WO2021013951A1 - Veterinary compositions comprising melatonin and their uses for ruminants - Google Patents

Abstract

The present invention concerns a veterinary composition comprising melatonin to be administered to ruminants. This composition comprising melatonin is particularly useful in a method for favoring mammary involution, improving milk yield, and/or treating ketosis, and/or improving glucose metabolism.

Description

Veterinary compositions comprising melatonin and their uses for ruminants

The present invention concerns a veterinary composition comprising melatonin to be administered to ruminants. This composition comprising melatonin is particularly useful in a method for favoring mammary involution, improving milk yield, and/or treating ketosis, and/or improving glucose metabolism.

Background of the invention

The transition from pregnant non-lactating to lactating represents a challenge for the majority of dairy cows because of a high incidence of metabolic diseases. The most frequent metabolic diseases include hypocalcemia and ketosis (especially in their subclinical forms). Subclinical ketosis and hypocalcemia can affect >40% of cows in a dairy herd with a cost of approximately 150 euros for each case.

Glucose metabolism is particularly affected in dairy cows during the transition from late pregnancy to early lactation. In late pregnancy and during early lactation, the udder is the most important glucose consumer (50% to 85% of whole body glucose consumption), because of the high energy needed for milk production. While glucose uptake in the mammary gland is insulin- independent, this does not occur in other peripheral tissues such as adipose, muscular, and reproductive tissue. This is particularly important considering that during the peripartum period there is a decreased peripheral insulin sensitivity to facilitate the biosynthetic activities of the mammary gland by preserving available glucose for lactose synthesis and by promoting mobilization of endogenous energy reserves through massive lipolysis. Under these conditions, lipolytic mechanisms in adipose tissue are activated and there is an important increase in non- esterified fatty acids (NEFA) and, if insufficient glucose precursors are available, ketone bodies (ketosis).

During early lactation, cows frequently undergo a negative energy balance followed by an increase in mobilization of body reserves. As a result, non-esterified fatty acids (NEFAs) are released from the adipose tissue into the bloodstream, which are metabolized primarily in the liver to ketone bodies. The increase in NEFAs and ketone bodies leads to an increase in formation of reactive oxygen species and hepatic steatosis. Subclinical ketosis is defined as concentrations of b-hydroxybutyrate (BHBA) >1.2 to 1.4 mmol/L and it is considered a gateway condition for other metabolic and infectious disorders such as metritis, mastitis and displaced abomasum.

Melatonin (5-methoxy-N-acetyltryptamine) is an indoleamine that was first isolated in the late fifties from the pineal gland (where is mainly produced) of a cow. It is produced by conversion of tryptophan onto serotine (5-hydroxytryptamine) which is then converted to melatonin by the hydroxyindole-O-methyltransferase. The retina sends circadian information (hours of light or dark exposure) to the suprachiasmatic nucleus of the hypothalamus, which during exposure to dark releases norepinephrine which stimulates the secretion of melatonin from the pinealocytes. Once released onto the blood stream, in humans, melatonin experiences a bi-exponential decay, with a fraction of melatonin having a half-life of about 2 min, and the remaining fraction about 20 min. The action of melatonin in the body is mediated by its direct stimulation of two melatonin membrane (MTi and MT2) receptors. These receptors are mainly located in the central nervous system, oocytes, mammary gland, retina, liver, kidney, skin, and the immune system. Interestingly, melatonin has been shown to exert a down-regulation on its receptors.

The exposure to short-day photoperiods (long hours of darkness) during the dry period (or pre- partum period) have been shown to increase melatonin secretion and in turn reduce circulating prolactin (PRL) and increase prolactin receptor (PRLr). These changes have led in some instances to subsequent improvements in milk yield (Velasco et ah, 2008). In Lacasse et al. (2014), a daily oral administration of 25 mg of melatonin per day during 8 weeks preceding calving resulted in a slight decrease in blood PRL concentrations in cows exposed to a long- day photoperiod (but did not reach the levels observed in cows exposed to a short-day photoperiod). Melatonin supplementation had, however, no effect on blood PRL concentrations, feed intake, or milk production after calving in multiparous cows, although blood PRL was greater after calving in both, primiparous cows exposed to a short-day photoperiod or a long-day photoperiod plus oral melatonin (Lacasse et al., 2014). Administration of melatonin implants to dairy cows moderately suppressed pre-partum blood PRL concentrations but did not affect milk production in the subsequent lactation (Garcia- Ispierto et al., 2013).

On the other hand, the increased expression of PRLr observed in the lymphocytes of animals exposed to short-day photoperiods have been linked with improved immune function. In addition, melatonin is a neuro-hormone that possesses powerful antioxidant properties and is capable of scavenging oxygenous and nitrogenous free radicals (Bibak et al., 2014) in both animals and plants and thus it may also improve immune and hepatic functions (Kleber et al, 2014). Some studies have, in fact, reported that short-day photoperiod at dry-off has resulted in improved immune response (Nelson et al., 1995).

Interestingly, melatonin is also produced in the gastrointestinal tract by enterochromaffin cells located in the epithelium of the digestive tract, and it has been related to gastrointestinal cell proliferation and regulation of food intake in several mammals (Bubenik et al., 1996). In fact, melatonin concentrations in the rumen, jejunum, and ileum can be 5 to 10 times greater than those found in blood (Bubenik et al., 1999) and its concentration in the gut is independent of the presence of the pineal gland (Bubenik and Brown, 1997). In general, melatonin decreases food intake in diurnal species such as mouse and humans. In cattle, exposure to darkness or dim light has been associated with decreased intake, but it has been associated with increased intake in dairy cattle during the dry period (Auchtung et al., 2005).

Melatonin seems to play a role in the glucose metabolism via altering insulin secretion and modulating the activity of insulin receptors, and thus it could also affect food intake through insulin-dependent mechanisms. However, as it occurs with intake, the bibliography in this regard is contradictory. For instance, melatonin injections in rats have been shown to reduce circulating levels of insulin, glucose, and triglycerides (R os-Lugo et al., 2010). However, melatonin has also been reported to increase the sensitivity of insulin-dependent glucose transporters (Ha et al., 2006). On the other hand, other authors have reported that administration of melatonin impairs insulin sensitivity and glucose tolerance (Rubio-Sastre et al., 2014), although Eckel et al. (2015) reported that the reduced insulin sensitivity is compensated by an increased amount of insulin secretion in response to glycaemia (which is contradictory to previous studies reporting decreased insulin secretion).

It has been found here that administration of melatonin to dairy cows at the day of halting milk removal and during most of the dry period improves glucose availability and metabolism in lactating cows and it also reduces some post-partum metabolic and health afflictions. Said administration composition to ruminants has also revealed to be particularly advantageous as it allows a significant improvement of milk yield, of mammary involution and/or remodeling, improvement of glucose metabolism as well as it has a positive impact on the treatment or prevention of ketosis, notably by diminishing blood beta-hydroxybutyrate (BHBA) concentrations. SUMMARY OF THE INVENTION

This invention relates to a veterinary composition comprising melatonin for improving milk yield, and/or promoting mammary involution, and/or treating ketosis and/or improving glucose metabolism, wherein the composition is administered via intramuscular, subcutaneous, intravaginal, intranasal administration or injection, wherein the dose administered ranges between 150 and 300 mg per ruminant.

According to the invention, this composition is administered in therapeutically effective amounts, as a single treatment, in order to increase milk yield, promote mammary involution, regenerate tissue in the mammary gland, and/or treating ketosis and/or improving glucose/insulin metabolism.

The composition thus provides a significant improvement of the welfare of ruminants, and is particularly useful in mammary involution in ruminants, in the treatment and/or the prevention of ketosis, and ultimately of intra-mammary inflammations and intra-mammary infections of ruminants.

The present invention further relates to methods for increasing milk yield, for inducing mammary involution or tissue regeneration of the mammary glands, or for improving glucose/insulin metabolism, in ruminants, as well as methods of prevention and/or treatment of ketosis, by administering said composition, in a single administration or injection.

The present invention lastly relates to kits comprising such veterinary composition.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1: Dry matter intake (kg/day) throughout the study (from -60 days to +56 days relative to calving) of control (black circle) and MEL (grey circle) cows on different treatments, as detailed in the examples.

Figure 2: Milk yield (kg/day) from day 1 to day 58 after calving of control (black circle) and MEL (grey circle) cows on different treatments, as detailed in the examples.

Figure 3: Glucose (mg/L) (A) (C) (E) and insulin (pg/L) (B) (D) (F) concentrations in blood as a response to a glucose tolerance test (GTT) as affected by day relative to calving and treatment (Control = Black circle; MEL = Grey and cross). Glucose infusion (GTT) at Day -50 (Fig. 3(A) and 3(B)) and Day -20 (Fig. 3(C) and 3(D)) and Day +10 (Fig. 3(E) and 3(F)) with respect to the calving day. Glucose and insulin concentration are measured several times (minutes) starting from glucose infusion. Blood was harvested at 0, 4, 8, 12, 18, 25, 35, 45, 60, 90, and 180 minutes (x axis) after glucose infusion (i.e. 0 minute) to determine glucose and insulin concentrations.

Figure 4: Relative gene expression of selected genes in the mammary gland as affected by physiological state and treatment (Shaded area denotes lactation). ■ 10 days after the day of halting milk removal (control); ® 30 days after calving (control); 1 10 days after the day of halting milk removal (melatonin treatment); ^ 30 days after calving (melatonin treatment). Figure 5: Blood NEFA concentration (mM) as affected by melatonin - control: black circle; Melatonin treatment: grey circle.

Figure 6: Relationships between blood BHBA (mM) at 30 DIM and body weight (BW) loss during the first 30 DIM as affected by treatment (Control: Black, MEL: Grey).

Figure 7: Blood growth hormone concentration (ng/mL) as affected by melatonin (Control: Black circle, MEL: Grey circle).

Figure 8: Blood prolactin concentration (ng/mL) as affected by melatonin (Control: Black circle, MEL: Grey circle).

DETAILED DESCRIPTION OF THE INVENTION

This invention relates to a veterinary composition comprising melatonin or use thereof for improving milk yield, and/or promoting mammary involution, and/or treating ketosis and/or improving glucose metabolism in a lactating ruminant, wherein the composition is administered via intramuscular, subcutaneous, intravaginal, or intranasal administration or injection, wherein the released melatonin dose ranges between 150 and 300 mg per ruminant, wherein the composition is administered to the lactating ruminant the first day of the dry period and the melatonin is released during at least 60 consecutive days starting from the said first day of dry period.

The invention also relates to a method for improving milk yield, and/or promoting mammary involution, and/or treating ketosis and/or improving glucose metabolism of a lactating ruminant, wherein a composition comprising melatonin is administered in a sufficient amount to the lactating ruminant via intramuscular, subcutaneous, intravaginal, or intranasal administration or injection, wherein the administered melatonin dose is from 150 to 300 mg per ruminant, and wherein the composition is administered to the lactating ruminant the first day of the dry period and the melatonin is released during at least 60 consecutive days starting from the said first day of dry period. This present invention further relates to the use of melatonin for the preparation of veterinary compositions to be administered to ruminants for improving milk yield, and/or promoting mammary involution, and/or treating ketosis and/or improving glucose metabolism of a ruminant, wherein the composition is administered via intramuscular, subcutaneous, intravaginal, or intranasal administration or injection, and wherein the administered melatonin dose is from 150 to 300 mg per ruminant, and wherein the composition is administered to the lactating ruminant the first day of the dry period and the melatonin is released during at least 60 consecutive days starting from the said first day of dry period.

This invention relates to the usage of the said veterinary composition administered in sufficient therapeutic quantities to lactating ruminants so that the quantity of released melatonin is from 150 to 300 mg, preferably from 170 to 250 mg, and more specifically at 216 mg, per gestating ruminant and for at least 60 consecutive days as of the first day of a dry period.

In ruminants, lactation generally lasts between 5 and 20 months. In dairy cows it is most often 10 months. After this lactation period, milking is stopped, generally suddenly, and the ruminant produces no more milk until calving, when the subsequent lactation begins. This period between the day of halting milk removal and the day of subsequent calving (and onset of lactation), known as the dry period, is generally set at about 1.5-2.5 months (i.e. 45-75 days) in dairy cows.

According to the invention, the melatonin containing composition is administered to the lactating ruminant the first day of the dry period, i.e. from the day of halting milk removal, and the melatonin is then released during at least 60 consecutive days starting from the said first day of dry period, and preferably the melatonin is released until the end of the dry period. More specifically, the melatonin release time period lasts from 60 days to 70 days.

According to the present invention, the composition is preferably administered during the gestation period of the ruminants.

The composition of the invention promotes milk yield, mammary involution and/or health, treats ketosis, or improves glucose metabolism of a ruminant. It acts on successive hormonal, physiological or morphological changes that improve the udder health and the overall welfare or health of the ruminants, not only during the dry period but also after calving. According to a particular embodiment, the composition is an extended-release melatonin composition, so as to administer from 150 to 300 mg of melatonin per ruminant for an extended period of time as detailed above. Preferably melatonin release occurs along a time period starting from the first day of the dry period and lasting until the end of the dry period of the lactating ruminant. More specifically, melatonin is released for a time period ranging from 60 consecutive days to 70 consecutive days.

The extended-release melatonin composition is preferably at least one implant, at least 3, 6, or 9, or 12 implants. Said at least one implant is preferably placed subcutaneously under the skin, by surgery or injection.

According to particular embodiment, the administered composition allows to have a daily blood melatonin concentration above 60 pg/mL, and preferably above 130 pg/mL.

The veterinary composition may further comprise pharmaceutical conventional ingredients for the preparation of liquid formulations to be used via intramuscular, subcutaneous, intravaginal or intranasal administration or injection. The injectable preparations are prepared by mixing melatonin as described previously with a solvent, a pH regulator, a buffer agent, a suspending agent, a solubilization agent, a stabilizer, an agent of tonicity and/or a preservative, and by transforming the mix with a traditional process for subcutaneous or intramuscular injection. Examples of solvents are oily solvents such as medium chain triglycerides in C8-C10, or a mix of capric acid, caprylic acid, and triglycerides, such as those marketed under the name of Mygliol®812. Injectable preparations can be freeze-dried according to a traditional process.

Examples of suspending agents include methylcellulose, polysorbate 80, hydroxyethyl cellulose, xanthan gum, sodium carboxymethyl cellulose, polyethylene sorbitan monolaureate. Examples of solubilization agents include ricin oil solidified by polyoxyethylene, polysorbate 80, nicotinamide, polyethoxylated sorbitan monolaurate, macrogol and ester ethyl fatty ricin acid. Besides, the stabilizing agent includes sodium sulfate, sodium metasulfate and ester, while the preservative includes methyl p-hydroxybenzoate, ethyl p-hydroxybenzoate, sorbic acid, benzyl alcohol, phenol, cresol and chlorocresol. One example of the agent of tonicity is mannitol. During the preparation of solutions and injectable suspensions, it is better to ensure that they are isotonic with blood. Advantageously, the veterinary composition of the present invention can be administered in association with standard treatments of mastitis of ruminants. Examples of standard care or prophylactic compositions of mastitis are local disinfectants for udders, antibiotics such as penicillins of group M, cephalosporine, gentamycin or colistine or even enzymes, such as lysozymes or muramidase.

The veterinary composition comprising melatonin is administered via intramuscular, subcutaneous, intravaginal, or intranasal administration or injection. Preferably, the composition is administered subcutaneously or intramuscularly, and more specifically subcutaneously.

According to the invention, the composition is for use to improve glucose metabolism, giving rise to a better glycemic status of the ruminant. More specifically, the composition is for use to improve glucose metabolism during the dry period and within the first 3 to 15 days after calving, more particularly during the transition period, i.e. from late pregnancy to early lactation. Interestingly, the composition used according to the invention allow to obtain a greater capacity of secreting greater amounts of insulin. Moreover, the composition used according to the invention allows to have blood glucose levels returned back to baseline values earlier and to have a greater production of insulin when compared to untreated ruminants (preferably cows). Furthermore, the composition used according to the invention allows to have an improvement of the response to insulin.

According to the invention, the composition is for use to improve milk yield. More specifically, the composition used according to the invention increases milk production, especially within the first consecutive 60 days in milk after calving of the treated ruminants. According to a particular embodiment, the use of the composition according to the invention reduces milk fat content. According to a particular embodiment, the use of the composition according to the invention reduces milk urea content, especially within the first 3 to 15 days after calving. Interestingly, this reduction of milk urea content is an indication of an improvement of the nitrogen metabolism, most likely due to a lower utilization of amino acids as glucose sources and/or lower mobilization of amino acid reserves in the body (i.e. albumin or muscle protein).

According to the invention, the composition is for use to promote mammary involution or mammary health of the ruminant. More specifically, the used composition according to the invention allows regeneration of the tissue of the mammary glands, in particular by lowering senescence or improving cell renovation at the mammary level. Such regeneration of the tissue is most likely due to a reduction of the expression of sirtl in the mammary gland after calving and/or reduction of somatic cell count during the first 30 days of lactation.

According to the invention, the composition is for use to treat or prevent ketosis, more preferably subclinical forms of ketosis. More specifically, the composition as used in the invention reduces ketone bodies or prevent apparition of ketone bodies in adipose tissue of the ruminant, and more specifically in mammary glands.

The veterinary composition according to the invention allows promotion of the welfare of the ruminants. More specifically, the melatonin treatment according to the invention allows to:

Increase feed intake, especially after about 4 weeks from calving;

Increase milk production, notably due the reduction of the expression of IGFBP5 in the mammary gland;

Reduce the content of urea in milk. Such lower milk urea content is an indication of an improvement of nitrogen metabolism (this improvement is most likely due to a lower utilization of amino acids as glucose sources and/or a lower mobilization of amino acid reserves in the body);

Improve the amount of insulin release against a glucose challenge or glucose tolerance test during the dry period, and improves insulin response after calving;

Increase the speed at which blood concentrations of insulin peak after a glucose challenge during the dry period;

Increase the speed to reach basal blood glucose concentrations after a glucose challenge, especially after calving;

Reduce blood concentrations of BHBA (an indication of better glucose availability of the ruminant, in particular cow, to metabolize NEFA);

Reduce the expression of IGFBP5 in the mammary gland;

Regenerate tissue of the mammary glands, in particular by lowering senescence or improving cell renovation at the mammary level. Such regeneration of the tissue is most likely due to a reduction of the expression of sirtl in the mammary gland after calving. Reduce somatic cell count during the first 30 days of lactation, which is an indication of improved udder health. Due to these advantageous effects, the composition according to the invention can be useful for the treatment and/or the prevention of intra-mammary diseases and/or infections of ruminants.

The ruminants according to the invention are herbivorous mammals chosen among bovine, ovine, caprine, or camelids, preferably ruminants are pregnant dairy cows or pregnant heifers.

Lastly, the invention discloses a veterinary kit comprising a veterinary composition as defined above, which is particularly useful to improve milk yield, and/or promote mammary involution, and/or treat ketosis and/or improve glucose metabolism in a ruminant. According to a specific embodiment, the kit further comprises an instruction sheet on the mode of operation and mode of administration of the veterinary composition, as detailed herein. More specifically the kit further comprises an instruction sheet on the mode of operation and mode of administration of the veterinary composition to improve milk yield, and/or promote mammary involution, and/or treat ketosis and/or improve glucose metabolism in a ruminant. The kit can further comprise means of administering the compositions subcutaneously, parenterally, vaginally, or nasally, as well as an instruction sheet concerning the mode of administration of the veterinary composition of the present invention. According to a particular embodiment, the kit comprises one or more implants containing melatonin, as detailed above.

The invention will be better understood by reference to the Experimental Details which follow, but those skilled in the art will readily appreciate that the specific experiments detailed are only illustrative, and are not meant to limit the invention as described herein, which is defined by the claims which follow thereafter.

Throughout this application, various references or publications are cited. Disclosures of these references or publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this invention pertains. It is to be noted that the transitional term“comprising”, which is synonymous with“including”, “containing” or“characterized by”, is inclusive or open-ended and does not exclude additional, un-recited elements or method steps.

EXAMPLES Methods

Thirty lactating cows («=15) with >220 days of pregnancy were split in 2 treatments: Control and Melatonin (MEL; 12 implants of 18 mg each, equivalent to a dose of 216 mg melatonin). All cows were fed and managed in the same manner in both treatments. The implants were placed the first day of the dry period, in the two sides of the neck of the cows in sets of 6 implants per puncturing spot. The melatonin was thus released from the first day of the dry period and in a time period which lasts from 60 days to 70 days. All lactating cows were dried- off between 15th of August and 26 of September, 2017, which coincided with the decreasing phase of the long-day photoperiod ranging from 13 h and 50 min to 11 h and 58 min of day light at the day of halting milk removal, and decreasing to 11 h and 44 min (on October 1st) and 10 h and 9 min (November 7th) at calving. Thus, cows in the current study were dried during the last part of the long-day and the beginning of the short-day photoperiod, and calved during a short-day photoperiod. The calving of the tested cows occurred between 45 and 75 days from the first day of the dry period, depending on each cow and/or farmer.

Measurements

• Initial body weight (BW)

• Individual feed intake throughout the study.

• Glucose tolerance test (on each cow per treatment) at: 10 days (d), and 40 d after the day of halting milk removal (Day -50 and Day -20 relative to calving, respectively) and 10 d after calving (Day +10). Basically, 200 mg/kg of BW of glucose were infused to each cow using a catheter and then blood was harvested at 0, 4, 8, 12, 18, 25, 35, 45, 60, 90, and 180 minutes after glucose infusion to determine glucose and insulin concentrations.

• Mammary biopsies (on 10 animals per treatment) at: 10 d before and 30 d after calving to determine expression of prolactin receptor ( PRLr ) and insulin growth factor-I binding protein 5 (IGFBP5 ) and markers of mammary involution and evolution (bax, cas3, sirtl , ki67, and pi 6).

• Blood samples at: 5, 30, 50 d after the day of halting milk removal and 1, 3, 10, and 30 d after calving to determine: insulin growth factor I (IGF-1), prolactin (PRL), and growth hormone. • Blood samples at: 0, 5, 30, 50 d after the day of halting milk removal and 1, 3, 10, and 30 d after calving to determine: non-esterifred fatty acids (NEFA) and B-OH-butyrate (BHBA).

• Uterine inflammation status (10 animals per treatment) on day 5 after calving via uterine biopsy to determine gene expression for interleukin 8 ( IL-8 ), interleukin 10 (IL-10), and interleukin IB ( IL-lfi ).

• Milk production (fat and protein) after calving during the first 60 days in milk (DIM).

Calculations and statistics

Insulin sensitivity was determined by two different methods. One method followed Galvin et al. (1992), who prosed the following equation:

Si = K_G/(AUClns/T)

where, K_G is the rate of glucose disappearance (i.e., the slope of the log of blood glucose concentration between 4 and 60 min following the GTT), AUCi_ns is the area under the curve of blood insulin concentration (subtracting the basal blood insulin levels) for the period between 4 and 60 min, and T is the difference between 4 and 60 (i.e., 56 min). The area under the curve of blood insulin concentrations relative to basal levels of insulin were calculated using the trapezoidal method.

The second method to determine insulin sensitivity and glucose effectiveness was based on a simplified alternative to the minimal model (Bergman, 1989) following Christoffersen et al. (2009). The glucose model consists of two differential equations:

dG/dt = -fo x [G( - G_b\ - X(t) x G{t) dX/dt = fo _x [/(/) - /_b] - fo x X(t) where, G is the concentration of blood glucose as a function of time and dG/dt is its rate of change, I is the concentration of insulin in the blood as a function of time, G_b is the basal concentration of blood glucose and fo is the basal concentration of blood insulin, X is the concentration of insulin in the blood as a function of time, and dX/dt is its rate of change. Lastly, fo, fo, and fo are parameters that model the rates of appearance and disappearance for glucose and insulin. Glucose effectiveness (the ability of glucose per se to stimulate its own uptake and to suppress its own production at a given insulin concentration) was also determined using the minimal model.

Lastly, the clearance rates of glucose (%/min; GCR) and glucose half-time (min, T1_/2) were computed following Jaakson et al. (2017) Data were analyzed using a mixed-effects model that accounted for the fixed effect of treatment, day of study, and their 2-way interaction, plus the random effect of cow and block (i.e., batch of cows included in the study according to their expected day of calving). Data that were monitored daily were analyzed using a compound symmetry structure. Data that were taken at different time points (not equally spaced) were analyzed using a SP(GAU) structure for unequally spaced observations.

Results

Table 1 shows the results for performance. Intake was affected by an interaction between treatment and day of study (Figure 1). Before calving there were no differences in dry matter intake (DMI) between Control and MEL cows; however, after calving, cows on MEL consumed more dry matter than Control after about 30 days in milk (Figure 1). As a result of numerical differences in DMI before calving and the greater DMI after about 30 DIM, MEL cows, overall, tended (P = 0.06) to consume more feed than Control cows.

Milk production tended (P = 0.08) to be greater in MEL than in Control cows. The evolution of milk production during the first 60 DIM is depicted in Figure 2. Milk protein content did not differ among treatments, but milk fat content was lower in MEL than in Control cows. As a result, there were no differences in Energy Corrected Milk (ECM) between the 2 treatments. Interestingly, milk urea at days 3 and 10 after calving was lower ( P < 0.05) in MEL than in Control cows (Table 1). A lower milk urea content is an indication of an improve nitrogen metabolism due to a lower utilization of amino acids as glucose sources and/or lower mobilization of amino acid reserves in the body (i.e. albumin or muscle protein).

Table 1. Performance of Holstein cows throughout transition as affected by melatonin.

Treatment -value¹

Control MEL SE T D TxD

DMI before calving, kg/d 13.9 Ϊ5T5 L96 024 <0.01 029 DMI after calving, kg/d 22.0 23.8 1.14 0.11 <0.01 0.03 Overall DMI, kg/d 18.8 20.4 0.84 0.06 <0.01 0.35 BW at calving, kg 664 686 16.8 0.41

BW loss in first 30 DIM, kg 25.9 35.8 9.56 0.33

Milk yield, kg/d 36.7 41.0 2.51 0.08 <0.01 0.17 Milk fat, % 3.87 3.62 0.10 0.04 <0.01 0.06 Milk protein, % 3.54 3.52 0.04 0.79 <0.01 0.96 ECM,² kg/d 39.3 42.7 2.53 0.19 <0.01 0.83 Milk urea,³ mg/1 157.3 86.0 19.4 0.04 0.93 0.72

¹ T: Effect of treatment; D: Effect of day; TxD: Effect of the interaction between treatment and day.

² ECM: Energy corrected milk calculated following (NRC, 2001).

³ Milk urea was determined only at days 3 and 10 after calving.

Blood glucose levels did not differ along the glucose tolerance tests (GTT) performed among different physiological stages nor treatments (Figure 3). However, insulin response was different in MEL than in Control cows (Table 2; Figure 3).

Table 2 shows the results from the GTT. All parameters, except for glucose effectiveness were influenced by the physiological state of the cow. Blood glucose basal levels were greater in MEL than in Control cows (Table 2), which indicates a better glycemic status in MEL than in Control cows. As expected, peak glucose concentrations after the GTT were lower (P < 0.01) after calving (130.1+2.85 mg/dl) than before calving (156.4+2.80 mg/dl), independently of treatment. Blood peak insulin concentrations tended to be greater (P = 0.06) in MEL than in Control cows, independently of physiological stage (Table 2), and interestingly, time to reach maximum blood insulin concentration was lower ( P < 0.05) in MEL than in Control cows. Thus, it appears that MEL cows had a greater capacity of secreting greater amounts of insulin and doing it faster than Control cows. However, despite this apparent improvement in insulin response, glucose clearance rate did no differ between treatments, although it was fastest (P < 0.01) 10 days after the day of halting milk removal (2.28%/min) and then decreased to 1.89%/min 20 d before and 10 d after calving. Similarly, glucose half-time increased (P < 0.01) from 31.6 min at 10 after the day of halting milk removal to about 40 min 20 d before and 10 d after calving (Table 2).

The AUC for glucose during the first 60 min of the GTT tended to be lower ( P = 0.06) in MEL than in Control cows. On the other hand, the AUC for blood insulin concentration also tended (P = 0.06) to be greater in MEL than in Control cows independently of physiological state. Time to reach basal glucose baseline was shorter (P < 0.05) in MEL than in Control cows (Table 2), but time to insulin base line did not differ among treatments. This would suggest that MEL cows responded effectively to the increased production of insulin elicited by the GTT. Insulin/glucose ratio was greater ( P < 0.05) in MEL than in Control cows regardless of physiological state, which would indicate that for every unit of glucose in blood, MEL cows produced more insulin. This is also seen by the tendency towards a greater peak of insulin concentration. A greater insulin/glucose ratio could be an indication of impaired insulin sensitivity, and in fact, the insulin sensitivity index (calculated following Galvin et al. (1992)) was lower (P < 0.05) in MEL than in Control cows, independently of physiological stage. However, MEL cows were able to return blood glucose levels back to baseline values earlier than Control cows did, and responded more quickly (P < 0.05) and with a tendency (P = 0.06) for a greater production of insulin. Furthermore, the response to insulin was better in MEL than in Control as the AUC for glucose tended (P = 0.06) to be lower in MEL than in Control cows.

Table 2. Blood glucose and insulin responses to a glucose tolerance test at -50, -40, and 10 d relative to calving as affected by treatments.

Treatment¹ P-value²

Control MEL SE T D TxD

Basal glucose³, mg/dl 56.1 60.1 1.45 0.02 <0.01 0.44

Basal insulin⁴, pg/l 0.54 0.61 0.06 0.38 <0.01 0.48

Peak glucose⁵, mg/dl 147.9 147.3 2.49 0.86 <0.01 0.60

Peak insulin⁶, pg/l 6.36 8.29 0.71 0.06 <0.01 0.59

CRglucose⁷, %/min 2.05 2.08 0.10 0.82 <0.01 0.38

Glucose Ti_/2⁸, min 36.4 35.8 1.97 0.83 <0.01 0.96

Time to insulin peak, min 10.49 8.03 0.92 0.01 <0.01 0.24

AUC60glucose⁹, mg/dlx60 min 2,110 I,858 126.2 0.06 0.05 0.34

AUC₆oinsulin¹⁰, pg/lx60 min 152.2 189.5 14.0 0.06 <0.01 0.89

Time to glucose baseline¹¹, min 76.1 61.9 7.6 0.04 0.04 0.64

Time to insulin baseline¹¹, min 86.1 85.6 6.42 0.95 0.01 0.57

_

¹ Control: Non supplemented; MEL: 260 mg of melatonin.

² T: effect of treatment; D: effect of day; TxD: interaction between treatment and day.

³ Average glucose concentration in blood samples taken before the start of the GTT.

⁴ Average insulin concentration in blood samples taken before the start of the GTT.

⁵ The maximum glucose concentration measured during the GTT.

⁶ The maximum insulin concentration measured during the GTT.

⁷ Clearance rate of glucose during the first 90 min calculated following Jaakson et al. (2017).

⁸ Glucose half-time calculated following Jaakson et al. (2017).

⁹ Area under the curve of glucose during the first 60 min of the GTT.

¹⁰ Area under the curve of insulin during the first 60 min of the GTT.

¹¹ Time to glucose or insulin baseline determined following Pires et al. (2007).

¹² Area under the curve of glucose/area under the curve of insulin.

¹³ Insulin sensitivity index derived using the approach from Galving et al. (1992).

¹⁴ Insulin sensitivity index derived using the minimal model. ¹⁵ Glucose effectiveness derived using the minimal model.

Table 3 shows the gene expression of selected cytokines in the uterus at 5 d after calving of 10 cows per treatment. MEL treatment increased, just numerically, the expression of ILlfi and IL8 in the uterus. These 2 compounds are pro-inflammatory cytokines. On the other hand, expression of IL10 was also numerically greater in MEL than in Control cows, which would balance any potential increase in pro-inflammatory state of the uterus.

Table 3. Expression (folds relative to the lowest expression in Control cows) of genes coding for pro- and anti-inflammatory cytokines in the uterus Holstein cows 5 d after calving as affected by melatonin.

Treatment

Control MEL SE¹ E-value

Interleukine-ΐb 0.04 0.55 0.81 0.12

Interleukin-8 0.11 0.84 0.59 0.13

Interleukin- 10 0.74 1.01 0.22 0.38

¹ SE for II- IB and P-8 are derived from log-transformed statistical analysis.

At the mammary level, the 5 genes evaluated were not affected by treatments (Table 4). As expected, though, expression of ki67 decreased and pi 6 and sirtl increased with the onset of lactation (Figure 4). The gene ki67 is involved in cell regeneration, whereas pi 6 and sirtl are proxies for cell senescence and cell survival, respectively. Interestingly, mammary expression of sirtl tended (P = 0.07) to be lower in MEL than in Control cows after calving, which would be an indication of lower senescence (or better cell renovation). The level of expression of bax (a gene associated with apoptosis) was similar at early dry period and early lactation, but that of casp3 (a gene also involved in apoptosis) increased after calving relative to the levels measured 10 d after the day of halting milk removal .

The administration of melatonin did not result in increased PRLr expression in the mammary gland after calving compared with Control (Figure 4). Blood PRL concentrations were not affected by treatments either (Table 5).

Similarly, the mammary expression of IGFBP5 was much greater at 30 DIM (1.12+0.09) than at 10 d after the day of halting milk removal (0.36+0.09). IGFBP5 acts inhibiting the action of IGF-I. Interestingly, the expression of IGFBP5 in the mammary gland in MEL cows was lower (P < 0.01) than in Control cows both before and after calving, with a tendency (P = 0.06) for this difference to be greater after calving. This may explain, in part, the increased milk production in MEL cows, as lower expression of IGFBP5 would allow for an increased activity of IGF-I in the mammary gland fostering an increased cell proliferation and secretory activity. The negative effects of IGFBP5 on milk production have been demonstrated by the fact that cows milked once a day have lower expression of IGFBP5 in the mammary gland than cows milked twice (Littlejohn et al., 2010; Boutinaud et al., 2013) or four times daily (Mumey et al., 2015). It is interesting to note that the decrease in expression of mammary IGFBP5 herein was independent of blood PRL, despite the fact that PRL has been reported to negatively affect IGFBP5 expression (Accorsi et al, 2002). Furthermore, the tendency for MEL cows to have lower expression for sirtl after calving than Control cows also supports the hypothesis that tissue regeneration in the mammary gland was greater in MEL than in Control cows.

Table 4. Expression (fold relative to the lowest expression in Control cows) of selected genes in the mammary gland of Holstein cows 10 d after the day of halting milk removal and 30 days after calving as affected by melatonin.

Treatment P-value¹

Control _ MEL _ SE T _ D TxD bax 1.00 1.04 0.07 0.72 0.86 0.56 H67 0.84 1.02 0.13 0.33 <0.01 0.44 pi 6 1.02 0.99 0.09 0.89 <0.01 0.63 sirtl 1.15 0.87 0.13 0.13 <0.01 0.07 casp3 1.09 1.00 0.17 0.72 <0.01 0.35

PRLr 1.03 0.87 0.11 0.31 0.08 0.45

IGFBP5 0.90 0.52 0.11 <0.01 <0.01 0.06

¹ T: Effect of treatment; D: Effect of day; TxD: Effect of the interaction between treatment and day.

Table 5. Selected blood parameters in Elolstein cows throughout transition as affected by melatonin.

Treatment P- value¹

Control MEL SE T D TxD

NEFA, m M 0.41 0.38 0.02 0.40 <0.01 0.09

IGF-1, ng/ml 114.8 118.6 10.4 0.80 <0.01 0.47

IGFBP3, pg/ml 15,299 15,116 3,568 0.97 <0.01 0.17

IGFBP5, pg/ml 45,109 46,669 2,128 0.42 <0.01 0.35

Growth hormone, ng/ml 24.1 22.1 1.31 0.08 <0.01 0.67 Prolactin, ng/ml 15.1 15.0 1.30 0.98 <0.01 0.93 b-OH-butyrate, mM 0.50 0.39 0.03 0.04 0.12 0.13

¹ T: Effect of treatment; D: Effect of day; TxD: Effect of the interaction between treatment and day.

There were no differences in blood NEFA concentrations between treatments (Table 3) but there was a weak tendency (P = 0.09) for MEL cows to have a greater peak of NEFA at calving (Figure 5). Interestingly, MEL cows had lower blood BHBA concentrations than Control cows, despite the fact the produced more milk. In fact, Figure 6 shows how MEL cows, despite they metabolized more BW during the first 30 DIM, they were able to metabolize all NEFA into CO2 and the amount of BHBA in blood did no increase (the contrary) as BW was mobilized. This observation could be explained by a greater glucose availability in MEL than in Control as indicated by the greater blood basal glucose levels in MEL cows.

Regarding IGF-1, IGFBP3, and IGFBP5, there were no differences between treatments.

Blood concentrations of growth hormone tended (P = 0.08) to be lower in MEL than in Control cows, independently of physiological state (Figure 7), and there were no differences in blood PRL concentrations (Table 4; Figure 8).

In summary, the tendency towards improvement milk yield observed herein in MEL could be explained by an improvement in the mammary remodeling because mammary remodeling was affected by melatonin supplementation (as indicated by sirtl and IGFBP5). PRL concentrations post-calving (and pre-calving) did not differ among treatments and no differences in PRLr expression in the mammary gland were observed herein. Therefore, it can be inferred that in the current study, the improved milk yield observed with MEL cows was mainly due to:

1) a decreased expression of IGFBP5 at the mammary gland, which most likely improved cell proliferation (resulting in a tendency for lower cell senescence in the mammary gland),

2) an improved glucose metabolism (with greater basal glucose levels and faster and more vigorous insulin responses to glycaemia), and

3) an increased supply of nutrients as a result of the observed increase in DMI after about 25 DIM (Figure 1). The increase in DMI could have been caused by an improved metabolism of glucose and decreased insulin sensitivity at the central nervous system. References

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Claims

1. A use of a veterinary composition comprising melatonin for improving milk yield, and/or promoting mammary involution, and/or treating ketosis and/or improving glucose metabolism in a lactating ruminant, wherein the composition is administered via intramuscular, subcutaneous, intravaginal or intranasal administration or injection, wherein the released melatonin dose ranges between 150 and 300 mg per ruminant, wherein the composition is administered to the lactating ruminant the first day of the dry period and the melatonin is released during at least 60 consecutive days starting from the said first day of dry period.

2. The use according to claim 1, wherein the quantity of released melatonin is from 170 to 250 mg, and more specifically at 216 mg.

3. The use according to the claim 1 or 2, wherein said composition is an extended-release melatonin composition.

4. The use according to any one of claims 1-3, wherein the administered composition allows to have a daily blood melatonin concentration above 60 pg/mL.

5. The use according to any one of claims 1-4, wherein the composition is administered subcutaneously or intramuscularly.

6. The use according to any one of the preceding claims, wherein the composition is an extended-release melatonin composition.

7. The use according to any one of the preceding claims, for the treatment and/or the prevention of intra-mammary diseases and/or infections of ruminants.

8. The use according to any one of the preceding claims, wherein the ruminants are gestating ruminants.

9. The use according to any one of the preceding claims, wherein the ruminants are herbivorous mammals chosen among bovines, ovine, caprine, or camelids, preferably the ruminants are pregnant dairy cows or heifers.

10. A veterinary composition comprising melatonin, for use in improving milk yield, and/or promoting mammary involution, and/or treating ketosis and/or improving glucose metabolism in a lactating ruminant, wherein the composition is administered via intramuscular, subcutaneous, intravaginal or intranasal administration or injection, wherein the released melatonin dose is from 150 to 300 mg per ruminant, wherein the composition is administered to the lactating ruminants the first day of the dry period and the melatonin is released during at least 60 consecutive days starting from the said first day of dry period.

11. The composition according to claim 10, wherein the composition is an extended-release melatonin composition.

12. A veterinary kit comprising a veterinary composition comprising melatonin, as defined in any one of the preceding claims, preferably for use in improving milk yield, and/or promoting mammary involution, and/or treating ketosis and/or improving glucose metabolism in a lactating ruminant.

13. Kit according to claim 12 further comprising an instruction sheet on the mode of operation and mode of administration of the veterinary composition.

14. Kit according to any one the claims 12 and 13, further comprising an instruction sheet on the mode of operation and mode of administration of the veterinary composition to improve milk yield, and/or promote mammary involution, and/or treat ketosis and/or improve glucose metabolism in a ruminant.