**3. Influence of metabolic hormones on prolactin secretion in lactation sheep**

### **3.1. Role of orexin**

Studies on the role of orexin A in the control of prolactin (PRL) and growth hormone (GH) secretion in rodents have produced inconsistent results. Orexin A may play a special role in animals' sensitivity such as sheep to the day length changes. The aim of the study was to determine the role of orexin A in the control of prolactin secretion and growth hormone in sheep during different photoperiods. In vitro studies were carried out on 10 Polish Longwool ewes on 30 days of lactation during long photoperiod (May, LD, n = 5) and short photoperiod (December, SD, n = 5). After rearing lambs to 30 days of age, ewes were decapitated and the pituitaries were dissected and then cut along the longitudinal fissure into two halves, so that each half contained the glandular and nervous parts. Pituitary glands were collected and divided along the longitudinal fissure into two halves. Glands were incubated for 3 hours at 37°C in Parker medium with addition of orexin A—experimental group or in medium alone—control group. During the following 3-hour incubation, medium was exchanged every 15 minutes and a sample of 1 ml was collected and immediately frozen at −80°C until assay. Prolactin concentrations in the medium were determined radioimmunologically (RIA).

In the long-day conditions (May), the pituitary explants of lactating sheep exhibited the strongest secretory activity during the first hour of incubation—significantly higher in orexintreated group (O1) than the control group (K1), (*P* < 0.01). During the second hour of the incubation, PRL concentration decreased and reached the similar values in both groups. During the third hour, PRL concentration in O1 group was again significantly higher than that noted in K1 group (*P* < 0.01). In the short-day conditions (December), PRL concentration was significantly higher in orexin-treated group O2 during the first hour of incubation than the value observed in the control group—K2 (*P* < 0.01). The inverse relationship in prolactin release was observed during the second hour of incubation (*P* < 0.01), however, during the third hour, PRL concentration was again significantly higher in O2 group than the concentration noted in K2 group (*P* < 0.05). Collective analysis of the data showed that PRL concentrations were higher in experimental groups (O1 and O2) than the concentrations noted in control groups (K1 and K2) under both the long (May) and short (December) photoperiods (**Figure 4**).

SDC and SDM ewes were at a similar level up to the second time of blood collection day (**Figure 3**). The only significant (*P* ≤ 0.001) rise in plasma GH secretion was recorded in the third week of lactation in the SDM group (12.23 ± 5.36 ng/ml) compared with the SDC

In conclusion, the long-term treatment with exogenous melatonin of early-lactating sheep reduced the PRL secretion during the increasing photoperiod, despite strong stimulation by suckling. Moreover, in nursing ewes, melatonin stimulated GH secretion during the short photoperiod. It can therefore be assumed that melatonin may be indirectly affected by the level

**3. Influence of metabolic hormones on prolactin secretion in lactation sheep**

Studies on the role of orexin A in the control of prolactin (PRL) and growth hormone (GH) secretion in rodents have produced inconsistent results. Orexin A may play a special role in animals' sensitivity such as sheep to the day length changes. The aim of the study was to determine the role of orexin A in the control of prolactin secretion and growth hormone in sheep during different photoperiods. In vitro studies were carried out on 10 Polish Longwool ewes on 30 days of lactation during long photoperiod (May, LD, n = 5) and short photoperiod (December, SD, n = 5). After rearing lambs to 30 days of age, ewes were decapitated and the pituitaries were dissected and then cut along the longitudinal fissure into two halves, so that each half contained the glandular and nervous parts. Pituitary glands were collected and divided along the longitudinal fissure into two halves. Glands were incubated for 3 hours at 37°C in Parker medium with addition of orexin A—experimental group or in medium alone—control group. During the following 3-hour incubation, medium was exchanged every 15 minutes and a sample of 1 ml was collected and immediately frozen at −80°C until assay. Prolactin concentrations in the medium were determined

In the long-day conditions (May), the pituitary explants of lactating sheep exhibited the strongest secretory activity during the first hour of incubation—significantly higher in orexintreated group (O1) than the control group (K1), (*P* < 0.01). During the second hour of the incubation, PRL concentration decreased and reached the similar values in both groups. During the third hour, PRL concentration in O1 group was again significantly higher than that noted in K1 group (*P* < 0.01). In the short-day conditions (December), PRL concentration was significantly higher in orexin-treated group O2 during the first hour of incubation than the value observed in the control group—K2 (*P* < 0.01). The inverse relationship in prolactin release was observed during the second hour of incubation (*P* < 0.01), however, during the third hour, PRL concentration was again significantly higher in O2 group than the concentration noted in K2 group (*P* < 0.05). Collective analysis of the data showed that PRL concentrations were higher in experimental groups (O1 and O2) than the concentrations noted in control groups (K1 and

K2) under both the long (May) and short (December) photoperiods (**Figure 4**).

of milk production in sheep, especially following the nursing period.

group (6.58 ± 2.36 ng/ml).

32 Current Topics in Lactation

**3.1. Role of orexin**

radioimmunologically (RIA).

**Figure 4.** Mean concentrations of prolactin in control and orexin A-treated pituitary explant cultures during long-day (LD) and short-day (SD) photoperiods. See text for statistical comparisons.

GH release from the pituitary explants during the long-day conditions was maintained on significantly higher level in orexin-treated group O1 than control K1 group (*P* < 0.05), throughout the whole period of the incubation. In contrast, during the short-day period, GH release from the explants was significantly less in orexin-treated group O2 than that in the control K2 group (*P* < 0.05). The suppressive effect of orexin was observed during 2 hours. Collective analysis of the data showed that GH concentrations were higher under long-day conditions than under short-day conditions (**Figure 5**).

**Figure 5.** Mean concentrations of growth hormone in control and orexin A-treated pituitary explant cultures during long-day (LD) and short-day (SD) photoperiods. See text for statistical comparisons.

The results of experiments performed on lactating sheep, i.e., animals with strong seasonality characteristics, are difficult to compare with others, however, in ewes as in rodents, orexin A is able to stimulate PRL secretion due to its direct effect on the lactotropic cells. The slight response of ovine pituitary glands to orexin during short days is probably due to the insensitivity of lactotropic cells to the orexin signal. The initiation and maintenance of lactation in sheep require the presence of many hormones, where PRL and GH seem to be the most important. Studies on lactating sheep showed that the ewes starting lactation during the period of increasing day length produced 50% more milk compared with sheep milked during the decreasing day length [15]. When June-lambed ewes were kept under artificial conditions of the long day (16L:8D), PRL level decreased as the natural length of a day became shorter. The fact that pituitary cells become refractory to over-repeated summer signal of the darkness hormone (melatonin) makes it impossible to lengthen lactation in sheep in the autumn-winter period [15]. Determining the role of orexins, especially orexin A, in regulating prolactin secretion may help to clarify the process of lactation maintenance in sheep, especially during the decreasing photoperiod. In conclusion, our results obtained on the pituitary explants demonstrated that the pituitary tissue of lactating sheep was sensitive to photoperiod and orexin A. We conclude that the secretion of PRL and GH from the ovine pituitary gland is negatively responsive to orexin A during SD, whereas orexin may stimulate PRL and GH secretion during LD. Further studies investigating orexin—PRL and GH interactions are needed.

#### **3.2. Role of TRH**

Recently, it was observed that TRH has a role to play in the initiation and maintenance of lactation in small ruminants. The aim of the performed study was to determine the impact of the TRH factor on secretion of prolactin in lactating sheep. In vitro studies were carried out on 10 animals. The pituitary gland of each sheep was collected at day 40 of lactation. In vitro incubations were performed on 12 microwell plates in Parker medium for 1 hour at 37°C. One half of the gland was incubated in pure Parker medium (control group), while the second (test group) half was incubated in Parker medium conditioned with exogenous TRH (TRH concentration—36 ug/100 ml medium). The medium was administered every 15 minutes and collected from the wells; in each case, 1 ml of medium was administered. The first 15 minutes served as blank and both halves of the pituitary remained in the same medium; the aim was to stabilize the secretory function of lactotropic cells. Prolactin measurements were made using RIA method. The tests carried out have demonstrated a stimulating impact of the TRH factor on secretion of prolactin. In the first 15 minutes of incubation, PRL concentration in the control group was 81.83 ± 11.4 pg/ml and was significantly (*P* ≤ 0.05) lower than the concentration (87.48 ± 11.6 pg/ml) observed in the test group. After 30 minutes of incubation, the control group showed significantly (*P* ≤ 0.05) lower prolactin level (74.04 ± 10.03 pg/ml) than the group with TRH-enriched medium (79.9 ± 10.6 pg/ml). After 45 minutes of incubation, the concentration of PRL in the control group was 59.66 ± 9.4 mg/ml and it was significantly (*P* ≤ 0.01) lower than that in the experimental group (10.2 ± 65.47 mg/ml) (**Figure 6**).

Role of Melatonin and the Biological Clock in Regulating Lactation in Seasonal Sheep http://dx.doi.org/10.5772/66208 35

**Figure 6.** Mean concentrations of prolactin in control and TRH-treated pituitary explant cultures during long-day. See text for statistical comparisons.

#### **3.3. Role of ghrelin**

The results of experiments performed on lactating sheep, i.e., animals with strong seasonality characteristics, are difficult to compare with others, however, in ewes as in rodents, orexin A is able to stimulate PRL secretion due to its direct effect on the lactotropic cells. The slight response of ovine pituitary glands to orexin during short days is probably due to the insensitivity of lactotropic cells to the orexin signal. The initiation and maintenance of lactation in sheep require the presence of many hormones, where PRL and GH seem to be the most important. Studies on lactating sheep showed that the ewes starting lactation during the period of increasing day length produced 50% more milk compared with sheep milked during the decreasing day length [15]. When June-lambed ewes were kept under artificial conditions of the long day (16L:8D), PRL level decreased as the natural length of a day became shorter. The fact that pituitary cells become refractory to over-repeated summer signal of the darkness hormone (melatonin) makes it impossible to lengthen lactation in sheep in the autumn-winter period [15]. Determining the role of orexins, especially orexin A, in regulating prolactin secretion may help to clarify the process of lactation maintenance in sheep, especially during the decreasing photoperiod. In conclusion, our results obtained on the pituitary explants demonstrated that the pituitary tissue of lactating sheep was sensitive to photoperiod and orexin A. We conclude that the secretion of PRL and GH from the ovine pituitary gland is negatively responsive to orexin A during SD, whereas orexin may stimulate PRL and GH secretion during LD. Further studies investigating orexin—PRL and GH interactions are

Recently, it was observed that TRH has a role to play in the initiation and maintenance of lactation in small ruminants. The aim of the performed study was to determine the impact of the TRH factor on secretion of prolactin in lactating sheep. In vitro studies were carried out on 10 animals. The pituitary gland of each sheep was collected at day 40 of lactation. In vitro incubations were performed on 12 microwell plates in Parker medium for 1 hour at 37°C. One half of the gland was incubated in pure Parker medium (control group), while the second (test group) half was incubated in Parker medium conditioned with exogenous TRH (TRH concentration—36 ug/100 ml medium). The medium was administered every 15 minutes and collected from the wells; in each case, 1 ml of medium was administered. The first 15 minutes served as blank and both halves of the pituitary remained in the same medium; the aim was to stabilize the secretory function of lactotropic cells. Prolactin measurements were made using RIA method. The tests carried out have demonstrated a stimulating impact of the TRH factor on secretion of prolactin. In the first 15 minutes of incubation, PRL concentration in the control group was 81.83 ± 11.4 pg/ml and was significantly (*P* ≤ 0.05) lower than the concentration (87.48 ± 11.6 pg/ml) observed in the test group. After 30 minutes of incubation, the control group showed significantly (*P* ≤ 0.05) lower prolactin level (74.04 ± 10.03 pg/ml) than the group with TRH-enriched medium (79.9 ± 10.6 pg/ml). After 45 minutes of incubation, the concentration of PRL in the control group was 59.66 ± 9.4 mg/ml and it was significantly (*P* ≤ 0.01)

lower than that in the experimental group (10.2 ± 65.47 mg/ml) (**Figure 6**).

needed.

**3.2. Role of TRH**

34 Current Topics in Lactation

The process of initiation and maintenance of lactation in sheep requires the presence of a number of hormones. The aim of this study was to determine the role of ghrelin in the regulation of prolactin secretion in lactating sheep, based on the culture of in vitro pituitary. The study was conducted in May—long-day period. Pituitary was collected from 10 sheep on day 30 of lactation and divided along the longitudinal grooves so that each contains half of the glandular part and nerves. Incubations were carried out in vitro pituitary in 12 well plates for 1 hour at 37°C. The control group was incubated in a clean Parker medium and experimented in medium supplemented with exogenous ghrelin. The concentration of prolactin in the medium was determined by RIA method. The study showed stimulatory effect of ghrelin on the secretion of prolactin. The tests demonstrated a modulating effect of ghrelin on secretion of prolactin. Significant (*P* ≤ 0.05) increase in prolactin secretion after 30 minutes of incubation in the test group (89.6 ± 18.1 mg/ml) compared with the control group (73.6 ± 17.4 mg/ml) was noted. After 45 minutes of incubation, the concentration (69 ± 15.2 mg/ml) of prolactin in the test group was significantly (*P* ≤ 0.05) lower than the concentration (77.2 ± 17.6 mg ml) in the control group. After 60 minutes, prolactin level was significantly lower at *P* ≤ 0.05 in the test group (46.3 ± 8.4 mg/ml) than that in the control group (51.8 ± 9.6 mg/ml) (**Figure 7**). The results of studies conducted have demonstrated a modulating impact of ghrelin on secretion of prolactin. While increase in prolactin secretion during the incubation period was observed, reduction in prolactin secretion has been recorded in the test group. Administration of exogenous ghrelin during the period of physiologically high prolactin concentration in lactating sheep has not given a clear answer as to whether ghrelin stimulates the secretion of prolactin. The results suggest, therefore, that ghrelin does not directly affect the secretion of prolactin from the pituitary. The hitherto obtained test results showed that the effects of ghrelin may be dependent on the species of animals. In the case of sheep, seasonal breeders, the mechanism of ghrelin activity is complicated. As revealed by the studies in lactating sheep, administration of exogenous ghrelin modulates the secretion of prolactin.

**Figure 7.** Mean concentrations of prolactin in control and ghrelin-treated pituitary explant cultures during long day. See text for statistical comparisons.

### **4. Summary**

In seasonal animals, the process of triggering and maintaining lactation requires numerous hormones. The interaction of growth factors and other hormones is necessary in processes such as mammogenesis, lactogenesis and galactopoiesis. Due to the proper synchronization of pregnancy and changes in the area of the mammary gland, the gland is ready for the production of milk at the moment the offspring is born. Mammogenesis is a phenomenon that requires the participation of a number of hormones, including prolactin (PRL), growth hormone (GH), estrogens, progesterone, oxytocin, placental lactogen (PL) and insulin-like growth factor (somatomedin, e.g., IGF1). The coparticipation of IGF and GH is necessary in coordinating the differentiation and proliferation of epithelial cells. The manner in which the growth factors stimulate or inhibit the growth of cells or their influence on the cell cycle is not fully understood. The role of IGF in particular stages of functioning of the mammary gland (mammogenesis, lactogenesis, galactopoiesis and desiccation), particularly in the case of ruminants, is highly complicated. Recently, attention has been given to the metabolic hormones, particularly the role of leptin, orexin and ghrelin in mammogenesis, lactogenesis and galactopoiesis, respectively. Due to the recently increased interest in sheep's milk products, an understanding of the endocrine mechanisms facilitating the maintenance of lactation during autumn and winter may contribute to the improved profitability and usefulness of sheep's milk.
