of oxytocin. Overcoming the lack of oxytocin release by injection of exogenous oxytocin for an extended time results in a reduction of the mammary response to endogenous oxytocin. In different production systems, it has to be verified that udder stimulation is sufficient to prevent disturbed milk ejection. Different brands of automatic milking systems induce a sufficient prestimulation of the udder, even if a few minutes are needed for a successful onset of the teat clusters. Specific breeds used for less intense milk production may need the presence of their calves for sufficient oxytocin release during milking. In conclusion, in all milk production systems, the maximal possible reduction of stress has to be targeted and proper udder prestimulation must be performed for an optimal milking of the cow by the farmer.

Key words: milk removal, milk ejection, oxytocin, mammary gland, dairy cow ©2008 American Society of Animal Science. All rights reserved.

INTRODUCTION

J. Anim. Sci. 2008. 86(Suppl. 1):15–20 doi:10.2527/jas.2007-0335

is only available if actively ejected. The ejection is induced by the neuropeptide oxytocin, which is released from the posterior pituitary in response to tactile teat stimulation by the calf, hand, or milking machine. During situations in which oxytocin release is totally lacking or reduced, milk ejection is inhibited, thus resulting in production loss. During milking, an empty cistern can lead to a disturbance of further milk removal. Therefore, the occurrence of milk ejection before the cistern is empty is important, and because of the limited cisternal size, adequate udder preparation in cows is more important than in small ruminants. Concomitantly, complete removal of the alveolar milk at each suckling or milking, which is only achieved with complete milk ejection, is a prerequisite to maintain a high level of milk synthesis and secretion throughout an ongoing lactation. Finally, milk that remains in the udder increases the risk for mammary infection, because residual milk is an exqui-

The milk in the udder of a dairy cow is mostly (80 to 100%) stored in the alveolar compartment and only up to 20% is stored in the cistern. In contrast, in small ruminants, such as sheep and goats, the cisternal fraction amounts to more than 50% (Bruckmaier and Blum, 1992), whereas water buffaloes have small cisterns that hold only approximately 5% of the total milk (Thomas et al., 2004). The cisternal milk fraction is available for machine milking or to the suckling calf before the occurrence of milk ejection. The alveolar milk, however,

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Presented at the Eighth International Workshop on the Biology of Lactation in Farm Animals held in Pirassununga, Brazil, August 21–23, 2006. 2 Corresponding author: [email protected] Received June 8, 2007. Accepted August 6, 2007.

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site substrate for microorganisms in the mammary gland.

MILK STORAGE AND MILK EJECTION Between milkings, the milk secreted by the epithelial cells accumulates in the alveoli and cisterns. The milk in the alveoli and small milk ducts (i.e., the alveolar fraction) is fixed by capillary forces and requires an active expulsion into the cistern, an event that is termed milk ejection or milk letdown. The cisternal fraction usually amounts to less than 20% of the stored milk after an interval of 12 h from the previous milking in dairy cows (Pfeilsticker et al., 1996). Shortly after milking, almost no cisternal milk is present (Knight et al., 1994; Bruckmaier and Hilger, 2001; Ayadi et al., 2003). Only several hours after the previous milk removal event is milk increasingly transferred into the cisternal compartment (Knight et al., 1994). Cisternal milk yield and fraction are highest at peak lactation and decrease toward the end of lactation (Pfeilsticker et al., 1996; Bruckmaier and Hilger, 2001). Thus, the least amount of milk available for milk removal before milk ejection is present after short intervals from the previous milking and in the late stages of lactation. Bruckmaier et al. (1994a) demonstrated a close positive correlation between cisternal size and lactation number (r = 0.90) and measured the largest cisternal fractions in the oldest cows. Milk ejection is essential to have the alveolar fraction available for milk removal.

Milk Ejection Milk ejection is the active transport of alveolar milk into the cisternal compartment. It consists of contraction of the myoepithelial cells that surround the mammary alveoli like a basket and transfer of the milk through the milk duct system. Milk ejection is an innate reflex that occurs in response to tactile stimulation of the mammary gland through a neuroendocrine reflex arc (Crowley and Armstrong, 1992). In response to elevated oxytocin concentrations in the blood, binding of oxytocin to oxytocin receptors on the myoepithelial cells causes alveolar contraction (Soloff et al., 1980). As a consequence, alveolar milk is forcefully shifted into the cisternal space. Alveolar milk ejection causes a rapid increase of pressure within the cistern (Bruckmaier and Blum, 1996) and an enlargement of the cisternal cavity size (Bruckmaier and Blum, 1992). If oxytocin is injected, the cistern is maximally distended within 3 min after i.v. injection of 5 IU of oxytocin, regardless of the quantity of milk stored in the udder (Caja et al., 2004). However, because of the limited cisternal space, not all alveolar milk can be ejected if milk is not removed simultaneously from the udder. Therefore, further milk is ejected during the course of suckling or milking (Bruckmaier et al., 1994b, 1997).

Stimulation Milk ejection occurs if the tactile udder stimulation is sufficient to elevate the blood oxytocin concentration above a threshold (Schams et al., 1984; Bruckmaier et al., 1994b). Even the stimulus of having the teat cup liner attached without pulsation is usually sufficient to evoke oxytocin release (Weiss et al., 2003). Hand milking induces a more pronounced release of oxytocin than machine milking (Gorewit et al., 1992). A stronger stimulus for oxytocin release is the suckling of a calf (Akers and Lefcourt, 1982; Lupoli et al., 2001). The strongest stimulus for oxytocin release is vaginal stimulation by air blown into the vagina (Schams et al., 1982; Bruckmaier and Blum, 1992). However, the intensity of stimulation affects oxytocin release but not the degree of milk ejection (Schams et al., 1982; Bruckmaier and Blum, 1996; Weiss et al., 2003). Therefore, the intensity of stimulation is much less important than the duration of the stimulus. Prestimulation before milk is withdrawn promotes early induction of milk ejection to avoid an interruption of milk flow during early milking. The alveolar milk is then already available before the cisternal milk is removed. Technical solutions from the milking machine industry provide different remedies either to avoid milk from being removed during a prestimulation phase or to reduce vacuum and the b-phase of pulsation (i.e., milk extraction) during the period of early milking before milk ejection. During central inhibition of milk ejection caused by emotional stress, the effect of stimulation is decreased. Thus, a stimulus with less strength (e.g., a teat cup liner attached without pulsation) may no longer be sufficient to release enough oxytocin to initiate milk ejection. However, a stimulus with more strength (e.g., vaginal stimulation) can cause sufficient release of oxytocin to induce milk ejection (Bruckmaier and Blum, 1992). Because of the distribution of milk within the cow’s udder, milking without prestimulation can cause a transient reduction or even total interruption of milk flow after removal of the cisternal fraction. This occurs if the cisternal milk is removed before milk ejection has started. Because of the large cisternal milk volume in small ruminants, the prestimulation is of less importance in these species. In contrast, prestimulation is specifically important in water buffaloes because of the very small cisternal size. If the cistern is empty before milk ejection occurs, milking is consequently performed on empty teats, which can cause a penetration of the milking vacuum into the cistern, collapsing of the cavities, and climbing of the cluster. This effect can cause reduced milkability during the remainder of milking, even after delayed milk ejection has finally occurred (Bruckmaier and Blum, 1996). In addition, optimization of milk flow positively influences the distribution of milk constituents in milk fractions during milking (Tancˇin et al., 2007). Johansson et al. (1952) showed that milk fat content continuously increases in the milk

Induction of milk ejection and milk removal

fractions during the process of milking, and this was confirmed by Ontsouka et al. (2003). Furthermore, the somatic cell count differs in the different milk fractions and is high in the cisternal fraction, lowest in the first alveolar fraction, and increases again during removal of the alveolar milk toward the end of milking (Sarikaya and Bruckmaier, 2006). This shows that a disturbance in oxytocin release and milk ejection, and a lack of removal of all milk fractions can alter the overall milk composition. The lag time from start of tactile teat stimulation until onset of milk ejection normally ranges from 40 s to more than 2 min and depends on the degree of udder filling (Bruckmaier et al., 1994b; Bruckmaier and Hilger, 2001). The degree of udder filling is low during the later stages of lactation and during short intervals from previous milk removal. During extremely low udder filling, milk ejection may occur only 3 min after the start of tactile teat stimulation. In one study, mean lag time from the start of stimulation until milk ejection commenced was 50 s in early lactation after a 12-h milking interval and 90 s in late lactation after a 4-h milking interval (Bruckmaier and Hilger, 2001). A field study in Italy by Sandrucci et al. (2007) also showed that proper udder preparation, including forestripping and predipping, resulted in a greater milk yield per milking, a shorter milking time, and less bimodality. Specifically, if the delay between the start of teat stimulation and attachment of the clusters did not exceed 60 s, then the milking routine was considered optimal. During the course of lactation, release of oxytocin at the start of milking is not reduced or delayed (Mayer et al., 1991). The delayed milk ejection during low degrees of udder filling is obviously due to a delayed response to the oxytocin at the level of the mammary gland. Much greater myoepithelial contraction is needed to eject milk out of an incompletely filled alveolus than out of a completely filled one. Greater contraction of the myoepithelial cells requires more time, and the period until alveolar milk appears in the cistern is extended. A long lag time until the onset of milk ejection results in very low amounts of cisternal milk at low degrees of udder filling, which increases the risk of milking of empty teats (Bruckmaier and Hilger, 2001). Interestingly, the lag time from the start of teat stimulation until milk ejection occurs is independent of the intensity of the stimulus. At a minimum stimulation, such as keeping a teat cup liner attached to the teat without pulsation, sufficient oxytocin is released to induce immediate milk ejection (Weiss et al., 2003). In particular, at low udder filling, an adequate prestimulation (i.e., a stimulation of the teats to induce milk ejection without simultaneously removing milk at the full vacuum level) helps to avoid the negative effects of milking on empty teats, such as tissue damage and climbing of the clusters, at the start of milking that influence the further course of milk removal (Weiss and Bruckmaier, 2005).

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Disturbance of Milk Ejection Under various conditions, milk ejection may be disturbed in dairy cows either at the site of oxytocin release from the posterior pituitary (central inhibition) or at the site of oxytocin action in the mammary gland (peripheral inhibition). Peripheral inhibition of milk ejection in cows can be experimentally induced by administration of α-adrenergic receptor agonists or an oxytocin receptor blocking agent (Bruckmaier et al., 1997). However, peripheral inhibition of milk ejection is limited to experimental approaches and occurs in the presence of normal oxytocin release (Bruckmaier et al., 1997; Inderwies et al., 2003). Central inhibition of milk ejection (i.e., inhibition of oxytocin release from the pituitary) occurs frequently in dairy practice during various types of emotional stress. For example, milking in unfamiliar surroundings has been demonstrated to result in an inhibition of milk ejection, which could be reversed by administering small dosages of exogenous oxytocin (Bruckmaier et al., 1993). The same effect occurs in primiparous cows milked for the very first time (Bruckmaier et al., 1992), as well as for cows being switched from suckling to machine milking (Tancˇin et al., 1995). When cows are repeatedly transferred to unfamiliar surroundings, oxytocin release reaches its normal level when the animals gradually acclimate to the procedure (Bruckmaier et al., 1996). Because milk ejection is a continuous process throughout suckling or milking (Bruckmaier et al., 1994b, 1997), an emotionally stress-free environment for the cow is crucial to achieve continuous oxytocin release throughout milk removal and, hence, complete emptying of milk from the udder. The absence of oxytocin release in unfamiliar surroundings is accompanied by elevated plasma levels of β-endorphin and cortisol (Bruckmaier et al., 1993, 1997). When cows acclimate to the new surroundings, the concentrations of these hormones decrease, whereas oxytocin release gradually returns to normal (Bruckmaier et al., 1996). These observations have led to the concept that endogenous opioid peptides play a role within the mechanisms causing the central inhibition of milk ejection. This hypothesis is supported by experiments in which oxytocin release and milk ejection could be inhibited by the exogenous opioid morphine, and this effect could be abolished by administration of naloxone, an opioid antagonist (Tancˇin et al., 2000a). On the other hand, naloxone application could not prevent the inhibition of oxytocin release in cows milked in unfamiliar surroundings (Wellnitz et al., 1997; Macˇuhova´ et al., 2002) or in primiparous cows shortly after parturition (Kraetzl et al., 2001). Therefore, the role of endogenous opioids in the regulation of milk ejection and the mechanism of inhibition of oxytocin release in cows remains unclear. However, naloxone administration causes an increased release of oxytocin during stress-free milking (Tancˇin et al., 2006). Although this effect was seen only during stress-free milking, it is possible that this increase can abolish a central

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inhibition of milk ejection in specific situations when the threshold of stimulatory effectiveness for oxytocin release is only slightly reduced. Cortisol does not seem to have an influence on the central inhibition of milk ejection, because intravenous administration of cortisol has no effect on milk ejection in cows (Mayer and Lefcourt, 1987). Plasma cortisol concentrations increase physiologically during normal machine milking (Gorewit et al., 1992; Bruckmaier et al., 1993) and hand milking (Gorewit et al., 1992) of cows. Although cortisol release usually occurs in response to a release of ACTH from the pituitary, the milking-related release of cortisol is apparently independent of ACTH release. Tancˇin et al. (2000b) observed that ACTH plasma concentrations did not change during milking, whereas cortisol increased. Release of cortisol from the adrenal cortex in response to ACTH is related to heart rate on the one hand and to the disturbance of milk ejection in unfamiliar surroundings on the other hand. In cows with more alveolar milk ejected during the first milking in unfamiliar surroundings, the adrenal response to ACTH challenge tended to be greater (Macˇuhova´ et al., 2002). Furthermore, the increase in cortisol levels during the first milking in unfamiliar surroundings, as compared with control milkings, tended to be less in cows with total inhibition of milk ejection during all relocations compared with cows that showed a similar positive reaction in oxytocin release in response to milking or after vaginal stimulation (Macˇuhova´ et al., 2002). Thus, animals with a greater adrenal sensitivity to ACTH or stress had less pronounced inhibition of milk ejection as a consequence of the greater oxytocin release (Macˇuhova´ et al., 2002). In addition, catecholamines do not cause central inhibition of milk ejection in cows because exogenously administered catecholamines cause an augmentation rather than a reduction of oxytocin release (Wellnitz et al., 1997; Bruckmaier and Blum, 1998). To examine the relationship between adrenal cortex sensitivity and the coping process during the changeover from conventional milking to an automatic milking system (AMS), an ACTH challenge experiment was performed independently of milking (Weiss et al., 2004). Cows that released more cortisol in response to the ACTH injection had a less enhanced heart rate and less disturbed milk ejection during the first milking in the new environment. Thus, the degree of central inhibition of milk ejection (e.g., the coping capacity toward the new milking environment) varies widely among cows. The course of adaptation to the novel milking environment can be predicted by testing the sensitivity of the adrenal cortex to ACTH. There is, however, no proof of a causal relationship between adrenal cortex sensitivity and the disturbance of milk ejection. In addition to avoiding as much stress as possible, maintaining an atmosphere during milking that is conducive to cow comfort is always necessary to promote sufficient oxytocin release. For example, feeding concentrates during milking enhances milking-related oxy-

tocin release and milk production (Svennersten et al., 1995). The release of oxytocin during milking is also influenced by light. A study with dairy cows showed greater milking-related oxytocin release during milking in a well-illuminated AMS because of the location and size of the window compared with less illumination on a farm with comparable management (Macˇuhova´ and Bruckmaier, 2004). Surely, this has no influence on the degree of milk ejection if oxytocin is released in a normal manner, but it may prevent the inhibition of milk ejection when oxytocin concentrations in blood do not reach threshold levels because of reduced stimulatory efficiency caused by stress factors. To overcome the central inhibition of milk ejection, it is common in dairy practice to inject cows with exogenous oxytocin before milking to alleviate disturbed milk ejection caused by lacking or reduced oxytocin release. However, this can result in reduced spontaneous milk removal if it is chronically applied because of the reduced contractibility of myoepithelial cells in the mammary gland (Macˇuhova´ et al., 2004a). Therefore, oxytocin therapy should be used very carefully.

DIFFERENT PRODUCTION SYSTEMS Conventional Milking Systems Gorewit et al. (1992) reported that hand milking induces a more pronounced oxytocin release than machine milking, although machine milking induces an oxytocin release sufficient to induce milk ejection and complete udder evacuation. Interestingly, the amplitude of oxytocin release during prestimulation and the first few minutes of milking does not differ between hand stimulation and stimulation by the teat cup liner (Bruckmaier and Blum, 1996). It can be assumed that the continuously similar stimulus of the milking machine throughout milking does not exhibit the same effect as the inconsistent stimulation during hand milking. However, because only a slight increase of oxytocin is needed to elicit milk ejection, the stimulus of the milking cluster is usually sufficient to induce complete milk ejection.

Robotic Milking The milking routine in an AMS differs considerably from that in conventional milking. In addition, intervals between milking are variable and may be as short as 6 h. Because of these characteristics of robotic milking, the milking routine must be specifically adapted to the physiological requirements of the cow. In all available AMS, the first action after the cow has entered is teat cleaning by means of automatic devices, which are different among brands of AMS. Teat-cleaning devices are either rotating brushes or devices to rinse the teat with water in a teat cup. Teats are not cleaned simultaneously but sequentially one by one. This teat cleaning has been shown to provide an

Induction of milk ejection and milk removal

excellent stimulatory effect on the teats, thus causing a sufficient release of oxytocin to induce milk ejection (Macˇuhova´ et al., 2003; Dzidic et al., 2004a,b). The sequential stimulation of individual teats does not cause a reduced release of oxytocin. The stimulation of one teat is sufficient to induce complete milk ejection (Bruckmaier et al., 2001). After teat cleaning, the teat cups are attached, and it may take some time (up to 1 min or longer) until the teat cups are attached. Bruckmaier et al. (2001) showed that a total interruption of tactile stimuli after prestimulation for several minutes caused a decrease in oxytocin concentrations during milking before sufficient milk ejection, and caused a reduced milk yield. However, even if the attachment of teat cups is occasionally not successful in an AMS, the action of the robotic devices on the udder maintains the release of oxytocin even if the attempts to attach the teat cups successfully requires up to 7 min, compared with the typical 1 to 2 min observed in non-AMS systems (Macˇuhova´ et al., 2004b). Jago et al. (2006) showed no differences in daily milk yield; instead, the same milk yield was obtained with a shorter duration of the milking procedure if brushing was not performed. The milking procedure seems to be very cow friendly as soon as the animals have adapted to the system. No stress could be detected, as determined by measuring milk cortisol, in AMS compared with milking in auto-tandem milking parlors (Hopster et al., 2002; Gygax et al., 2006).

Milking with Calf Presence Some breeds of cows that are used for less intense milk production but are better adapted to particular climates in specific areas are milked in the presence of their calves for a stimulatory effect on oxytocin release. For example, Kaskous et al. (2006) showed that during milking of Shami cattle in Syria without the presence of their calves, no oxytocin release could be detected, which led to lower milk yields, increased residual milk fractions, and a higher content of protein and lactose in the milk. These effects did not disappear within 3 mo of milking after cows became acclimated to this procedure. This result shows that not all breeds are suitable for exclusive machine milking without the presence of their calves. However, this does not apply for most dairy cows used for more intense milk production, in which the presence of the calf can decrease the secretion of oxytocin release during milking (Akers and Lefcourt, 1984; Tancˇin et al., 2001). In conclusion, in all milk production systems, milk ejection follows the same mechanisms. A disturbance in milk ejection leads to a loss of milk and increases the health risk for the cow. Therefore, in all production systems the greatest possible reduction of stress has to be aimed for and proper udder prestimulation must be performed for optimal milking of the cow by the farmer.

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LITERATURE CITED Akers, R. M., and A. M. Lefcourt. 1982. Milking- and suckling-induced secretion of oxytocin and prolactin in parturient dairy cows. Horm. Behav. 16:87–93. Akers, R. M., and A. M. Lefcourt. 1984. Effect of presence of calf on milking-induced release of prolactin and oxytocin during early lactation of dairy cows. J. Dairy Sci. 67:115–122. Ayadi, M., G. Caja, X. Such, and C. H. Knight. 2003. Use of ultrasonography to estimate cistern size and milk storage at different milking intervals in the udder of dairy cows. J. Dairy Res. 70:1–7. Bruckmaier, R. M., and J. W. Blum. 1992. B-Mode ultrasonography of mammary glands of cows, goats and sheep during alpha- and beta-adrenergic agonist and oxytocin administration. J. Dairy Res. 59:151–159. Bruckmaier, R. M., and J. W. Blum. 1996. Simultaneous recording of oxytocin release, milk ejection and milk flow during milking of dairy cows with and without prestimulation. J. Dairy Res. 63:201–208. Bruckmaier, R. M., and J. W. Blum. 1998. Oxytocin release and milk removal in ruminants. J. Dairy Sci. 81:939–949. Bruckmaier, R. M., and M. Hilger. 2001. Milk ejection in dairy cows at different degrees of udder filling. J. Dairy Res. 68:369–376. Bruckmaier, R. M., J. Macˇuhova´, and H. H. D. Meyer. 2001. Specific aspects of milk ejection in robotic milking: A review. Livest. Prod. Sci. 72:169–176. Bruckmaier, R. M., H. U. Pfeilsticker, and J. W. Blum. 1996. Milk yield, oxytocin and β-endorphin gradually normalize during repeated milking in unfamiliar surroundings. J. Dairy Res. 63:191–200. Bruckmaier, R. M., E. Rothenanger, and J. W. Blum. 1994a. Measurement of mammary gland cistern size and determination of the cisternal milk fraction in dairy cows. Milchwissenschaft 49:543–546. Bruckmaier, R. M., D. Schams, and J. W. Blum. 1992. Aetiology of disturbed milk ejection in parturient primiparous cows. J. Dairy Res. 59:479–498. Bruckmaier, R. M., D. Schams, and J. W. Blum. 1993. Milk removal in familiar and unfamiliar surroundings: Concentrations of oxytocin, prolactin, cortisol and β-endorphin. J. Dairy Res. 60:449–456. Bruckmaier, R. M., D. Schams, and J. W. Blum. 1994b. Continuously elevated concentrations of oxytocin during milking are necessary for complete milk removal in dairy cows. J. Dairy Res. 61:323–334. Bruckmaier, R. M., O. Wellnitz, and J. W. Blum. 1997. Inhibition of milk ejection in cows by oxytocin receptor blockade, α-adrenergic receptor stimulation and in unfamiliar surroundings. J. Dairy Res. 64:15–25. Caja, G., M. Ayadi, and C. H. Knight. 2004. Changes in cisternal compartment based on stage of lactation and time since milk ejection in the udder of dairy cows. J. Dairy Sci. 87:2409–2415. Crowley, W. R., and W. E. Armstrong. 1992. Neurochemical regulation of oxytocin secretion in lactation. Endocr. Rev. 13:33–65. Dzidic, A., J. Macˇuhova´, and R. M. Bruckmaier. 2004a. Effects of cleaning duration and water temperature on oxytocin release and milk removal in an automatic milking system. J. Dairy Sci. 87:4163–4169. Dzidic, A., D. Weiss, and R. M. Bruckmaier. 2004b. Oxytocin release, milk ejection and milking characteristics in a single stall automatic milking system. Livest. Prod. Sci. 86:61–68. Gorewit, R. C., K. Svennersten, W. R. Butler, and K. Uvna¨s-Moberg. 1992. Endocrine responses in cows milked by hand and machine. J. Dairy Sci. 75:443–448. Gygax, L., I. Neuffer, C. Kaufmann, R. Hauser, and B. Wechsler. 2006. Milk cortisol concentration in automatic milking systems compared with auto-tandem milking parlors. J. Dairy Sci. 89:3447–3454. Hopster, H., R. M. Bruckmaier, J. T. Van der Werf, S. M. Korte, J. Macˇuhova´, G. Korte-Bouws, and C. G. van Reenen. 2002. Stress

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