**2. The effect of diverse photoperiod and exogenous melatonin on the secretion of prolactin under** *in vivo* **conditions**

According to Misztal et al. (1999) [19] the modulating effect of melatonin on the secretion of prolactin can be exerted via two various mechanisms. The first one refers to the circadian rhythm and applies probably only to the prolactin stored in lactotrph cells of the pituitary gland. Tuberalin - a factor produced in the infundibular part of the gland probably triggers the expression of the PRL gene in lactotroph cells [20]. The daily secretion of prolactin is also controlled by the dopaminergic system because even the short-lasting growth of prolactin under the influence of melatonin is observed only in a situation when the activity of the dopaminergic system is weakened [21]. It must be stressed that the daily rhythm of prolactin displays a high seasonal variability; in the spring a higher concentration of the hormone is observed in morning and evening hours, and in the summer the daily secretion peak falls at night. In the autumn the rhythm has a two-phase character, like in the spring, whereas the concentration rises in morning and evening hours. In the winter, though, no specific release of prolactin is observed at all.

The other mechanism for regulation of the prolactin secretion is related to its circannual secretion rhythm, when melatonin, owing to its lipophilic, exerts a direct effect on lactotroph cells of the pituitary gland and, accordingly, on the secretion of prolactin [19,22,23]. Under natural conditions the maximum PRL concentration in the sheep's bloodstream is recorded in the long-day period; whereas at this time the melatonin level drops. The lowest level of prolactin is observed during short days, when the melatonin level is the highest [24,25] Shortening of the day length or prolonged administration of melatonin in the period of physiologically increased concentration of prolactin leads to a reduced secretion of this hormone [1,13]. Seasonal changes in the prolactin secretion in the lactation period in sheep undoubtedly affect milk yields. Rhythmical changes of the level of prolactin and melatonin throughout the year were observed mainly in barren sheep and rams; however, studies carried out in the group of sheep used for dairy production confirmed the presence of the seasonal rhythm of these two hormones. The experiments demonstrated a definite influence of the day length on the parameters of ewes' milk yields. Mothers entering lactation in the period of shortening days yielded 50% less milk as compared to ewes milked in the longday period. The drop in milk yields in the shortening photoperiod resulted from the change in the prolactin secretion. The highest PRL concentration in sheep milked in the long-day period was identified in May 312.6 ± 45.2 ng/ml, at this time the concentration of melatonin amounted to 33.5± 11.2 ng/ml. As lactation progressed and days became shorter, the concentration of prolactin declined, and that of melatonin increased (table 1).

122 Prolactin

the biological clock [10,11]. It is confirmed via seasonal changes of the activity of the hypothalamic-pituitary axis in animals kept under permanent light conditions. Thanks to constant and cyclical factors physiological processes can be synchronized with a relevant season of the year. The synthesis of melatonin is a biochemical signal informing the organism about changing light conditions [12,13,14]. Numerous studies go to show the existence of a molecular mechanism involved in deciphering of the melatonin signal which is found in the SCN (Suprachiasmatic Nucleus) and in the PT (Pars Tuberalis). Both in the SCN and PT there are several dozens of genes of the biological clock such as *Per1, Per2* or *Cry 1, Cry2*, which are associated with each other [15,16]. The melatonin profile changing in a 24-hour cycle affects the rhythmical changes in the expression of the clock genes, which is reflected via their different amounts of mRNA in the PT and SCN. The maximum expression of the *Cry1* gene occurs during the dusk period parallel to the growth of the melatonin concentration, whereas the expression of the *Per1* gene is induced at dawn [17,18].

**2. The effect of diverse photoperiod and exogenous melatonin on the** 

According to Misztal et al. (1999) [19] the modulating effect of melatonin on the secretion of prolactin can be exerted via two various mechanisms. The first one refers to the circadian rhythm and applies probably only to the prolactin stored in lactotrph cells of the pituitary gland. Tuberalin - a factor produced in the infundibular part of the gland probably triggers the expression of the PRL gene in lactotroph cells [20]. The daily secretion of prolactin is also controlled by the dopaminergic system because even the short-lasting growth of prolactin under the influence of melatonin is observed only in a situation when the activity of the dopaminergic system is weakened [21]. It must be stressed that the daily rhythm of prolactin displays a high seasonal variability; in the spring a higher concentration of the hormone is observed in morning and evening hours, and in the summer the daily secretion peak falls at night. In the autumn the rhythm has a two-phase character, like in the spring, whereas the concentration rises in morning and evening hours. In the winter, though, no

The other mechanism for regulation of the prolactin secretion is related to its circannual secretion rhythm, when melatonin, owing to its lipophilic, exerts a direct effect on lactotroph cells of the pituitary gland and, accordingly, on the secretion of prolactin [19,22,23]. Under natural conditions the maximum PRL concentration in the sheep's bloodstream is recorded in the long-day period; whereas at this time the melatonin level drops. The lowest level of prolactin is observed during short days, when the melatonin level is the highest [24,25] Shortening of the day length or prolonged administration of melatonin in the period of physiologically increased concentration of prolactin leads to a reduced secretion of this hormone [1,13]. Seasonal changes in the prolactin secretion in the lactation period in sheep undoubtedly affect milk yields. Rhythmical changes of the level of prolactin and melatonin throughout the year were observed mainly in barren sheep and rams; however, studies carried out in the group of sheep used for dairy production confirmed the presence of the

**secretion of prolactin under** *in vivo* **conditions** 

specific release of prolactin is observed at all.


**Table 1.** Changes in the concentration of melatonin and prolactin in sheep milked in the long-day period

As regards sheep lambed and milked in the short-day period and kept under the natural photoperiod conditions the highest level of prolactin was observed in August, i.e. 124.0 ± 48.8 ng/ml. In the first month of milking the concentration of prolactin in sheep corresponded to its seasonal rhythm and declined in subsequent months (table 2).


**Table 2.** Changes in the concentration of melatonin and prolactin in sheep milked in the short-day period

As the light day shortened, the secretion of PRL declined as the level of the hormone in September was lower by 15% as compared to the concentration observed in August. A distinct drop in the prolactin level was observed in the period of the last two months of lactation, i.e. in October and November, and amounted respectively to 30.8 ± 9.7 ng/ml and 16.8 ± 4.1 ng/ml. The low concentration of prolactin already in the first month of milking and systematic growth of the melatonin secretion in the period of shortening days exerted an impact on the parameters of sheep milk yields, causing a drop of ewes' milk yields. In the group of sheep kept under artificially simulated long-day conditions, 16 hours of light and 8 hours of darkness (16L:8D), in the period from August to November, the concentration of PRL already in the second month of milking, i.e. from September to November, decreased. In October and November a sharp drop in the concentration of prolactin was observed. The parameters of milk yields of sheep kept under conditions of artificially extended light day decreased proportionately to the drop in the concentration of prolactin (table 3).

The Effect of Physiological and Environmental Factors on the Prolactin Profile in Seasonally Breeding Animals 125

In the first month of milking, in May, the highest concentration of prolactin was identified in sheep kept under natural day length conditions, with its level amounting to 220.52 ± 8.1 ng/ml. As lactation progressed and days shortened, the secretion of prolactin dropped and in the last two months it amounted respectively to 125.7 ± 9.21 ng/ml and 84.3 ± 4.4 ng/ml. Use of subcutaneous melatonin implants in ewes caused a drop in the concentration of prolactin. Already in the first month of milking, in May, the concentration of prolactin amounted to 160.8 ± 5.6 ng/ml. In subsequent 60 days of lactation (June) a further bigger decrease of the concentration of prolactin was recorded, reaching the level of 82.8 ± 4.2 ng/ml. As lactation progressed and melatonin implants started to exert their effects, a further drop in the secretion of prolatin was observed. In the group of sheep exposed to the effects of artificially simulated short-day conditions (16D:8L), the highest concentration of prolactin was identified in May, equaling 192.8 ± 9.3ng/ml. In subsequent months of lactation keeping sheep under 16D:8L conditions caused a drop in the secretion of prolactin. In the fourth and fifth month of milking the secretion of prolactin decreased again, reaching in August the level of 78.97± 8.63 ng/ml, and in September 52.83 ± 2.73 ng/ml. The studies revealed that simulation of the long signal of melatonin in the spring and summer period

In studies carried out to date no differences have been identified as to the amount of milk obtained in the period of lambs rearing. In the early lactation period sucking is an important factor stimulating the PRL secretion in the mother's organism [27]. The sucking impulse induces release of serotonin [28] and oxytocin in the central nervous system (CNS) which give rise to the release of prolactin from the pituitary gland into the peripheral blood [29]. Experiments conducted on lactating rat females demonstrated that intravenous administration of specific oxytocin antagonist (desGly-NH2-d(CH2)5[D-Tyr2,Thr4]OVT) completely inhibits the release of prolactin triggered by the sucking impulse [30]. Another important compound stimulating the release of prolactin induced by sucking is represented by salsolinol produced by the dopaminergic system in the lactation period. Infusion of exogenous salsolinol into the CNS in the group of lactating sheep gave rise to the release of

With that in mind, further experiments were carried out, which aimed at determining changes in the secretion of prolactin in sheep feeding lambs in the period of lengthening and shortening days, and verifying the hypothesis that melatonin can modify the secretion of prolactin despite strong stimulation of the mammary gland by sucking in the early lactation period [32]. Results of these studies demonstrated that administration of exogenous melatonin to ewes feeding lambs in the long-day period caused a significant drop in the concentration of prolactin. The identified changes in the PRL concentration in sheep entering lactation in the long-day period are comparable with the studies by Rhind et al. (1991) [33] which proved that in mothers rearing lambs in the period of lengthening days the concentration of prolatin rises. The studies conducted showed that despite intensive sucking the secretion of prolactin in sheep with melatonin implants dropped significantly. It must be stressed that the melatonin signal, acting as a marker of the biochemical biological clock, is evolutionarily so strong that

the secretion of prolactin is reduced despite the stimulating sucking impulse.

contributes to disturbance of the endogenous rhythm of prolactin.

prolactin into blood [31].


**Table 3.** Changes in the concentration of melatonin and prolactin in sheep kept under simulated longday conditions -16L:8D

In the short-day period even in lactating sheep it is highly difficult to maintain an appropriately high concentration of prolactin. Studies carried out by Molik et al. (2007) [26] demonstrated that in sheep under artificially extended long-day conditions (16L:8D) it is impossible to maintain a high concentration of prolactin as well as to maintain lactation during shortening days.

Subsequent studies conducted on sheep milked in the long-day period showed that introduction of exogenous melatonin and initiation of artificial short-day conditions (16D:8L - 16 hours of darkness 8 hours of light) in the long-day period gave rise to reduction of the prolactin concentration (table 4).


**Table 4.** Changes in the concentration of prolactin in sheep exposed to the effects of exogenous melatonin and simulated short-day conditions 16D:8L

Melatonin

Prolactin

day conditions -16L:8D

during shortening days.

Months

Control group milked in the long

day period

Sheep with melatonin implants

Sheep group under artificial short-day

conditions 16D:8L

prolactin concentration (table 4).

220.5

melatonin and simulated short-day conditions 16D:8L

group of sheep kept under artificially simulated long-day conditions, 16 hours of light and 8 hours of darkness (16L:8D), in the period from August to November, the concentration of PRL already in the second month of milking, i.e. from September to November, decreased. In October and November a sharp drop in the concentration of prolactin was observed. The parameters of milk yields of sheep kept under conditions of artificially extended light day

Months August September October November

pg/ml 60.4 19.8 17.6 7.6 4.4 1.1 17.0 5.5

ng/ml 132.7 37.4 147.9 22.4 84.3 12.5 38.3 15.2

**Table 3.** Changes in the concentration of melatonin and prolactin in sheep kept under simulated long-

In the short-day period even in lactating sheep it is highly difficult to maintain an appropriately high concentration of prolactin. Studies carried out by Molik et al. (2007) [26] demonstrated that in sheep under artificially extended long-day conditions (16L:8D) it is impossible to maintain a high concentration of prolactin as well as to maintain lactation

Subsequent studies conducted on sheep milked in the long-day period showed that introduction of exogenous melatonin and initiation of artificial short-day conditions (16D:8L - 16 hours of darkness 8 hours of light) in the long-day period gave rise to reduction of the

> Concentracion of prolactin ng/ml May June July August September *x* SE *x* SE *x* SE *x* SE *x* SE

> <sup>2</sup>8.1 199.1 5.6 137.8 9.4 125.7 9.2 84.3 4.4

160.8 5.6 155.3 5.4 117.1 9.3 84.6 11.3 60.3 4.7

192.8 9.3 82.8 4.2 163 6.2 78.6 8.6 52.8 2.7

**Table 4.** Changes in the concentration of prolactin in sheep exposed to the effects of exogenous

*x* SE *x* SE *x* SE *x* SE

decreased proportionately to the drop in the concentration of prolactin (table 3).

In the first month of milking, in May, the highest concentration of prolactin was identified in sheep kept under natural day length conditions, with its level amounting to 220.52 ± 8.1 ng/ml. As lactation progressed and days shortened, the secretion of prolactin dropped and in the last two months it amounted respectively to 125.7 ± 9.21 ng/ml and 84.3 ± 4.4 ng/ml. Use of subcutaneous melatonin implants in ewes caused a drop in the concentration of prolactin. Already in the first month of milking, in May, the concentration of prolactin amounted to 160.8 ± 5.6 ng/ml. In subsequent 60 days of lactation (June) a further bigger decrease of the concentration of prolactin was recorded, reaching the level of 82.8 ± 4.2 ng/ml. As lactation progressed and melatonin implants started to exert their effects, a further drop in the secretion of prolatin was observed. In the group of sheep exposed to the effects of artificially simulated short-day conditions (16D:8L), the highest concentration of prolactin was identified in May, equaling 192.8 ± 9.3ng/ml. In subsequent months of lactation keeping sheep under 16D:8L conditions caused a drop in the secretion of prolactin. In the fourth and fifth month of milking the secretion of prolactin decreased again, reaching in August the level of 78.97± 8.63 ng/ml, and in September 52.83 ± 2.73 ng/ml. The studies revealed that simulation of the long signal of melatonin in the spring and summer period contributes to disturbance of the endogenous rhythm of prolactin.

In studies carried out to date no differences have been identified as to the amount of milk obtained in the period of lambs rearing. In the early lactation period sucking is an important factor stimulating the PRL secretion in the mother's organism [27]. The sucking impulse induces release of serotonin [28] and oxytocin in the central nervous system (CNS) which give rise to the release of prolactin from the pituitary gland into the peripheral blood [29]. Experiments conducted on lactating rat females demonstrated that intravenous administration of specific oxytocin antagonist (desGly-NH2-d(CH2)5[D-Tyr2,Thr4]OVT) completely inhibits the release of prolactin triggered by the sucking impulse [30]. Another important compound stimulating the release of prolactin induced by sucking is represented by salsolinol produced by the dopaminergic system in the lactation period. Infusion of exogenous salsolinol into the CNS in the group of lactating sheep gave rise to the release of prolactin into blood [31].

With that in mind, further experiments were carried out, which aimed at determining changes in the secretion of prolactin in sheep feeding lambs in the period of lengthening and shortening days, and verifying the hypothesis that melatonin can modify the secretion of prolactin despite strong stimulation of the mammary gland by sucking in the early lactation period [32]. Results of these studies demonstrated that administration of exogenous melatonin to ewes feeding lambs in the long-day period caused a significant drop in the concentration of prolactin. The identified changes in the PRL concentration in sheep entering lactation in the long-day period are comparable with the studies by Rhind et al. (1991) [33] which proved that in mothers rearing lambs in the period of lengthening days the concentration of prolatin rises. The studies conducted showed that despite intensive sucking the secretion of prolactin in sheep with melatonin implants dropped significantly. It must be stressed that the melatonin signal, acting as a marker of the biochemical biological clock, is evolutionarily so strong that the secretion of prolactin is reduced despite the stimulating sucking impulse.

Introduction of melatonin implants for sheep rearing lambs in the short-day period did not cause significant changes in the profile of the prolactin secretion. By analyzing the profile of the PRL secretion in both sheep groups, a conclusion can be drawn that in sheep lambed in November the concentration of prolactin in the first control sample drawn was lower by 50% as compared to the control sample drawn in March. In the long-day period the concentration of prolactin in the control group increased, and the secretion of melatonin decreased. In the autumn and winter period, though, as natural conditions set in, the concentration of melatonin increased and the level of prolactin dropped. The extending signal of melatonin observed in the autumn and winter period causes under natural conditions a decrease of the concentration of prolactin in sheep [34,35].

The Effect of Physiological and Environmental Factors on the Prolactin Profile in Seasonally Breeding Animals 127

Secretion of prolactin µg/ml March August November *x* SE *x* SE *x* SE

whereas the experimental group (G2) was incubated in a medium with exogenous melatonin. The experiments performed demonstrated that administration of exogenous melatonin in the long-day period caused a decrease of the secretion of prolactin in the pars

(G1) 145.4 21.2 65.4 15.7 13.1 2.8

melatonin (G2) 105.0 11.4 45.4 9.2 13.7 2.2

**Table 5.** Effects of the day length and exogenous melatonin on the secretion of prolactin under in vitro

The concentration of prolactin in the period of lengthening days (March) in the control group was at the level of 145.45 ± 21.2 µg/ml, whereas in the group incubated with melatonin it was lower and amounted to 105.06 ± 11.4 µg/ml. In the period of shortening days (August), the concentration of prolactin in the medium with exogenous melatonin was lower, equaling 45.4 ± 9.2 g/ml, as compared to the control group, being at the level of 65.4 ± 15.7 µg/ml. The lowest concentration of prolactin both in the control group and experimental one was recorded in the short-day period. The experiments conducted confirmed the seasonal rhythm of prolactin, because the highest concentration of this hormone was observed in the period of lengthening days (145.45 ± 21.2 µg/ml). At the same time the lowest one was recorded in the short-day period (13.1 ± 2.8 µg/ml). The experiments conducted under in vitro conditions showed that in the long-day period melatonin exerted a strong inhibitory effect on the secretion of prolactin. The studies confirmed also a direct influence of melatonin on cells of the sheep's pars tuberalis. It was demonstrated that the secretory activity of lactotroph cells of the pituitary gland under in vitro conditions is closely linked to the day length, and at the same time to the secretion of melatonin. It must be stressed at this point that the obtained seasonal distribution of prolactin concentrations in lactating sheep resembles a seasonal rhythm of the prolactin secretion in barren sheep [19]. However, it should be noted that in the group of lactating sheep the secretion of prolactin is much more intensive. Melatonin administered on the 30th

day of lactation contributed to the reduction of the prolactin secretion [40].

**4. The role of orexins and leptin in the regulation of the secretion of** 

The process of entering and maintenance of lactation in sheep involves a great number of hormones, with prolactin playing a key role. In recent years, though, a focus has been placed on orexins and their role in the secretion of prolactin in sheep. In 1998 a new group of

tuberalis (table 5)

Group

Parst tuberalis – control group

Parst tuberalis – with

**prolactin in sheep** 

conditions.
