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

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

neuropeptides was identified. It was demonstrated that they exert a significant influence on food intake, and in this manner they were dubbed orexins (Greek *orexis* – appetite) [41].

The Effect of Physiological and Environmental Factors on the Prolactin Profile in Seasonally Breeding Animals 129

corresponds with the published pattern for rodents. Results of the studies confirmed also that the orexin gene expression is sensitive to changes in the day length (higher during shorter days). However, no starving-induced changes were identified, which questions the

In the light of the presented results of the studies orexins can be perceived as a link integrating the processes of maintaining energy, hormonal and reproductive balance. Orexin A can be of special importance to animals exhibiting sensitivity to changes in the day length, which include sheep. The process of entering and maintenance of lactation in the animals involves a number of factors, whereas the key role is attributed to prolactin and growth hormone. Unfortunately, mechanisms regulating secretion of these compounds in sheep in the lactation period still remain unclear. It is certain, though, that defining functions of orexins - above all - in the regulation of the prolactin secretion will make it possible to understand the process of maintaining lactation in sheep, in particular in the short-day period. Thus, the purpose of the initiated studies was to determine the role of orexin A in the regulation of the prolactin secretion in sheep under different day-length conditions in in

The experiments were performed on 15 sheep. Pituitary glands were taken from sheep on the 30th day of lactation in the long-day period (May n=5), in the period of shortening days (August n=5) and in the short-day period (December n=5). The pituitaries taken were divided along the sulcus into two halves so that each one contained the adenohypophysis and the neurohypophysis. *In vitro* incubation was held during 3 hours in the Parker medium at a temperature of 370C. The control group (G1) was incubated in a pure medium, whereas the experimental group (G2) was incubated in a medium with exogenous orexin A. Injection of exogenous orexin caused an increase of prolactin secretion already in the first hour of

(G1) 401.8 6.0 344.6 8.2 155.8 3.6

(G2) 451.6 9.4 346.5 7.0 196.3 4.7

In the control group in the second hour of incubation the concentration of prolactin was lower (344.6±8.2 µg/ml) than in the group with orexin (346.5±7.0 µg/ml). But in the third hour of incubation the concentration of prolactin in the experimental group was higher by 40.5 µg/ml than in the control group. In general, the PRL concentration in the long-day period during three-hour incubation in the group with orexin A equaled (331±15.2ng/ml) and was higher as compared to the control group (300.0±16.6 ng/ml). The experiments

**Table 6.** Effects of orexin on the secretion of prolactin in the long-day period

Secretion of prolactin µg/ml Hours of incubation First hour Second hour Third hour *x* SE *x* SE *x* SE

key role of orexins in the regulation of appetite in sheep [51,52].

vitro experiments.

incubation (451.6±9.4 µg/ml) (table 6).

Group

Parst tuberalis – control group

Parst tuberalis – with orexin A

Orexins derive from the same precursor – prepro-orexin, but they are a product of different posttranslational modifications. It is a protein built of 130 amino acid residues (mouse, rat) or 131 amino acid residues (human). In terms of the amino acid sequence a rat's preproorexin displays 83% homology with a human protein and 95% with a mouse's protein [41]. Two forms of the newly discovered compounds are distinguished: orexin A (OxA) and orexin B (OxB), which are molecules whose amino acid composition is similar to the gut hormone - secretin [42]. Orexin A and orexin B bind specifically and activate two GPCR receptors (G-protein coupled receptors), which – before discovery of the ligands - were called orphan receptors, and now they are known as orexin receptors – 1 and – 2 (Ox1R and Ox2R). Studies carried out in 2000 by Date et al. [43] confirmed that matrix RNA of Ox1R and Ox2R exhibits strong expression in the middle, anterior (adenohypophysis) and posterior (neurohypophysis) lobe of the rats' pituitary. At the same time it was noticed that in the anterior part of the pituitary gland Ox1R is subject to stronger expression than Ox2R. The observations go to show that the rat's pituitary is a gland capable of receiving the orexin signal.

Studies related to the synthesis of orexins and distribution of their receptors reveal that they are found in numerous brain sections and confirm the theory, according to which these compounds are regulatory proteins active in the central nervous system [41,44].

Experiments conducted on rats demonstrated that administration of exogenous orexin causes increased intake of food by animals [41]. OxA stimulates appetite in a dosedependent manner and this effect prevails even for up to 4 hours after injection. The effect of OxB injection does not prevail as long as that of OxA; after two hours stimulation of food intake is low. The longer effects of OxA probably are attributable to the molecular structure, thanks to which it is more resistant than OxB to an attack of inactivating peptidases [41].The experiments also displayed functions of orexins – other than the metabolic one. It was confirmed that orexins play a modulating role in the regulation of the hypothalamicpituitary-thyroid axis [45], - adrenal axis [46] and – gonadal axis [47].

Results of *in vivo* studies conducted in 2000 by Russell et al. [48] showed for the first time that injection of orexin A reduced the level of prolactin in rats' blood. Moreover, it was concluded that the mechanism is not connected with the activity of TIDA neutrons synthesizing dopamine (PRL inhibitor). In *in vitro* experiments it was proven that OxA exerts a direct influence on the secretory activity of explants of the hypothalamus and pituitary gland. Orexin A is also described as an inhibitor of the PRL secretion by lactotroph cells of the pituitary anterior lobe by Dusza and Ciereszko (2007) [49]. However, results of studies carried out on immature female rats under primary culture conditions are completely different as they reveal a statistically significant growth of the prolactin secretion depending on a dose and time of incubation of pituitary glands [50].

The orexin gene expression in ruminants was defined for the first time only in 2002 and it was shown that the location of the mRNA of prepro-orexin in the hypothalamus in sheep corresponds with the published pattern for rodents. Results of the studies confirmed also that the orexin gene expression is sensitive to changes in the day length (higher during shorter days). However, no starving-induced changes were identified, which questions the key role of orexins in the regulation of appetite in sheep [51,52].

128 Prolactin

signal.

neuropeptides was identified. It was demonstrated that they exert a significant influence on food intake, and in this manner they were dubbed orexins (Greek *orexis* – appetite) [41].

Orexins derive from the same precursor – prepro-orexin, but they are a product of different posttranslational modifications. It is a protein built of 130 amino acid residues (mouse, rat) or 131 amino acid residues (human). In terms of the amino acid sequence a rat's preproorexin displays 83% homology with a human protein and 95% with a mouse's protein [41]. Two forms of the newly discovered compounds are distinguished: orexin A (OxA) and orexin B (OxB), which are molecules whose amino acid composition is similar to the gut hormone - secretin [42]. Orexin A and orexin B bind specifically and activate two GPCR receptors (G-protein coupled receptors), which – before discovery of the ligands - were called orphan receptors, and now they are known as orexin receptors – 1 and – 2 (Ox1R and Ox2R). Studies carried out in 2000 by Date et al. [43] confirmed that matrix RNA of Ox1R and Ox2R exhibits strong expression in the middle, anterior (adenohypophysis) and posterior (neurohypophysis) lobe of the rats' pituitary. At the same time it was noticed that in the anterior part of the pituitary gland Ox1R is subject to stronger expression than Ox2R. The observations go to show that the rat's pituitary is a gland capable of receiving the orexin

Studies related to the synthesis of orexins and distribution of their receptors reveal that they are found in numerous brain sections and confirm the theory, according to which these

Experiments conducted on rats demonstrated that administration of exogenous orexin causes increased intake of food by animals [41]. OxA stimulates appetite in a dosedependent manner and this effect prevails even for up to 4 hours after injection. The effect of OxB injection does not prevail as long as that of OxA; after two hours stimulation of food intake is low. The longer effects of OxA probably are attributable to the molecular structure, thanks to which it is more resistant than OxB to an attack of inactivating peptidases [41].The experiments also displayed functions of orexins – other than the metabolic one. It was confirmed that orexins play a modulating role in the regulation of the hypothalamic-

Results of *in vivo* studies conducted in 2000 by Russell et al. [48] showed for the first time that injection of orexin A reduced the level of prolactin in rats' blood. Moreover, it was concluded that the mechanism is not connected with the activity of TIDA neutrons synthesizing dopamine (PRL inhibitor). In *in vitro* experiments it was proven that OxA exerts a direct influence on the secretory activity of explants of the hypothalamus and pituitary gland. Orexin A is also described as an inhibitor of the PRL secretion by lactotroph cells of the pituitary anterior lobe by Dusza and Ciereszko (2007) [49]. However, results of studies carried out on immature female rats under primary culture conditions are completely different as they reveal a statistically significant growth of the prolactin secretion

The orexin gene expression in ruminants was defined for the first time only in 2002 and it was shown that the location of the mRNA of prepro-orexin in the hypothalamus in sheep

compounds are regulatory proteins active in the central nervous system [41,44].

pituitary-thyroid axis [45], - adrenal axis [46] and – gonadal axis [47].

depending on a dose and time of incubation of pituitary glands [50].

In the light of the presented results of the studies orexins can be perceived as a link integrating the processes of maintaining energy, hormonal and reproductive balance. Orexin A can be of special importance to animals exhibiting sensitivity to changes in the day length, which include sheep. The process of entering and maintenance of lactation in the animals involves a number of factors, whereas the key role is attributed to prolactin and growth hormone. Unfortunately, mechanisms regulating secretion of these compounds in sheep in the lactation period still remain unclear. It is certain, though, that defining functions of orexins - above all - in the regulation of the prolactin secretion will make it possible to understand the process of maintaining lactation in sheep, in particular in the short-day period. Thus, the purpose of the initiated studies was to determine the role of orexin A in the regulation of the prolactin secretion in sheep under different day-length conditions in in vitro experiments.

The experiments were performed on 15 sheep. Pituitary glands were taken from sheep on the 30th day of lactation in the long-day period (May n=5), in the period of shortening days (August n=5) and in the short-day period (December n=5). The pituitaries taken were divided along the sulcus into two halves so that each one contained the adenohypophysis and the neurohypophysis. *In vitro* incubation was held during 3 hours in the Parker medium at a temperature of 370C. The control group (G1) was incubated in a pure medium, whereas the experimental group (G2) was incubated in a medium with exogenous orexin A. Injection of exogenous orexin caused an increase of prolactin secretion already in the first hour of incubation (451.6±9.4 µg/ml) (table 6).



In the control group in the second hour of incubation the concentration of prolactin was lower (344.6±8.2 µg/ml) than in the group with orexin (346.5±7.0 µg/ml). But in the third hour of incubation the concentration of prolactin in the experimental group was higher by 40.5 µg/ml than in the control group. In general, the PRL concentration in the long-day period during three-hour incubation in the group with orexin A equaled (331±15.2ng/ml) and was higher as compared to the control group (300.0±16.6 ng/ml). The experiments

demonstrated that irrespective of how the secretory capabilities of cells deteriorated in the course of incubation, exogenous orexin A stimulates the PRL secretion in the long-day period in lactating ewes (53).

The Effect of Physiological and Environmental Factors on the Prolactin Profile in Seasonally Breeding Animals 131

Results obtained in the third hour of incubation revealed that the concentration of prolactin in the control group equaled (65.7±5.9 pg/ml), whereas in the group treated with exogenous orexin it amounted to (79.9±7.0 pg/ml). Despite the fact that secretory capabilities of cells deteriorated in the course of incubation, the PRL concentration in the examined samples

The experiments conducted under in vitro conditions confirmed that the annual rhythm of the PRL secretion in sheep is characterized by a higher concentration in the long-day period

Orexin exerts its effects on the PRL secretion in sheep, like in case of rodents, thanks to the presence of specific receptors Ox1R and Ox2R in the cytoplasmatic membrane of cells. Their existence in the sheep's adenohypophysis was described in 2002 by Xu et al.[54], who identified a high homology of the structure of a gene encoding sheep's Ox1R and a gene of the receptor in a rat (87%) and human being (89%). The presence of both of these forms of orexin receptors in the sheep's pituitary gland was also confirmed by Zhang et al. in 2004

Studies carried out to date with a view to explaining the relations between orexin and prolactin have been conducted mainly on rats. Thus, results of experiments on sheep in the period of lactation offer new information about the role of orexins in seasonally breeding animals. The conducted experiments proved that orexin A exerts a greater stimulating effect on the level of the PRL secretion in the summer season than in the winter season. The weaker response of sheep's pituitaries to orexin during short days as compared to the response during long days is a phenomeon of resistance of lactotroph cells to the orexin signal. Such a reaction of endocrine cells can be explained by the seasonal rhythm of biosynthesis and secretion of orexin in sheep, regulated by the photoperiod. It was proven that under conditions of shortening light days the expression of the orexin precursor gene in these ruminants is on a higher level than during long days [51]. As a result, an increased concentration of the mRNA of prepro-orexin in the winter season is observed, followed by an increased concentration of orexin in the organism. It is probable that at this time the saturation degree of orexin receptors of lactotroph cells with endogenous ligand is so high that adding exogenous orexin A does not cause such a distinct response as that occurring during long days. While attempting to identify interactions between the secretion of prolactin and growth hormone and the level of melatonin in lactating sheep an analogous phenomenon was noticed. Despite the fact that ewes lambed in June were kept under artificial long-day conditions, as the day became shorter, a drop in the PRL level was observed. In consequence, it was concluded that the secretion of the hormones retained its endogenous seasonal rhythm. The effect of the loss of the pituitary cells' sensitivity to the summer signal of the hormone of darkness repeating for too long does not allow for

(summer) and lower concentration in the short-day period (winter).

extension of sheep's lactation to the autumn and winter period [57].

In experiments conducted in 2006 it was proven for the first time that leptin exerts an effect on the melatonin secretion both under *in vitro* [57] and *in vivo* [58] conditions. It was observed that the day length (photoperiod) is one of the factors which modulate the effects

grew.

[55].

Studies conducted in the period of shortening days (August) showed that administration of orexin A can modulate prolactin secretion (table 7).


**Table 7.** Effects of orexin on the secretion of prolactin in the period of shortening days (August)

While analyzing changes in the prolactin secretion in sheep during 3-hour incubation, the highest secretory activity of cells was observed in the first two hours of the experiment. In the first hour of incubation the concentration of prolactin in the group incubated with orexin A equaled (40.6±13.5 µg/ml) and was higher as opposed to the control group (38.05±12.1 µg/ml). The concentration of prolactin in the second hour of the experiment was still higher than that in the control group and equaled respectively (38.7±10.1, 32.1±9.6 µg/ml). In the third hour of the experiment it was demonstrated that in the experimental group the concentration of prolactin was higher (28.6±9.7 µg/ml) as compared to the control group (19.2±7.3 µg/ml).

In the period of shortening days, when the concentration of prolactin under natural conditions is reduced, administration of orexin A caused a growth of the prolactin secretion; similarly, injection of exogenous orexin A in the short-day period caused an increase of the prolactin secretion (table 8)


**Table 8.** Effects of orexin on the secretion of prolactin in the short-day period (December)

In the first hour of incubation of the pituitaries in the short-day period it was observed that the prolactin secretion in the experimental group increased (158.9±9.2 pg/ml) as contrasted with the control group (135.1±8.4 pg/ml). In the second hour of incubation it was recorded that the concentration of prolactin in the experimental group grew (164.8±7.5 pg/ml), whereas in the control group the concentration of prolactin amounted to (107.8±4.0 pg/ml).

The Effect of Physiological and Environmental Factors on the Prolactin Profile in Seasonally Breeding Animals 131

Results obtained in the third hour of incubation revealed that the concentration of prolactin in the control group equaled (65.7±5.9 pg/ml), whereas in the group treated with exogenous orexin it amounted to (79.9±7.0 pg/ml). Despite the fact that secretory capabilities of cells deteriorated in the course of incubation, the PRL concentration in the examined samples grew.

130 Prolactin

period in lactating ewes (53).

Group

(19.2±7.3 µg/ml).

prolactin secretion (table 8)

Group

orexin A can modulate prolactin secretion (table 7).

demonstrated that irrespective of how the secretory capabilities of cells deteriorated in the course of incubation, exogenous orexin A stimulates the PRL secretion in the long-day

Studies conducted in the period of shortening days (August) showed that administration of

Parst tuberalis – control group 38.05 12.1 32.1 9.6 19.2 7.3 Parst tuberalis – with orexin A 40.6 13.5 38.7 10.1 28.6 9.7 **Table 7.** Effects of orexin on the secretion of prolactin in the period of shortening days (August)

While analyzing changes in the prolactin secretion in sheep during 3-hour incubation, the highest secretory activity of cells was observed in the first two hours of the experiment. In the first hour of incubation the concentration of prolactin in the group incubated with orexin A equaled (40.6±13.5 µg/ml) and was higher as opposed to the control group (38.05±12.1 µg/ml). The concentration of prolactin in the second hour of the experiment was still higher than that in the control group and equaled respectively (38.7±10.1, 32.1±9.6 µg/ml). In the third hour of the experiment it was demonstrated that in the experimental group the concentration of prolactin was higher (28.6±9.7 µg/ml) as compared to the control group

In the period of shortening days, when the concentration of prolactin under natural conditions is reduced, administration of orexin A caused a growth of the prolactin secretion; similarly, injection of exogenous orexin A in the short-day period caused an increase of the

Parst tuberalis – control group 135.1 8.4 107.8 7.5 65.7 5.9 Parst tuberalis – with orexin A 158.9 9.2 164.8 7.5 79.9 7

In the first hour of incubation of the pituitaries in the short-day period it was observed that the prolactin secretion in the experimental group increased (158.9±9.2 pg/ml) as contrasted with the control group (135.1±8.4 pg/ml). In the second hour of incubation it was recorded that the concentration of prolactin in the experimental group grew (164.8±7.5 pg/ml), whereas in the control group the concentration of prolactin amounted to (107.8±4.0 pg/ml).

**Table 8.** Effects of orexin on the secretion of prolactin in the short-day period (December)

Secretion of prolactin µg/ml Hours of incubation First hour Second hour Third hour *x* SE *x* SE *x* SE

Secretion of prolactin µg/ml Hours of incubation First hour Second hour Third hour *x* SE *x* SE *x* SE The experiments conducted under in vitro conditions confirmed that the annual rhythm of the PRL secretion in sheep is characterized by a higher concentration in the long-day period (summer) and lower concentration in the short-day period (winter).

Orexin exerts its effects on the PRL secretion in sheep, like in case of rodents, thanks to the presence of specific receptors Ox1R and Ox2R in the cytoplasmatic membrane of cells. Their existence in the sheep's adenohypophysis was described in 2002 by Xu et al.[54], who identified a high homology of the structure of a gene encoding sheep's Ox1R and a gene of the receptor in a rat (87%) and human being (89%). The presence of both of these forms of orexin receptors in the sheep's pituitary gland was also confirmed by Zhang et al. in 2004 [55].

Studies carried out to date with a view to explaining the relations between orexin and prolactin have been conducted mainly on rats. Thus, results of experiments on sheep in the period of lactation offer new information about the role of orexins in seasonally breeding animals. The conducted experiments proved that orexin A exerts a greater stimulating effect on the level of the PRL secretion in the summer season than in the winter season. The weaker response of sheep's pituitaries to orexin during short days as compared to the response during long days is a phenomeon of resistance of lactotroph cells to the orexin signal. Such a reaction of endocrine cells can be explained by the seasonal rhythm of biosynthesis and secretion of orexin in sheep, regulated by the photoperiod. It was proven that under conditions of shortening light days the expression of the orexin precursor gene in these ruminants is on a higher level than during long days [51]. As a result, an increased concentration of the mRNA of prepro-orexin in the winter season is observed, followed by an increased concentration of orexin in the organism. It is probable that at this time the saturation degree of orexin receptors of lactotroph cells with endogenous ligand is so high that adding exogenous orexin A does not cause such a distinct response as that occurring during long days. While attempting to identify interactions between the secretion of prolactin and growth hormone and the level of melatonin in lactating sheep an analogous phenomenon was noticed. Despite the fact that ewes lambed in June were kept under artificial long-day conditions, as the day became shorter, a drop in the PRL level was observed. In consequence, it was concluded that the secretion of the hormones retained its endogenous seasonal rhythm. The effect of the loss of the pituitary cells' sensitivity to the summer signal of the hormone of darkness repeating for too long does not allow for extension of sheep's lactation to the autumn and winter period [57].

In experiments conducted in 2006 it was proven for the first time that leptin exerts an effect on the melatonin secretion both under *in vitro* [57] and *in vivo* [58] conditions. It was observed that the day length (photoperiod) is one of the factors which modulate the effects of leptin directly in the sheep's pineal gland, with an inhibitory effect of leptin on melatonin secreted by explants of the pineal gland in the period of lengthening days. However, during shortening days leptin stimulated the melatonin secretion by the gland explants [57]. Results of the *in vitro* experiments were confirmed in *in vivo* experiments*,* in which leptin was injected into the 3rd brain chamber. They demonstrated that exogenous leptin has an inhibitory effect on the concentration of melatonin during long days and a stimulating dosedependent effect – during short days [58].

The Effect of Physiological and Environmental Factors on the Prolactin Profile in Seasonally Breeding Animals 133

2-week interval intraventricular infusion of Ringer-Locke's fluid – control, recombinant sheep leptin (roleptin) – in a dose of 0.5 µg/kg body weight (Leptin 1), and roleptin in a dose of 1.0 ug/kg body weight (Leptin 2). On the day of the experiment animals were put in individual cattle crushes, the Ringer-Locke's fluid (control) and roleptin were injected intraventricularly for the first time directly after drawing time-zero blood samples and in the 60th and 120th minute of the 6-hour experiment. During the experiment blood samples were drawn every 15 minutes. Blood was poured into test tubes containing 100 l of heparin

During lengthening days the total PRL concentration in the plasma of control sheep was significantly higher (*P* < 0.001) than during shortening days (132.28 19.87 vs. 44.41 8.27 ng/ml). Intraventricular infusions of two doses of exogenous leptin reduced the concentration of prolactin during SD (*P* < 0.001) as compared to the values observed in control ewes. It was concluded that the day length is one of the factors which modulate the effects of leptin on the prolactin secretion in sheep. It was observed that central infusions of leptin into the 3rd brain chamber significantly decreased the concentration of prolactin in sheep during short days in a dose-dependent manner; whereas the effect was opposite

In subsequent experiments a test of the hypothesis about season-dependent effects of leptin and orexin B on the endocrine activity of sheep was performed. Six weeks prior to the start of the experiments ewes were subjected to an ovariectomy procedure, and subcutaneous estradiol implants were inserted into each of them. Three weeks prior to the initiation of the planned experiments metal stainless cannulas were inserted surgically into the third brain chamber of ewes (using the stereotactic method by Traczyk and Przekop [1963] (59). Like in the previous experiment, the tests were started at the sunset, and were continued during subsequent 6 hours. The experiments were performed so that each ewe received in oneweek interval intraventricular infusion: 1. Control group (CG) - Ringer-Locke's fluid (pH=7.4); 2. Experimental group 1 (Gr 1) – recombinant sheep leptin (PLR Laboratory, Israel) in a dose of 0.5 g/kg, the dose was selected based on experiments [58]; 3. Experimental group 3 (Gr 3) – orexins B (PolyPeptides Laboratories, Strasbourg, France) in a dose of 0,3 µg/kg., 4. Experimental group 5 (Gr 5) - superantagonists of leptin (D23L/L39A/D40A/F41A; PLR Laboratory Israel) in a dose of 50 µg/kg, and then orexin B in a dose used previously. In the control group and experimental group 1, 2, and 3 infusions of selected factors were performed three times, every 60 minutes from the start of the experiment, in group 5 the leptin antagonist was infused twice, in the zero and 1st hour of the experiment, and ghrelin/orexin B was infused 15 and 60 minutes after administration of leptin antagonist. During the 6-hour experiment blood samples were drawn every 15 minutes. The average concentration of melatonin in the plasma of ewes from the control group was higher (*P*< 0.001) during LD as compared to the values recorded in animals during short days (87.28 1.2 vs. 59.70 3.1 pg/ml). In the seasons of lengthening days exogenous recombinant sheep leptin significantly reduced the concentration of melatonin (*P*< 0.001) in relation to the values observed in ewes from the control group, whereas during a short day it significantly increased (*P*< 0.01) the concentration of melatonin. Orexin B did not have any impact on the

solution (1000 IU/ml).

during a long photoperiod.

Seasonal insensitivity to leptin is observed in sheep in the spring and summer period, making it possible for the ruminants to make energy reserves, and despite increasing fatness intake of food in sheep is not reduced. Increased intake of food and a growth of the body weight during long days – LD (*Long Days*) spring - summer – is characterized by a high concentration of leptin in blood plasma, which "loses" then its anorectic features. In the short-day period – SD (*Short Days*) autumn - winter, when the availability of food dwindles, the sensitivity of centres regulating food intake in the hypothalamus to the concentration of leptin returns to normal. This paradox is explained by leptin resistance occurring during LD, whereas the neuroendocrinological basis of leptin resistance has not been fully understood yet. One of the phenomena underlying leptin resistance is auto-supression of the transfer of signal from the receptor to the cell centre resulting from the leptin-induced expression of SOCS-3 factors, being inhibitors of cytokine sygnalisation [58]. The phenomenon is observed in particular in the area of ventromedial hyopothalamus [58], where long-form receptors of leptin Ob-Rb are found in the highest concentration.

Orexigenic and anorexigenic systems are linked in terms of morphology and functionality. It confirms the hypothesis about the existence of a nervous network regulating hunger, located in nervous centres of the hypothalamus. The area of receptors and nerve endings for orexigenic factors overlaps with the area of receptors for anorexigenic factors, which also reveals mutual interactions of the two systems. In animals with strong seasonal breeding characteristics the network of the above described relations overlaps additionally with effects of the photoperiod, which by way of a biological signal in the form of melatonin interferes with the developed relations. The suprachiasmatic nucleus (SCN), which is a part of the biological clock, generates signals adjusting food intake to the circadian rhythm. This element is considerably weakened in the primates, and in particular in humans, but it is very efficient in ruminants. The objective of the conducted experiments was to investigate interactions between the day length and leptin and orexin B in sheep under *in vivo* conditions.

The experiments were carried out on 24 sheep of the Polish sheep breed. The first stage of experiments (n=12) was conducted in the period of lengthening days (spring – summer). The second stage (n=12) was held under conditions of shortening days (autumn – winter). Three weeks prior to the initiation of the planned experiments metal stainless cannulas were inserted surgically into the third brain chamber of ewes (using the stereotactic method by Traczyk and Przekop [1963] [59]). The tests were started at the sunset, and were continued during subsequent 6 hours. The experiments were performed so that each ewe received in a

conditions.

dependent effect – during short days [58].

leptin Ob-Rb are found in the highest concentration.

of leptin directly in the sheep's pineal gland, with an inhibitory effect of leptin on melatonin secreted by explants of the pineal gland in the period of lengthening days. However, during shortening days leptin stimulated the melatonin secretion by the gland explants [57]. Results of the *in vitro* experiments were confirmed in *in vivo* experiments*,* in which leptin was injected into the 3rd brain chamber. They demonstrated that exogenous leptin has an inhibitory effect on the concentration of melatonin during long days and a stimulating dose-

Seasonal insensitivity to leptin is observed in sheep in the spring and summer period, making it possible for the ruminants to make energy reserves, and despite increasing fatness intake of food in sheep is not reduced. Increased intake of food and a growth of the body weight during long days – LD (*Long Days*) spring - summer – is characterized by a high concentration of leptin in blood plasma, which "loses" then its anorectic features. In the short-day period – SD (*Short Days*) autumn - winter, when the availability of food dwindles, the sensitivity of centres regulating food intake in the hypothalamus to the concentration of leptin returns to normal. This paradox is explained by leptin resistance occurring during LD, whereas the neuroendocrinological basis of leptin resistance has not been fully understood yet. One of the phenomena underlying leptin resistance is auto-supression of the transfer of signal from the receptor to the cell centre resulting from the leptin-induced expression of SOCS-3 factors, being inhibitors of cytokine sygnalisation [58]. The phenomenon is observed in particular in the area of ventromedial hyopothalamus [58], where long-form receptors of

Orexigenic and anorexigenic systems are linked in terms of morphology and functionality. It confirms the hypothesis about the existence of a nervous network regulating hunger, located in nervous centres of the hypothalamus. The area of receptors and nerve endings for orexigenic factors overlaps with the area of receptors for anorexigenic factors, which also reveals mutual interactions of the two systems. In animals with strong seasonal breeding characteristics the network of the above described relations overlaps additionally with effects of the photoperiod, which by way of a biological signal in the form of melatonin interferes with the developed relations. The suprachiasmatic nucleus (SCN), which is a part of the biological clock, generates signals adjusting food intake to the circadian rhythm. This element is considerably weakened in the primates, and in particular in humans, but it is very efficient in ruminants. The objective of the conducted experiments was to investigate interactions between the day length and leptin and orexin B in sheep under *in vivo*

The experiments were carried out on 24 sheep of the Polish sheep breed. The first stage of experiments (n=12) was conducted in the period of lengthening days (spring – summer). The second stage (n=12) was held under conditions of shortening days (autumn – winter). Three weeks prior to the initiation of the planned experiments metal stainless cannulas were inserted surgically into the third brain chamber of ewes (using the stereotactic method by Traczyk and Przekop [1963] [59]). The tests were started at the sunset, and were continued during subsequent 6 hours. The experiments were performed so that each ewe received in a 2-week interval intraventricular infusion of Ringer-Locke's fluid – control, recombinant sheep leptin (roleptin) – in a dose of 0.5 µg/kg body weight (Leptin 1), and roleptin in a dose of 1.0 ug/kg body weight (Leptin 2). On the day of the experiment animals were put in individual cattle crushes, the Ringer-Locke's fluid (control) and roleptin were injected intraventricularly for the first time directly after drawing time-zero blood samples and in the 60th and 120th minute of the 6-hour experiment. During the experiment blood samples were drawn every 15 minutes. Blood was poured into test tubes containing 100 l of heparin solution (1000 IU/ml).

During lengthening days the total PRL concentration in the plasma of control sheep was significantly higher (*P* < 0.001) than during shortening days (132.28 19.87 vs. 44.41 8.27 ng/ml). Intraventricular infusions of two doses of exogenous leptin reduced the concentration of prolactin during SD (*P* < 0.001) as compared to the values observed in control ewes. It was concluded that the day length is one of the factors which modulate the effects of leptin on the prolactin secretion in sheep. It was observed that central infusions of leptin into the 3rd brain chamber significantly decreased the concentration of prolactin in sheep during short days in a dose-dependent manner; whereas the effect was opposite during a long photoperiod.

In subsequent experiments a test of the hypothesis about season-dependent effects of leptin and orexin B on the endocrine activity of sheep was performed. Six weeks prior to the start of the experiments ewes were subjected to an ovariectomy procedure, and subcutaneous estradiol implants were inserted into each of them. Three weeks prior to the initiation of the planned experiments metal stainless cannulas were inserted surgically into the third brain chamber of ewes (using the stereotactic method by Traczyk and Przekop [1963] (59). Like in the previous experiment, the tests were started at the sunset, and were continued during subsequent 6 hours. The experiments were performed so that each ewe received in oneweek interval intraventricular infusion: 1. Control group (CG) - Ringer-Locke's fluid (pH=7.4); 2. Experimental group 1 (Gr 1) – recombinant sheep leptin (PLR Laboratory, Israel) in a dose of 0.5 g/kg, the dose was selected based on experiments [58]; 3. Experimental group 3 (Gr 3) – orexins B (PolyPeptides Laboratories, Strasbourg, France) in a dose of 0,3 µg/kg., 4. Experimental group 5 (Gr 5) - superantagonists of leptin (D23L/L39A/D40A/F41A; PLR Laboratory Israel) in a dose of 50 µg/kg, and then orexin B in a dose used previously. In the control group and experimental group 1, 2, and 3 infusions of selected factors were performed three times, every 60 minutes from the start of the experiment, in group 5 the leptin antagonist was infused twice, in the zero and 1st hour of the experiment, and ghrelin/orexin B was infused 15 and 60 minutes after administration of leptin antagonist. During the 6-hour experiment blood samples were drawn every 15 minutes. The average concentration of melatonin in the plasma of ewes from the control group was higher (*P*< 0.001) during LD as compared to the values recorded in animals during short days (87.28 1.2 vs. 59.70 3.1 pg/ml). In the seasons of lengthening days exogenous recombinant sheep leptin significantly reduced the concentration of melatonin (*P*< 0.001) in relation to the values observed in ewes from the control group, whereas during a short day it significantly increased (*P*< 0.01) the concentration of melatonin. Orexin B did not have any impact on the concentration of melatonin during a short day, but it caused a significant growth (*P*< 0.05) of the concentration of melatonin on a long day.

The Effect of Physiological and Environmental Factors on the Prolactin Profile in Seasonally Breeding Animals 135

*The Kielanowski Institute of Animal Physiology and Nutrition, Polish Academy of Sciences, Poland* 

This research was supported by projects MNiSZW NN311245033 and DS/KHiOK/3242/2010.

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**Author details** 

Tomasz Misztal

**6. References** 

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**Acknowledgement** 

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During a lengthening day the total PRL concentration in the plasma of sheep from the control group was markedly higher (*P*< 0.001) as contrasted with shortening days (132.28 19.87 vs. 44.41 8.27 ng/ml). Intraventricular infusions of exogenous leptin reduced the concentration of prolactin during SD (*P*< 0.001) as compared to the values observed in ewes from the control group. The average concentration of endogenous orexin was higher (P< 0.01) during short days (0.59 0.05 ng/ml) as opposed to LD (0.39 0.01 ng/ml) in the control group of sheep. Exogenous orexin caused a growth (*P*< 0.05) of the plasma concentration of endogenous orexin respectively on a long and short day (0.62 0.01 ng/ml and 0.71 0.03 ng/ml), whereas the effects of leptin reduced (*P*< 0.05) the concentration of orexin during LD and SD. Based on the conducted experiments it was concluded that orexin B and anorectic hormone – leptin directly interact closely with each other, regulating not only the processes of metabolism but influencing jointly release of melatonin and prolactin, and the interactions additionally depend on the prevalent photoperiod.

#### **5. Summary**

So far it has been believed that milk yields in mammals are determined by genetic and environmental factors. In recent years, though, a special focus has been placed on light, being the modulator of the prolactin level. In farm animals changes of the light day play a very important role as they determine their yields. The length of the light day, and in particular the melatonin profile, is of special importance in sheep as they determine reproductive processes, in which lactation is the last stage of reproductive physiology. Experiments carried out on sheep demonstrated that both the melatonin profile and prolactin profile retain features of a seasonal rhythm depending on the day length. The synthesis of melatonin by the pineal gland is a biochemical signal informing the organism about the break of the day or night. This hormone regulates activities of numerous organs. Until now the activity of melatonin has been associated with the impact on the reproductive system. Experiments carried out in recent years have shown that melatonin can modulate the level of prolactin. Under natural conditions the maximum concentration of prolactin in sheep blood is observed in the long-day period, and at this time the level of melatonin drops. The lowest concentration of prolactin is recorded during short days, when the level of melatonin is the highest. Shortening of the day length or long-term injection of melatonin in the period of physiologically increased concentration of prolactin leads to a reduced secretion of this hormone. Lactation in sheep involves a number of hormones, and for that reason in recent years a special focus has been placed on the role of orexins in the regulation of the prolactin secretion. Experiments conducted demonstrated that orexin A exerts a greater stimulating effect on the level of the PRL secretion in the summer season than in the winter season. The weaker reaction of sheep's pituitaries to orexin during short days as compared to the response during long days is a phenomenon of resistance of lactotroph cells to the orexin signal. Such a reaction of endocrine cells can be explained by the seasonal rhythm of biosynthesis and secretion of orexin in sheep, regulated by the photoperiod.

The Effect of Physiological and Environmental Factors on the Prolactin Profile in Seasonally Breeding Animals 135
