Domestic Animal Endocrinology 47 (2014) 47–54
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Effect of premilking stimulation and milking frequency on milking-induced prolactin release in lactating dairy cows P. Lacasse*, S. Ollier Dairy and Swine Research and Development Centre, Agriculture and Agri-Food Canada, Sherbrooke, Quebec J1M 0C8, Canada
a r t i c l e i n f o
a b s t r a c t
Article history: Received 22 October 2013 Received in revised form 19 November 2013 Accepted 24 November 2013
Four experiments were conducted to investigate the factors controlling prolactin (PRL) release at milking. Each experiment used 9 dairy cows in mid-lactation in a 3 3 Latin square design. Experiment 1 evaluated the effect of premilking stimulation. The milking unit was attached after 0, 20, or 120 s of manual stimulation. Blood samples were collected from 20 min before to 60 min after milking-unit attachment. The peak value and total PRL release (area under the curve) were not affected by the treatments, but the 120-s stimulation hastened PRL release. Stimulation (20 or 120 s) increased the b-endorphin peak value (P ¼ 0.02), but the magnitudes of PRL and b-endorphin releases were not correlated. Experiment 2 evaluated the effect of milking frequency. Cows were milked twice, at 7 AM and 7 PM; 3 times, at 7 AM, 1 PM, and 7 PM; or 7 times, at 7 AM, 9 AM, 11 AM, 1 PM, 3 PM, 5 PM, and 7 PM. The amount of PRL released at the 7 PM milking decreased as the number of milkings increased (P < 0.01), and peak values were smaller with 7 milkings than with 2 and 3 milkings (P < 0.05). Beta-endorphin release was not affected by milking frequency and not correlated with the magnitude of PRL release. Experiment 3 evaluated the effect of manual stimulation between milkings on milking-induced PRL release. Cows received no stimulation; 5 stimulations (5 min each), at 9 AM, 11 AM, 1 PM, 3 PM, and 5 PM; or 1 stimulation at 5 PM. Manual stimulation reduced (P < 0.5) the amount of PRL released and the maximum PRL concentration at the 7 PM milking, but no difference were found between 1 and 5 stimulations. Manual stimulation did not affect the amount of cortisol released but did impair milk ejection. Experiment 4 evaluated the effect of milking frequency on the PRL release induced by manual stimulation. Cows were milked at 7 AM only; at 7 AM, 9 AM, 11 AM, 1 PM, 3 PM, and 5 PM; or at 7 AM and 5 PM. All cows then received manual stimulation at 7 PM. Milking every 2 h or once 2 h before manual stimulation reduced the amount of PRL released and the maximum PRL concentration but did not affect cortisol release. In conclusion, the length of premilking stimulation has no significant effect on milking-induced PRL release, but increasing milking frequency reduces the amount of PRL released at milking. This effect is due not to the number of milkings or the amount of milk harvested during the milking but to the interval since the preceding milking. Crown Copyright Ó 2014 Published by Elsevier Inc. All rights reserved.
Keywords: Lactation Milking frequency b-Endorphin Cortisol
1. Introduction
* Corresponding author. Dairy and Swine Research and Development Centre, Agriculture and Agri-Food Canada, 2000 College Street, Sherbrooke, Quebec J1M 0C8, Canada. Tel.: þ1 819 780 7236; fax: þ1 819 564 5507. E-mail address: [email protected] (P. Lacasse).
Biological mechanisms exist to adjust milk output to demand. Some evidence, including photoperiod, bovine somatotropin administration, gestation status, and stage of lactation, suggests that milk production is regulated primarily systemically, whereas other evidence, such as
0739-7240/$ – see front matter Crown Copyright Ó 2014 Published by Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.domaniend.2013.11.007
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milking frequency and milk stasis, suggests that milk production is also regulated locally at the level of the mammary gland. Milking and suckling induce an important release of hormones in the female circulation and, therefore, provide a link between local and systemic regulation of milk production. Prolactin (PRL), as its name implies, is the most important hormone for the control of lactation. In most mammals, suppression of PRL with bromocriptine strongly inhibits lactation [1,2]. Although the role of PRL in the control of ruminant lactation has been controversial, studies that involve the present authors found that administration of a specific and potent PRL release inhibitor, quinagolide, decreased milk production in dairy cows [3,4] and that PRL injection attenuated the inhibitor effect [5]. Even though the basal level of PRL has not been clearly associated with the level of milk production, milkinginduced PRL release has been correlated with the level of milk production in both PRL-inhibited and control cows. In addition, the effect of PRL inhibition on milk production was found to be modulated by milking frequency [3]. Therefore, factors that affect PRL concentration, especially milking-induced PRL release, are likely to play an important role in the control of milk production. Milking- and suckling-induced PRL release is a poorly understood neuroendocrine reflex. The b-endorphin antagonist naloxone [6] and dopamine agonists suppress milking-induced PRL release [3,7], suggesting that b-endorphin and dopamine are important players for the control of this reflex. This reflex is not related to the milk harvest itself, because PRL release can be induced in nonlactating animals by nipple stimulation [8]. Therefore, the objective of the experiments in the present study was to determine the effects of premilking stimulation and milking frequency on milking-induced PRL release. 2. Materials and methods All the experiments were conducted at Agriculture and Agri-Food Canada’s Dairy and Swine Research and Development Centre (Sherbrooke, QC, Canada) in accordance with the guidelines of the Canadian Council on Animal Care. Cows were kept in a tie-stall barn and were normally milked twice a day with a 12-h milking interval. 2.1. Experimental design 2.1.1. Effect of premilking stimulation on milking-induced prolactin release Experiment 1 used 9 cows in mid-lactation (171 6 DIM) in a 3 3 Latin square design in which the milking unit was attached a) without prior manual stimulation of the mammary gland, b) after 20 s of manual stimulation, or c) after 120 s of manual stimulation. Cows were milked at approximately 8 AM at the stall under the close supervision of a milkman and followed by teat dipping. On each experimental day, blood samples were collected before, during, and after the morning milking (20, 10, 2, 0, 3, 5, 7, 10, 15, 20, 25, 30, 40, and 60 min relative to the start of milking) in Vacutainer tubes without additives (BD, Mississauga, ON, Canada) from a Silastic catheter (i.d. 1.02 mm,
o.d. 2.16 mm; Dow Corning Corp, Midland, MI, USA) inserted into the jugular vein. In addition, blood samples were collected 9 times (2, 1, 0, 1, 2, 3, 5, 7, and 10 min relative to the start of milking) in EDTA-coated Vacutainer tubes that contained aprotinin. All blood tubes without additives were left for approximately 2 h at room temperature for clotting before centrifugation (1,900 g 4 C, 15 min). Then, the serum was stored at 20 C until determination of PRL concentration. All blood tubes that contained EDTA were placed on ice immediately after collection and centrifuged (1,900 g, 4 C, 15 min). Then, the plasma was stored at 80 C until b-endorphin determination. The 3 sampling days were separated from each other by 1 d of rest. 2.1.2. Effect of milking frequency on milking-induced prolactin release Experiment 2 used 9 cows in mid-lactation (178 6 DIM) in a 3 3 Latin square design in which they were milked on the sampling day at a) 7 AM and 7 PM (2); b) 7 AM, 1 PM, and 7 PM (3); or c) 7 AM, 9 AM, 11 AM, 1 PM, 3 PM, 5 PM, and 7 PM (7). Cows were milked at the stall under the close supervision of a milkman. The mammary gland was washed for about 25 sec, and the milking units were attached immediately after. Blood samples were collected at the 7 PM milking as described for Exp. 1, with 2 exceptions: the samples at 2 and 1 min were omitted, and a sampled was collected in an EDTA-coated tube at 10 min. The 3 sampling days were separated from each other by 1 d of rest. 2.1.3. Effect of manual stimulation of the mammary gland between milkings on milking-induced prolactin release Experiment 3 used 9 cows in mid-lactation (170 5 DIM) in a 3 3 Latin square design in which all the cows were milked 2 times, 12 h apart (7 AM and 7 PM). The treatments were a) no manual stimulation between milkings; b) 5 manual stimulations at 2-h intervals, starting 2 h after the morning milking (at 9 AM, 11 AM, 1 PM, 3 PM, and 5 PM); and c) 1 manual stimulation at 5 PM. The milkings were performed as described in experiment 2, and the manual stimulations were performed by rubbing the udder and teats vigorously with bare hands for 5 min. At the 7 PM milking, blood samples were collected in uncoated Vacutainer tubes at 20, 10, 0, 3, 5, 7, 10, 15, 20, 25, 30, 40, and 60 min relative to the start of milking. Serum samples were prepared as described for Exp. 1 and stored at 20 C until determination of PRL and cortisol concentrations. The 3 sampling days were separated from each other by 1 d of rest. 2.1.4. Effect of milking frequency on prolactin release induced by manual stimulation Experiment 4 used 9 cows in mid-lactation (177 5 DIM) in a 3 3 Latin square design in which all the cows were milked at 7 AM and manually stimulated at 7 PM. The treatments were a) no additional milking; b) 5 additional milkings at 2-h intervals, starting 2 h after the morning milking (at 9 AM, 11 AM, 1 PM, 3 PM, and 5 PM); and c) 1 additional milking at 5 PM. Blood samples were collected at the 7 PM manual stimulation. Milking, manual stimulation,
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and blood sampling were performed as described for Exp. 3. The 3 sampling days were separated from each other by 1 d of rest. 2.2. Analyses The concentration of PRL in serum was measured by RIA as described by Bernier-Dodier et al [9]. Bovine PRL, rabbit antiserum specific for bovine PRL, and goat anti-rabbit gamma globulin were purchased from the National Hormone and Peptide Program (Harbor-UCLA Medical Center, Torrance, CA, USA). The intra-assay and interassay CVs were, respectively, 4.7% and 7.4% for the first 2 experiments and 4.3% and 5.2% for the last 2 experiments. The plasma concentration of b-endorphin was measured by RIA with the use of a commercial kit (Phoenix Pharmaceuticals, Inc, Burlingame, CA, USA) according to the manufacturer’s instructions. Before analysis, peptides were extracted from plasma with the use of a SEP-COLUMN that contained 200 mg of C18 (Phoenix Pharmaceuticals, Inc) as recommended by the manufacturer. The intra-assay and interassay CVs were 5.9% and 11.8%, respectively. The serum concentration of cortisol was determined by ELISA with the use of a commercial kit (R&D Systems, Inc, Minneapolis, MN, USA) according to the manufacturer’s instructions. The intra-assay and interassay CVs were 4.2% and 9.2%, respectively. 2.3. Statistical analysis The amounts of PRL, cortisol, and b-endorphin released into the blood during milking or manual stimulation were calculated by determining the areas under the curves (AUCs). Basal and peak concentrations of these hormones were also calculated. The data were analyzed as triple Latin squares by ANOVA by using the MIXED procedure of the SAS software program (SAS Institute Inc, Cary, NC, USA). Cows were assigned randomly to each Latin square, and sampling days and cows were considered as blocks. The model includes the effects of treatments, sampling days, and cows. Preplanned orthogonal contrasts were used to compare treatments or, for Exp. 2, the linear and quadratic effects of the number of milkings. Other treatment comparisons were performed with the Tukey–Kramer adjustment. The relationships between PRL and b-endorphin secretions and between PRL and cortisol secretions were evaluated with the CORR procedure of SAS (SAS Institute Inc). 3. Results Manual stimulation of the mammary gland (Exp. 1) did not affect the amount of PRL released or the peak PRL concentration, with average PRL amounts of 1,578 110, 1,711 110, and 1,740 110 ng·min/mL and average peak concentrations of 89.3 12.8, 80.0 12.8, and 104.8 12.8 ng/mL for manual stimulation of 0, 20, and 120 s, respectively. Nevertheless, the 120-s stimulation hastened the release of PRL, and the time to peak value was shorter (P ¼ 0.02; Fig. 1A) than for the other treatments. Mammary gland stimulation before milking (20 or 120 s) increased
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the b-endorphin peak value (P ¼ 0.02) and tended to increase the AUC (P ¼ 0.08) in comparison with no stimulation (Fig. 1B). Again, manual stimulation hastened the release of b-endorphin (P < 0.01); for some animals, the peak value occurred before milking-unit attachment. However, the magnitudes of PRL and b-endorphin releases were not correlated (r ¼ 0.11, P ¼ 0.6). The effect of milking frequency on milking-induced PRL and b-endorphin releases (Exp. 2) is presented in Figure 2. Prolactin release decreased as the number of milkings increased (P ¼ 0.01), with averages of 1,650 106, 1,430 106, and 1,183 106 ng·min/mL for the cows in the 2, 3, and 7 treatments, respectively. Peak values were smaller in 7 than in 2 (P ¼ 0.04) and 3 (P ¼ 0.04). No significant effect of milking frequency was found on b-endorphin AUC (P ¼ 0.19), and a trend was observed for peak values to increase with the number of milkings (P ¼ 0.08). The amounts of PRL and b-endorphin released were not correlated (r ¼ 0.09; P ¼ 0.7). As expected, milk production at the 7 PM milking declined with the number of milkings, with averages of 14.7 0.6, 6.9 0.6, and 2.1 0.6 kg for 2, 3, and 7, respectively. Despite large differences in milk production, the duration of the 7 PM milking was only marginally shorter in 3 than in 2 (P ¼ 0.03), with averages of 6.03 0.24, 5.03 0.26, and 5.34 0.26 min for 2, 3, and 7, respectively. No effect of treatments was found on total milk production for the day (data not shown). The effect on milking-induced PRL and cortisol releases of manual stimulation of the mammary gland for 5 min every 2 h between the morning and evening milkings or once 2 h before the evening milking (Exp. 3) is presented in Figure 3. Manual stimulation reduced the amount of PRL released and the maximum PRL concentration at the evening milking (Table 1). For both variables, no difference was found between 1 and 5 stimulations. Conversely, manual stimulation did not affect the amount of cortisol released or the maximum cortisol concentration (Table 1). The amounts of PRL and cortisol released were not correlated (r ¼ 0.06; P ¼ 0.7). The duration of the evening milking was not affected by the treatments, but the quantity of milk harvested was lower (P ¼ 0.01; Table 1) when the cows had been manually stimulated during the day and lower with 5 stimulations than with 1 stimulation (P ¼ 0.01). However, the amount of milk harvested at the next morning’s milking was greater in the cows that had been stimulated the day before (Table 1), with the result that 24-h milk production did not differ (P > 0.5) among the 3 treatments. The effect of milking frequency on the PRL and cortisol releases induced by manual stimulation (Exp. 4) is presented in Figure 4. Milking every 2 h or once 2 h before manual stimulation reduced the amount of PRL released and the maximum PRL concentration but did not affect cortisol release (Table 2). Again, the amounts of PRL and cortisol released were not correlated (r ¼ 0.05; P ¼ 0.8). 4. Discussion Manual stimulation of the mammary gland before milking hastened PRL release into the blood but did not change the amount of PRL during and after milking. Similarly,
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Fig. 1. Effect of premilking manual stimulation of the mammary gland on milking-induced releases of prolactin (A) and b-endorphin (B) in the blood of dairy cows (Exp. 1). The milking unit was attached after manual stimulation of 0 s (A), 20 s (-), or 120 s (:). Data are presented as unadjusted means SEM. 0 min ¼ start of milking.
Bruckmaier and Blum [10] reported that manual prestimulation of the mammary gland induced an earlier increase in oxytocin concentration but did not affect the overall amount of oxytocin released. Premilking stimulation, although important for inducing a rapid milk ejection, is not an efficient way to increase milking-induced PRL release. The results of Exp. 2 show that the magnitude of the milking-induced PRL release decreases when the number of milkings in a day increases. Karg and Schams [11] observed a reduction in PRL release when several milkings and sucklings were performed within a few hours. However, this effect could be related to the degree of udder filling at the time of milking, which may influence milking time, responsiveness to stimulation, or both. In Exp. 3, no milk was harvested after the morning milking in any of the treatments, with the result that the degree of udder filling was similar for each one. In Exp. 4, the degree of udder filling was different, but the stimulation intensity was standardized by replacing the evening milking by a 5-min manual stimulation. In both experiments, inhibition of PRL release was maintained, indicating that the degree of udder filling, the amount of milk harvested, and the length
of milking were not the main factors that caused the inhibition. In addition, one 5-min manual stimulation 2 h before milking had the same effect on PRL release as 5 manual stimulations (Exp. 3), as did 1 milking 2 h before stimulation in comparison with 5 milkings (Exp. 4). These results indicate that the interval since the preceding milking is more important than the number of milkings. Overall, these experiments show that short intervals between milkings cause a depression of the PRL response to milking. The main physiological control of PRL secretion is exerted by the inhibitory action of dopamine on the lactotrophs of the anterior pituitary [12]. Dopamine is secreted in the hypothalamus through the tuberoinfundibular dopamine (TIDA) pathway and reaches the pituitary through a portal vascular system. It has been proposed that suckling reduces the activity of the TIDA neurons, enabling the release of PRL into the blood stream [13]. The mechanism or mechanisms responsible for inhibition of milking-induced PRL release with short milking intervals are unknown. Intuitively, it can be postulated that refractoriness occurs at the level of the mammary gland, the pituitary gland, or the hypothalamus.
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Fig. 2. Effect of milking frequency on milking-induced releases of prolactin (A) and b-endorphin (B) in the blood of dairy cows at the evening milking (7 PM; Exp. 2). On the sampling day, the cows were milked at 7 AM and 7 PM (2; A); at 7 AM, 1 PM, and 7 PM (3; -); or at 7 AM, 9 AM, 11 AM, 1 PM, 3 PM, 5 PM, and 7 PM (7; :). Data are presented as unadjusted means SEM. 0 min ¼ start of milking.
The establishment of mammary gland refractoriness to stimulation is supported by the gradual loss of PRL response during long milking or manual stimulation [14]. In addition, the oxytocin response decreases when milking frequency increases [15]. Nevertheless, the cortisol and b-endorphin responses were not affected in the present study, suggesting no loss of sensitivity of the mammary gland skin to tactile stimulation. A reduction in PRL response is also observed in situations in which no repeated tactile stimulation occurs. A previous study found that the PRL response to thyrotropin-releasing hormone was reduced with multiple injections [11]. Moreover, the presence of the calf with the cow reduces the milkinginduced PRL release even if suckling is not allowed [16]. Therefore, the establishment of mammary gland refractoriness to stimulation cannot be ruled out but does not appear to be the main causal factor of the inhibition of PRL release with short milking intervals. At the level of the pituitary gland, a depletion of PRL reserves and a reduction in sensitivity to PRL-releasing signals can be envisioned. However, the quantity of PRL released after injection of thyrotropin-releasing hormone [11] or the dopamine antagonist fluphenazine [7] was
observed to be far greater than that induced by milking, indicating that PRL reserves are not a limiting factor. In addition, a 5-min manual stimulation did not affect the PRL response to the dopamine antagonist domperidone 60 min later (P. Lacasse, X. Zhao, and S. Ollier, 2013). Thus, it seems unlikely that inhibition of milking-induced PRL release with short milking intervals is due to a reduction in pituitary gland responsiveness. Prolactin secretion is normally tightly regulated by a short-loop negative-feedback mechanism, whereby PRL stimulates the secretion by TIDA neurons of dopamine in the pituitary portal blood, which inhibits PRL secretion. It is therefore tempting to attribute the decreased PRL response to milking to a direct feedback of PRL on TIDA neurons. In rodents, however, the TIDA neurons were found to become unresponsive to PRL in late gestation and during lactation, disrupting the short-loop feedback mechanism [17]. Accordingly, Tucker et al [18] did not observe any inhibition of milking-induced PRL release in cows after infusion of PRL at 1 or 3 mg/h for 2.5 h. Nevertheless, inhibition was observed with administration of supraphysiological doses of PRL [18,19]. As observed in Exps. 3 and 4, milking also induces glucocorticoid release. In rodents, dexamethasone
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Fig. 3. Effect of manual stimulation of the mammary gland between milkings on milking-induced releases of prolactin (A) and cortisol (B) in the blood of dairy cows at the evening milking (7 PM; Exp. 3). The cows were milked at 7 AM and 7 PM with no manual stimulation (A), 5 manual stimulations (at 9 AM, 11 AM, 1 PM, 3 PM, and 5 PM; :), or 1 manual stimulation (at 5 PM; -) between milkings. Data are presented as unadjusted means SEM. 0 min ¼ start of milking.
but the hypothalamus. Similar information is not available in bovines, but it is possible that the cortisol release at milking transiently decreases TIDA responsiveness to inhibitory inputs, reducing the PRL response to a subsequent milking stimulus.
pretreatment inhibits suckling-induced PRL release [20,21]. Given that dexamethasone is able to block PRL release when implanted in the hypothalamus but does not affect domperidone-induced PRL release [20], it appears that the site of action of glucocorticoids is not the pituitary gland
Table 1 Effect of manual stimulation of the mammary gland between milkings on milking-induced PRL and cortisol releases and on milk production at the evening milking (7 PM) and the next morning’s milking (7 AM; Exp. 3). Treatment
PRL AUC, ng·min/mL PRL peak, ng/mL Cortisol AUC, ng·min/mL Cortisol peak, ng/mL Milk, kg (7 PM milking) Milking, min (7 PM milking) Milk, kg (7 AM milking)
SEM
0S
5S
1S
1,726 91.0 177.3 8.1 17.7 6.4 18.2
1,179 56.2 179.3 8.8 13.8 6.1 20.6
1,263 67.8 200.6 8.3 16.3 6.3 20.1
141 12.8 25.8 1.1 1.2 0.4 1.4
P value T
0S vs 1S þ 5S
1S vs 5S
0.03 0.08 0.38 0.72 0.01 0.77 0.05
0.01 0.04 0.44 0.60 0.01 0.56 0.02
0.70 0.40 0.18 0.55 0.01 0.57 0.55
Abbreviations: AUC, area under the curve; PRL, prolactin. The cows were milked at 7 AM and 7 PM with no manual stimulation (0S), 5 manual stimulations (at 9 AM, 11 AM, 1 PM, 3PM, and 5 PM; 5S), or 1 manual stimulation (at 5 PM; 1S) between milkings. Data are presented as least square means.
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Fig. 4. Effect of milking frequency on releases of prolactin (A) and cortisol (B) induced by manual stimulation (Exp. 4). The cows were milked at 7 AM (A); at 7 AM, 9 AM, 11 AM, 1 PM, 3 PM, and 5 PM (:); or at 7 AM and 7 PM (-). All cows were then manually stimulated for 5 min at 7 PM. Data are presented as unadjusted means SEM. 0 min ¼ start of manual stimulation.
The results of Exps. 1 and 2 confirm that milking induces
b-endorphin release in cattle, but the magnitude of that release is not correlated with that of PRL. Although the opiate receptor antagonist naloxone was found to suppress suckling-induced PRL release in rats [6] and goats [22], Tancin et al [23] reported no effects of naloxone or morphine on milking-induced PRL release in cows. Therefore, opioids do not appear to be involved in the release of PRL during milking in cows.
Unexpectedly, the evening milking duration in Exp. 2 was similar for all milking frequencies, despite that different quantities of milk were harvested. Moreover, the quantity of milk harvested at the evening milking in Exp. 3 was reduced by a 5-min manual stimulation 2 h before milking. Because most of the missing milk was harvested at the next milking, it can be concluded that milk ejection was impaired by manual stimulation. Bruckmaier and Hilger [24] reported that milk ejection was delayed with short
Table 2 Effect of milking frequency on the PRL and cortisol releases induced by manual stimulation (Exp. 4). Treatment
PRL AUC, ng·min/mL PRL peak, ng/mL Cortisol AUC, ng·min/mL Cortisol peak, ng/mL
SEM
1
6
2
1,986 146.1 178.5 7.6
1,215 76.2 164.6 7.0
1,402 73.6 162.4 7.1
228 25.5 21.4 0.8
P value T
1 vs 2 þ 6
1 vs 6
0.04 0.01 0.21 0.37
0.01 0.004 0.14 0.17
0.46 0.90 0.80 0.86
Abbreviations: AUC, area under the curve; PRL, prolactin. The cows were milked at 7 AM only (1); at 7 AM, 9 AM, 11 AM, 1 PM, 3 PM, and 5 PM (6); or at 7 AM and 7 PM (2). All cows were then manually stimulated for 5 min at 7 PM. Data are presented as least square means.
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milking intervals and provided some evidence that the delay was related to the lower degree of udder filling. The fact that impairment of milk ejection can occur despite similar degrees of udder filling (as in Exp. 3) does not support this interpretation. Given that frequent milking has been reported to reduce oxytocin release in ewes [15], frequent milking might be the cause of impaired milk ejection with short milking intervals. In conclusion, the length of prestimulation has no significant effect on milking-induced PRL release. Increasing milking frequency reduces the amount of PRL released at milking. This effect is due not to the number of milkings or the amount of milk harvested during the milking but to the interval since the preceding milking. Although milking induces b-endorphin secretion, opioids do not appear to be involved in the release of PRL during milking in cows. Milking-induced cortisol secretion was not affected by milking frequency. Further research is needed to determine how milking interval affects PRL release. Acknowledgments We thank the following people from Agriculture and AgriFood Canada (Sherbrooke, QC, Canada): Lisette St-James, Liette Veilleux, Véronique Roy, Philippe Bernier-Dodier, Caroline Roy, Amélie Piot, Pauline Maisonnasse, Martin Beaumont, and Dominique Poulin for providing technical assistance, and the dairy barn staff for taking care of the cows. We thank Mary Varcoe, from the Translation Bureau, Public Works and Government Services Canada, for her careful editing of this manuscript. We also thank the National Hormone and Peptide Program and A.F. Parlow for providing the bovine prolactin and antibodies. This research was financially supported by Agriculture and Agri-Food Canada.
References [1] Taylor JC, Peaker M. Effects of bromocriptine on milk secretion in the rabbit. J Endocrinol 1975;67:313–4. [2] Flint DJ, Gardner M. Evidence that growth hormone stimulates milk synthesis by direct action on the mammary gland and that prolactin exerts effects on milk secretion by maintenance of mammary deoxyribonucleic acid content and tight junction status. Endocrinology 1994;135:1119–24. [3] Lacasse P, Lollivier V, Bruckmaier RM, Boisclair YR, Wagner GF, Boutinaud M. Effect of the prolactin-release inhibitor quinagolide on lactating dairy cows. J Dairy Sci 2011;94:1302–9.
[4] Ollier S, Zhao X, Lacasse P. Effect of prolactin-release inhibition on milk production and mammary gland involution at drying-off in cows. J Dairy Sci 2013;96:335–43. [5] Lacasse P, Lollivier V, Dessauge F, Bruckmaier RM, Ollier S, Boutinaud M. New developments on the galactopoietic role of prolactin in dairy ruminants. Domest Anim Endocrinol 2012;43: 154–60. [6] Selmanoff M, Gregerson KA. Suckling-induced prolactin release is suppressed by naloxone and simulated by beta-endorphin. Neuroendocrinology 1986;42:255–9. [7] Ahmadzadeh A, Barnes MA, Gwazdauskas FC, Akers RM. Dopamine antagonist alters serum cortisol and prolactin secretion in lactating Holstein cows. J Dairy Sci 2006;89:2051–5. [8] Akers RM, Lefcourt AM. Teat stimulation-induced prolactin release in non-pregnant and pregnant Holstein heifers. J Endocrinol 1983; 96:433–42. [9] Bernier-Dodier P, Girard CL, Talbot BG, Lacasse P. Effect of dry period management on mammary gland function and its endocrine regulation in dairy cows. J Dairy Sci 2011;94:4922–36. [10] Bruckmaier RM, Blum JW. Simultaneous recording of oxytocin release, milk ejection and milk flow during milking of dairy cows with and without prestimulation. J Dairy Res 1996;63:201–8. [11] Karg H, Schams D. Prolactin release in cattle. J Reprod Fertil 1974; 39:463–72. [12] Torre DL, Falorni A. Pharmacological causes of hyperprolactinemia. Ther Clin Risk Manag 2007;3:929–51. [13] Li C, Chen P, Smith MS. Neuropeptide Y and tuberoinfundibular dopamine activities are altered during lactation: role of prolactin. Endocrinology 1999;140:118–23. [14] Tucker HA. Hormonal response to milking. J Anim Sci 1971;32:137– 41. [15] Negrão JA, Marnet PG, Labussière J. Effect of milking frequency on oxytocin release and milk production in dairy ewes. Small Ruminant Res 2001;39:181–7. [16] Akers RM, Lefcourt AM. Effect of presence of calf on milkinginduced release of prolactin and oxytocin during early lactation of dairy cows. J Dairy Sci 1984;67:115–22. [17] Grattan DR, Steyn FJ, Kokay IC, Anderson GM, Bunn SJ. Pregnancyinduced adaptation in the neuroendocrine control of prolactin secretion. J Neuroendocrinol 2008;20:497–507. [18] Tucker HA, Convey EM, Koprowski JA. Milking-induced release of endogenous prolactin in cows infused with exogenous prolactin. Proc Soc Exp Biol Med 1973;142:72–5. [19] Plaut K, Bauman DE, Agergaard N, Akers RM. Effect of exogenous prolactin administration on lactational performance of dairy cows. Domest Anim Endocrinol 1987;4:279–90. [20] Bartha L, Nagy GM, Kiem DOT, Fekete MIK, Makara GB. Inhibition of suckling-induced prolactin release by dexamethasone. Endocrinology 1991;129:635–40. [21] Horváth KM, Bánky Z, Tóth BE, Nagy GM, Halász B. Dual role of glucocorticoids in suckling-induced prolactin secretion. Endocrine 2001;15:287–90. [22] Bruna FA, De Di Nasso EG, Soaje M, Deis RP, Carón RW. Participation of opioid peptides in sucking-induced oxytocin and prolactin secretions in lactating goats. Reprod Domest Anim 2010;45:796–802. [23] Tancin V, Schams D, Kraetzl WD. The effect of morphine and naloxone on the release of prolactin during machine milking in dairy cows. J Dairy Res 2003;70:277–82. [24] Bruckmaier RM, Hilger M. Milk ejection in dairy cows at different degrees of udder filling. J Dairy Res 2001;68:369–76.
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Effect of premilking stimulation and milking frequency on milking-induced prolactin release in lactating dairy cows P. Lacasse*, S. Ollier Dairy and Swine Research and Development Centre, Agriculture and Agri-Food Canada, Sherbrooke, Quebec J1M 0C8, Canada
a r t i c l e i n f o
a b s t r a c t
Article history: Received 22 October 2013 Received in revised form 19 November 2013 Accepted 24 November 2013
Four experiments were conducted to investigate the factors controlling prolactin (PRL) release at milking. Each experiment used 9 dairy cows in mid-lactation in a 3 3 Latin square design. Experiment 1 evaluated the effect of premilking stimulation. The milking unit was attached after 0, 20, or 120 s of manual stimulation. Blood samples were collected from 20 min before to 60 min after milking-unit attachment. The peak value and total PRL release (area under the curve) were not affected by the treatments, but the 120-s stimulation hastened PRL release. Stimulation (20 or 120 s) increased the b-endorphin peak value (P ¼ 0.02), but the magnitudes of PRL and b-endorphin releases were not correlated. Experiment 2 evaluated the effect of milking frequency. Cows were milked twice, at 7 AM and 7 PM; 3 times, at 7 AM, 1 PM, and 7 PM; or 7 times, at 7 AM, 9 AM, 11 AM, 1 PM, 3 PM, 5 PM, and 7 PM. The amount of PRL released at the 7 PM milking decreased as the number of milkings increased (P < 0.01), and peak values were smaller with 7 milkings than with 2 and 3 milkings (P < 0.05). Beta-endorphin release was not affected by milking frequency and not correlated with the magnitude of PRL release. Experiment 3 evaluated the effect of manual stimulation between milkings on milking-induced PRL release. Cows received no stimulation; 5 stimulations (5 min each), at 9 AM, 11 AM, 1 PM, 3 PM, and 5 PM; or 1 stimulation at 5 PM. Manual stimulation reduced (P < 0.5) the amount of PRL released and the maximum PRL concentration at the 7 PM milking, but no difference were found between 1 and 5 stimulations. Manual stimulation did not affect the amount of cortisol released but did impair milk ejection. Experiment 4 evaluated the effect of milking frequency on the PRL release induced by manual stimulation. Cows were milked at 7 AM only; at 7 AM, 9 AM, 11 AM, 1 PM, 3 PM, and 5 PM; or at 7 AM and 5 PM. All cows then received manual stimulation at 7 PM. Milking every 2 h or once 2 h before manual stimulation reduced the amount of PRL released and the maximum PRL concentration but did not affect cortisol release. In conclusion, the length of premilking stimulation has no significant effect on milking-induced PRL release, but increasing milking frequency reduces the amount of PRL released at milking. This effect is due not to the number of milkings or the amount of milk harvested during the milking but to the interval since the preceding milking. Crown Copyright Ó 2014 Published by Elsevier Inc. All rights reserved.
Keywords: Lactation Milking frequency b-Endorphin Cortisol
1. Introduction
* Corresponding author. Dairy and Swine Research and Development Centre, Agriculture and Agri-Food Canada, 2000 College Street, Sherbrooke, Quebec J1M 0C8, Canada. Tel.: þ1 819 780 7236; fax: þ1 819 564 5507. E-mail address: [email protected] (P. Lacasse).
Biological mechanisms exist to adjust milk output to demand. Some evidence, including photoperiod, bovine somatotropin administration, gestation status, and stage of lactation, suggests that milk production is regulated primarily systemically, whereas other evidence, such as
0739-7240/$ – see front matter Crown Copyright Ó 2014 Published by Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.domaniend.2013.11.007
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milking frequency and milk stasis, suggests that milk production is also regulated locally at the level of the mammary gland. Milking and suckling induce an important release of hormones in the female circulation and, therefore, provide a link between local and systemic regulation of milk production. Prolactin (PRL), as its name implies, is the most important hormone for the control of lactation. In most mammals, suppression of PRL with bromocriptine strongly inhibits lactation [1,2]. Although the role of PRL in the control of ruminant lactation has been controversial, studies that involve the present authors found that administration of a specific and potent PRL release inhibitor, quinagolide, decreased milk production in dairy cows [3,4] and that PRL injection attenuated the inhibitor effect [5]. Even though the basal level of PRL has not been clearly associated with the level of milk production, milkinginduced PRL release has been correlated with the level of milk production in both PRL-inhibited and control cows. In addition, the effect of PRL inhibition on milk production was found to be modulated by milking frequency [3]. Therefore, factors that affect PRL concentration, especially milking-induced PRL release, are likely to play an important role in the control of milk production. Milking- and suckling-induced PRL release is a poorly understood neuroendocrine reflex. The b-endorphin antagonist naloxone [6] and dopamine agonists suppress milking-induced PRL release [3,7], suggesting that b-endorphin and dopamine are important players for the control of this reflex. This reflex is not related to the milk harvest itself, because PRL release can be induced in nonlactating animals by nipple stimulation [8]. Therefore, the objective of the experiments in the present study was to determine the effects of premilking stimulation and milking frequency on milking-induced PRL release. 2. Materials and methods All the experiments were conducted at Agriculture and Agri-Food Canada’s Dairy and Swine Research and Development Centre (Sherbrooke, QC, Canada) in accordance with the guidelines of the Canadian Council on Animal Care. Cows were kept in a tie-stall barn and were normally milked twice a day with a 12-h milking interval. 2.1. Experimental design 2.1.1. Effect of premilking stimulation on milking-induced prolactin release Experiment 1 used 9 cows in mid-lactation (171 6 DIM) in a 3 3 Latin square design in which the milking unit was attached a) without prior manual stimulation of the mammary gland, b) after 20 s of manual stimulation, or c) after 120 s of manual stimulation. Cows were milked at approximately 8 AM at the stall under the close supervision of a milkman and followed by teat dipping. On each experimental day, blood samples were collected before, during, and after the morning milking (20, 10, 2, 0, 3, 5, 7, 10, 15, 20, 25, 30, 40, and 60 min relative to the start of milking) in Vacutainer tubes without additives (BD, Mississauga, ON, Canada) from a Silastic catheter (i.d. 1.02 mm,
o.d. 2.16 mm; Dow Corning Corp, Midland, MI, USA) inserted into the jugular vein. In addition, blood samples were collected 9 times (2, 1, 0, 1, 2, 3, 5, 7, and 10 min relative to the start of milking) in EDTA-coated Vacutainer tubes that contained aprotinin. All blood tubes without additives were left for approximately 2 h at room temperature for clotting before centrifugation (1,900 g 4 C, 15 min). Then, the serum was stored at 20 C until determination of PRL concentration. All blood tubes that contained EDTA were placed on ice immediately after collection and centrifuged (1,900 g, 4 C, 15 min). Then, the plasma was stored at 80 C until b-endorphin determination. The 3 sampling days were separated from each other by 1 d of rest. 2.1.2. Effect of milking frequency on milking-induced prolactin release Experiment 2 used 9 cows in mid-lactation (178 6 DIM) in a 3 3 Latin square design in which they were milked on the sampling day at a) 7 AM and 7 PM (2); b) 7 AM, 1 PM, and 7 PM (3); or c) 7 AM, 9 AM, 11 AM, 1 PM, 3 PM, 5 PM, and 7 PM (7). Cows were milked at the stall under the close supervision of a milkman. The mammary gland was washed for about 25 sec, and the milking units were attached immediately after. Blood samples were collected at the 7 PM milking as described for Exp. 1, with 2 exceptions: the samples at 2 and 1 min were omitted, and a sampled was collected in an EDTA-coated tube at 10 min. The 3 sampling days were separated from each other by 1 d of rest. 2.1.3. Effect of manual stimulation of the mammary gland between milkings on milking-induced prolactin release Experiment 3 used 9 cows in mid-lactation (170 5 DIM) in a 3 3 Latin square design in which all the cows were milked 2 times, 12 h apart (7 AM and 7 PM). The treatments were a) no manual stimulation between milkings; b) 5 manual stimulations at 2-h intervals, starting 2 h after the morning milking (at 9 AM, 11 AM, 1 PM, 3 PM, and 5 PM); and c) 1 manual stimulation at 5 PM. The milkings were performed as described in experiment 2, and the manual stimulations were performed by rubbing the udder and teats vigorously with bare hands for 5 min. At the 7 PM milking, blood samples were collected in uncoated Vacutainer tubes at 20, 10, 0, 3, 5, 7, 10, 15, 20, 25, 30, 40, and 60 min relative to the start of milking. Serum samples were prepared as described for Exp. 1 and stored at 20 C until determination of PRL and cortisol concentrations. The 3 sampling days were separated from each other by 1 d of rest. 2.1.4. Effect of milking frequency on prolactin release induced by manual stimulation Experiment 4 used 9 cows in mid-lactation (177 5 DIM) in a 3 3 Latin square design in which all the cows were milked at 7 AM and manually stimulated at 7 PM. The treatments were a) no additional milking; b) 5 additional milkings at 2-h intervals, starting 2 h after the morning milking (at 9 AM, 11 AM, 1 PM, 3 PM, and 5 PM); and c) 1 additional milking at 5 PM. Blood samples were collected at the 7 PM manual stimulation. Milking, manual stimulation,
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and blood sampling were performed as described for Exp. 3. The 3 sampling days were separated from each other by 1 d of rest. 2.2. Analyses The concentration of PRL in serum was measured by RIA as described by Bernier-Dodier et al [9]. Bovine PRL, rabbit antiserum specific for bovine PRL, and goat anti-rabbit gamma globulin were purchased from the National Hormone and Peptide Program (Harbor-UCLA Medical Center, Torrance, CA, USA). The intra-assay and interassay CVs were, respectively, 4.7% and 7.4% for the first 2 experiments and 4.3% and 5.2% for the last 2 experiments. The plasma concentration of b-endorphin was measured by RIA with the use of a commercial kit (Phoenix Pharmaceuticals, Inc, Burlingame, CA, USA) according to the manufacturer’s instructions. Before analysis, peptides were extracted from plasma with the use of a SEP-COLUMN that contained 200 mg of C18 (Phoenix Pharmaceuticals, Inc) as recommended by the manufacturer. The intra-assay and interassay CVs were 5.9% and 11.8%, respectively. The serum concentration of cortisol was determined by ELISA with the use of a commercial kit (R&D Systems, Inc, Minneapolis, MN, USA) according to the manufacturer’s instructions. The intra-assay and interassay CVs were 4.2% and 9.2%, respectively. 2.3. Statistical analysis The amounts of PRL, cortisol, and b-endorphin released into the blood during milking or manual stimulation were calculated by determining the areas under the curves (AUCs). Basal and peak concentrations of these hormones were also calculated. The data were analyzed as triple Latin squares by ANOVA by using the MIXED procedure of the SAS software program (SAS Institute Inc, Cary, NC, USA). Cows were assigned randomly to each Latin square, and sampling days and cows were considered as blocks. The model includes the effects of treatments, sampling days, and cows. Preplanned orthogonal contrasts were used to compare treatments or, for Exp. 2, the linear and quadratic effects of the number of milkings. Other treatment comparisons were performed with the Tukey–Kramer adjustment. The relationships between PRL and b-endorphin secretions and between PRL and cortisol secretions were evaluated with the CORR procedure of SAS (SAS Institute Inc). 3. Results Manual stimulation of the mammary gland (Exp. 1) did not affect the amount of PRL released or the peak PRL concentration, with average PRL amounts of 1,578 110, 1,711 110, and 1,740 110 ng·min/mL and average peak concentrations of 89.3 12.8, 80.0 12.8, and 104.8 12.8 ng/mL for manual stimulation of 0, 20, and 120 s, respectively. Nevertheless, the 120-s stimulation hastened the release of PRL, and the time to peak value was shorter (P ¼ 0.02; Fig. 1A) than for the other treatments. Mammary gland stimulation before milking (20 or 120 s) increased
49
the b-endorphin peak value (P ¼ 0.02) and tended to increase the AUC (P ¼ 0.08) in comparison with no stimulation (Fig. 1B). Again, manual stimulation hastened the release of b-endorphin (P < 0.01); for some animals, the peak value occurred before milking-unit attachment. However, the magnitudes of PRL and b-endorphin releases were not correlated (r ¼ 0.11, P ¼ 0.6). The effect of milking frequency on milking-induced PRL and b-endorphin releases (Exp. 2) is presented in Figure 2. Prolactin release decreased as the number of milkings increased (P ¼ 0.01), with averages of 1,650 106, 1,430 106, and 1,183 106 ng·min/mL for the cows in the 2, 3, and 7 treatments, respectively. Peak values were smaller in 7 than in 2 (P ¼ 0.04) and 3 (P ¼ 0.04). No significant effect of milking frequency was found on b-endorphin AUC (P ¼ 0.19), and a trend was observed for peak values to increase with the number of milkings (P ¼ 0.08). The amounts of PRL and b-endorphin released were not correlated (r ¼ 0.09; P ¼ 0.7). As expected, milk production at the 7 PM milking declined with the number of milkings, with averages of 14.7 0.6, 6.9 0.6, and 2.1 0.6 kg for 2, 3, and 7, respectively. Despite large differences in milk production, the duration of the 7 PM milking was only marginally shorter in 3 than in 2 (P ¼ 0.03), with averages of 6.03 0.24, 5.03 0.26, and 5.34 0.26 min for 2, 3, and 7, respectively. No effect of treatments was found on total milk production for the day (data not shown). The effect on milking-induced PRL and cortisol releases of manual stimulation of the mammary gland for 5 min every 2 h between the morning and evening milkings or once 2 h before the evening milking (Exp. 3) is presented in Figure 3. Manual stimulation reduced the amount of PRL released and the maximum PRL concentration at the evening milking (Table 1). For both variables, no difference was found between 1 and 5 stimulations. Conversely, manual stimulation did not affect the amount of cortisol released or the maximum cortisol concentration (Table 1). The amounts of PRL and cortisol released were not correlated (r ¼ 0.06; P ¼ 0.7). The duration of the evening milking was not affected by the treatments, but the quantity of milk harvested was lower (P ¼ 0.01; Table 1) when the cows had been manually stimulated during the day and lower with 5 stimulations than with 1 stimulation (P ¼ 0.01). However, the amount of milk harvested at the next morning’s milking was greater in the cows that had been stimulated the day before (Table 1), with the result that 24-h milk production did not differ (P > 0.5) among the 3 treatments. The effect of milking frequency on the PRL and cortisol releases induced by manual stimulation (Exp. 4) is presented in Figure 4. Milking every 2 h or once 2 h before manual stimulation reduced the amount of PRL released and the maximum PRL concentration but did not affect cortisol release (Table 2). Again, the amounts of PRL and cortisol released were not correlated (r ¼ 0.05; P ¼ 0.8). 4. Discussion Manual stimulation of the mammary gland before milking hastened PRL release into the blood but did not change the amount of PRL during and after milking. Similarly,
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Fig. 1. Effect of premilking manual stimulation of the mammary gland on milking-induced releases of prolactin (A) and b-endorphin (B) in the blood of dairy cows (Exp. 1). The milking unit was attached after manual stimulation of 0 s (A), 20 s (-), or 120 s (:). Data are presented as unadjusted means SEM. 0 min ¼ start of milking.
Bruckmaier and Blum [10] reported that manual prestimulation of the mammary gland induced an earlier increase in oxytocin concentration but did not affect the overall amount of oxytocin released. Premilking stimulation, although important for inducing a rapid milk ejection, is not an efficient way to increase milking-induced PRL release. The results of Exp. 2 show that the magnitude of the milking-induced PRL release decreases when the number of milkings in a day increases. Karg and Schams [11] observed a reduction in PRL release when several milkings and sucklings were performed within a few hours. However, this effect could be related to the degree of udder filling at the time of milking, which may influence milking time, responsiveness to stimulation, or both. In Exp. 3, no milk was harvested after the morning milking in any of the treatments, with the result that the degree of udder filling was similar for each one. In Exp. 4, the degree of udder filling was different, but the stimulation intensity was standardized by replacing the evening milking by a 5-min manual stimulation. In both experiments, inhibition of PRL release was maintained, indicating that the degree of udder filling, the amount of milk harvested, and the length
of milking were not the main factors that caused the inhibition. In addition, one 5-min manual stimulation 2 h before milking had the same effect on PRL release as 5 manual stimulations (Exp. 3), as did 1 milking 2 h before stimulation in comparison with 5 milkings (Exp. 4). These results indicate that the interval since the preceding milking is more important than the number of milkings. Overall, these experiments show that short intervals between milkings cause a depression of the PRL response to milking. The main physiological control of PRL secretion is exerted by the inhibitory action of dopamine on the lactotrophs of the anterior pituitary [12]. Dopamine is secreted in the hypothalamus through the tuberoinfundibular dopamine (TIDA) pathway and reaches the pituitary through a portal vascular system. It has been proposed that suckling reduces the activity of the TIDA neurons, enabling the release of PRL into the blood stream [13]. The mechanism or mechanisms responsible for inhibition of milking-induced PRL release with short milking intervals are unknown. Intuitively, it can be postulated that refractoriness occurs at the level of the mammary gland, the pituitary gland, or the hypothalamus.
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Fig. 2. Effect of milking frequency on milking-induced releases of prolactin (A) and b-endorphin (B) in the blood of dairy cows at the evening milking (7 PM; Exp. 2). On the sampling day, the cows were milked at 7 AM and 7 PM (2; A); at 7 AM, 1 PM, and 7 PM (3; -); or at 7 AM, 9 AM, 11 AM, 1 PM, 3 PM, 5 PM, and 7 PM (7; :). Data are presented as unadjusted means SEM. 0 min ¼ start of milking.
The establishment of mammary gland refractoriness to stimulation is supported by the gradual loss of PRL response during long milking or manual stimulation [14]. In addition, the oxytocin response decreases when milking frequency increases [15]. Nevertheless, the cortisol and b-endorphin responses were not affected in the present study, suggesting no loss of sensitivity of the mammary gland skin to tactile stimulation. A reduction in PRL response is also observed in situations in which no repeated tactile stimulation occurs. A previous study found that the PRL response to thyrotropin-releasing hormone was reduced with multiple injections [11]. Moreover, the presence of the calf with the cow reduces the milkinginduced PRL release even if suckling is not allowed [16]. Therefore, the establishment of mammary gland refractoriness to stimulation cannot be ruled out but does not appear to be the main causal factor of the inhibition of PRL release with short milking intervals. At the level of the pituitary gland, a depletion of PRL reserves and a reduction in sensitivity to PRL-releasing signals can be envisioned. However, the quantity of PRL released after injection of thyrotropin-releasing hormone [11] or the dopamine antagonist fluphenazine [7] was
observed to be far greater than that induced by milking, indicating that PRL reserves are not a limiting factor. In addition, a 5-min manual stimulation did not affect the PRL response to the dopamine antagonist domperidone 60 min later (P. Lacasse, X. Zhao, and S. Ollier, 2013). Thus, it seems unlikely that inhibition of milking-induced PRL release with short milking intervals is due to a reduction in pituitary gland responsiveness. Prolactin secretion is normally tightly regulated by a short-loop negative-feedback mechanism, whereby PRL stimulates the secretion by TIDA neurons of dopamine in the pituitary portal blood, which inhibits PRL secretion. It is therefore tempting to attribute the decreased PRL response to milking to a direct feedback of PRL on TIDA neurons. In rodents, however, the TIDA neurons were found to become unresponsive to PRL in late gestation and during lactation, disrupting the short-loop feedback mechanism [17]. Accordingly, Tucker et al [18] did not observe any inhibition of milking-induced PRL release in cows after infusion of PRL at 1 or 3 mg/h for 2.5 h. Nevertheless, inhibition was observed with administration of supraphysiological doses of PRL [18,19]. As observed in Exps. 3 and 4, milking also induces glucocorticoid release. In rodents, dexamethasone
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Fig. 3. Effect of manual stimulation of the mammary gland between milkings on milking-induced releases of prolactin (A) and cortisol (B) in the blood of dairy cows at the evening milking (7 PM; Exp. 3). The cows were milked at 7 AM and 7 PM with no manual stimulation (A), 5 manual stimulations (at 9 AM, 11 AM, 1 PM, 3 PM, and 5 PM; :), or 1 manual stimulation (at 5 PM; -) between milkings. Data are presented as unadjusted means SEM. 0 min ¼ start of milking.
but the hypothalamus. Similar information is not available in bovines, but it is possible that the cortisol release at milking transiently decreases TIDA responsiveness to inhibitory inputs, reducing the PRL response to a subsequent milking stimulus.
pretreatment inhibits suckling-induced PRL release [20,21]. Given that dexamethasone is able to block PRL release when implanted in the hypothalamus but does not affect domperidone-induced PRL release [20], it appears that the site of action of glucocorticoids is not the pituitary gland
Table 1 Effect of manual stimulation of the mammary gland between milkings on milking-induced PRL and cortisol releases and on milk production at the evening milking (7 PM) and the next morning’s milking (7 AM; Exp. 3). Treatment
PRL AUC, ng·min/mL PRL peak, ng/mL Cortisol AUC, ng·min/mL Cortisol peak, ng/mL Milk, kg (7 PM milking) Milking, min (7 PM milking) Milk, kg (7 AM milking)
SEM
0S
5S
1S
1,726 91.0 177.3 8.1 17.7 6.4 18.2
1,179 56.2 179.3 8.8 13.8 6.1 20.6
1,263 67.8 200.6 8.3 16.3 6.3 20.1
141 12.8 25.8 1.1 1.2 0.4 1.4
P value T
0S vs 1S þ 5S
1S vs 5S
0.03 0.08 0.38 0.72 0.01 0.77 0.05
0.01 0.04 0.44 0.60 0.01 0.56 0.02
0.70 0.40 0.18 0.55 0.01 0.57 0.55
Abbreviations: AUC, area under the curve; PRL, prolactin. The cows were milked at 7 AM and 7 PM with no manual stimulation (0S), 5 manual stimulations (at 9 AM, 11 AM, 1 PM, 3PM, and 5 PM; 5S), or 1 manual stimulation (at 5 PM; 1S) between milkings. Data are presented as least square means.
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Fig. 4. Effect of milking frequency on releases of prolactin (A) and cortisol (B) induced by manual stimulation (Exp. 4). The cows were milked at 7 AM (A); at 7 AM, 9 AM, 11 AM, 1 PM, 3 PM, and 5 PM (:); or at 7 AM and 7 PM (-). All cows were then manually stimulated for 5 min at 7 PM. Data are presented as unadjusted means SEM. 0 min ¼ start of manual stimulation.
The results of Exps. 1 and 2 confirm that milking induces
b-endorphin release in cattle, but the magnitude of that release is not correlated with that of PRL. Although the opiate receptor antagonist naloxone was found to suppress suckling-induced PRL release in rats [6] and goats [22], Tancin et al [23] reported no effects of naloxone or morphine on milking-induced PRL release in cows. Therefore, opioids do not appear to be involved in the release of PRL during milking in cows.
Unexpectedly, the evening milking duration in Exp. 2 was similar for all milking frequencies, despite that different quantities of milk were harvested. Moreover, the quantity of milk harvested at the evening milking in Exp. 3 was reduced by a 5-min manual stimulation 2 h before milking. Because most of the missing milk was harvested at the next milking, it can be concluded that milk ejection was impaired by manual stimulation. Bruckmaier and Hilger [24] reported that milk ejection was delayed with short
Table 2 Effect of milking frequency on the PRL and cortisol releases induced by manual stimulation (Exp. 4). Treatment
PRL AUC, ng·min/mL PRL peak, ng/mL Cortisol AUC, ng·min/mL Cortisol peak, ng/mL
SEM
1
6
2
1,986 146.1 178.5 7.6
1,215 76.2 164.6 7.0
1,402 73.6 162.4 7.1
228 25.5 21.4 0.8
P value T
1 vs 2 þ 6
1 vs 6
0.04 0.01 0.21 0.37
0.01 0.004 0.14 0.17
0.46 0.90 0.80 0.86
Abbreviations: AUC, area under the curve; PRL, prolactin. The cows were milked at 7 AM only (1); at 7 AM, 9 AM, 11 AM, 1 PM, 3 PM, and 5 PM (6); or at 7 AM and 7 PM (2). All cows were then manually stimulated for 5 min at 7 PM. Data are presented as least square means.
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milking intervals and provided some evidence that the delay was related to the lower degree of udder filling. The fact that impairment of milk ejection can occur despite similar degrees of udder filling (as in Exp. 3) does not support this interpretation. Given that frequent milking has been reported to reduce oxytocin release in ewes [15], frequent milking might be the cause of impaired milk ejection with short milking intervals. In conclusion, the length of prestimulation has no significant effect on milking-induced PRL release. Increasing milking frequency reduces the amount of PRL released at milking. This effect is due not to the number of milkings or the amount of milk harvested during the milking but to the interval since the preceding milking. Although milking induces b-endorphin secretion, opioids do not appear to be involved in the release of PRL during milking in cows. Milking-induced cortisol secretion was not affected by milking frequency. Further research is needed to determine how milking interval affects PRL release. Acknowledgments We thank the following people from Agriculture and AgriFood Canada (Sherbrooke, QC, Canada): Lisette St-James, Liette Veilleux, Véronique Roy, Philippe Bernier-Dodier, Caroline Roy, Amélie Piot, Pauline Maisonnasse, Martin Beaumont, and Dominique Poulin for providing technical assistance, and the dairy barn staff for taking care of the cows. We thank Mary Varcoe, from the Translation Bureau, Public Works and Government Services Canada, for her careful editing of this manuscript. We also thank the National Hormone and Peptide Program and A.F. Parlow for providing the bovine prolactin and antibodies. This research was financially supported by Agriculture and Agri-Food Canada.
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