Evaluation of the influence of melatonin implants during the gestation period in sheep from a selenium-deficient region

Santiago Andrés Department of Animal Medicine, Faculty of Veterinary Sciences, University of Extremadura, Campus Universitario, 10071, Cáceres, Spain.

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Joaquín Sánchez Department of Animal Medicine, Faculty of Veterinary Sciences, University of Extremadura, Campus Universitario, 10071, Cáceres, Spain.

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Antonio Jiménez Department of Animal Medicine, Faculty of Veterinary Sciences, University of Extremadura, Campus Universitario, 10071, Cáceres, Spain.

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Abstract

Objective—To evaluate the possible effect of melatonin implants on blood glutathione peroxidase (GSHPx) activity and in the prevention of selenium (Se)-responsive disorders in sheep from an Se-deficient region.

Design—Randomized controlled clinical trial.

Animals—100 Merino ewes.

Procedures—Ewes of the same age, parity, body weight, body condition, and reproductive and health history were randomly allotted to 1 of 2 groups (control and implanted) of 50 sheep each. Treatment consisted of implants of melatonin (18 g) administered SC in the pinna of the right ear 6 weeks prior to introduction of rams. The control group did not receive implants. Hematologic and serum biochemical analyses were performed at various points before and after treatment, in addition to determinations of erythrocyte mean corpuscular fragility (MCF) and blood GSHPx activity. The incidence of Se-responsive disorders in lambs was recorded in both groups.

Results—Hematologic and serum biochemical analyses yielded values within respective reference ranges for both groups. Significant differences between groups were evident in MCF at early mating (lower in the implanted group vs the control group) and in blood GSHPx activity at early mating, gestation, and early lambing (higher in the implanted group vs the control group). There were significantly fewer lambs with nutritional myodystrophy in the implanted versus the control group.

Conclusions and Clinical Relevance—Use of melatonin implants in sheep may improve reproductive performance and yield an earlier start of breeding season. The stimulating effect of melatonin on GSHPx activity may protect against oxidative damage during the first stage of gestation.

Abstract

Objective—To evaluate the possible effect of melatonin implants on blood glutathione peroxidase (GSHPx) activity and in the prevention of selenium (Se)-responsive disorders in sheep from an Se-deficient region.

Design—Randomized controlled clinical trial.

Animals—100 Merino ewes.

Procedures—Ewes of the same age, parity, body weight, body condition, and reproductive and health history were randomly allotted to 1 of 2 groups (control and implanted) of 50 sheep each. Treatment consisted of implants of melatonin (18 g) administered SC in the pinna of the right ear 6 weeks prior to introduction of rams. The control group did not receive implants. Hematologic and serum biochemical analyses were performed at various points before and after treatment, in addition to determinations of erythrocyte mean corpuscular fragility (MCF) and blood GSHPx activity. The incidence of Se-responsive disorders in lambs was recorded in both groups.

Results—Hematologic and serum biochemical analyses yielded values within respective reference ranges for both groups. Significant differences between groups were evident in MCF at early mating (lower in the implanted group vs the control group) and in blood GSHPx activity at early mating, gestation, and early lambing (higher in the implanted group vs the control group). There were significantly fewer lambs with nutritional myodystrophy in the implanted versus the control group.

Conclusions and Clinical Relevance—Use of melatonin implants in sheep may improve reproductive performance and yield an earlier start of breeding season. The stimulating effect of melatonin on GSHPx activity may protect against oxidative damage during the first stage of gestation.

Melatonin use for reproductive purposes has become common in sheep farming around the world.1 Melatonin is a peptide produced by the pineal gland, which directly controls the secretion of gonadotropin-releasing hormone by the hypothalamus and luteinizing hormone by the hypophysis. The amount of melatonin released is proportional to the duration of the photoperiod. An increase in blood melatonin concentration, in response to progressive shortening of summer days, is responsible for initiating the breeding season in sheep.2 Therefore, use of melatonin implants would allow the breeding season to start earlier, resulting in an increase in numbers of pregnant ewes (pregnancy rate) and lambs per ewe lambing (degree of prolificacy).3

In addition to its effects on reproduction, melatonin has other functions, including protection of cellular structures against oxidative damage attributable to free radicals. Melatonin stimulates activity of some enzymes such as glutathione reductase or GSHPx, which metabolizes reduced glutathione to its oxidized form and converts hydrogen peroxide to water. In doing so, melatonin reduces generation of hydroxyl radicals, the most toxic of oxygen-based radicals.4

Glutathione peroxidase is an Se-dependent enzyme. Determination of its activity in blood is considered a biological indicator of Se uptake by animals. In fact, measurement of GSHPx activity is more useful for determining Se requirements than is concentration of Se.5 Antioxidant activity in animals that consume Sedeficient rations (eg, ruminants grazing on pastures with a low Se content) may be limited, and such animals could develop clinical signs of deficiency, progressing to death. Thus, prevention of Se deficiency is one of the goals of sheep production in many parts of the world. Prophylactic methods usually include addition of Se to rations or SC administration of fast-release (sodium selenite) or long-acting (barium selenate) Se salts. Provision of orally administered Se to livestock in range settings is difficult, whereas SC administration of Se-containing drugs is time-consuming and may have adverse effects.6 The purpose of the study reported here was to evaluate the effects of melatonin implants on blood GSHPx activity and in the prevention of Se-responsive disorders in sheep from Se-deficient areas.

Material and Methods

Animals—One hundred Merino ewes were used in the experiment. Ages ranged between 3 and 5 years, and parities ranged from second to fifth. Body weights, body condition (evaluated by means of a 5-point scale), and reproductive and health histories of ewes were similar.

Site of study—The research was conducted at 1 commercial farm, which was situated in the southwest of Spain at 5°45'W and 39°30'N, with an elevation of 500 m above sea level. Annual mean minimum and maximum temperatures in the area were 10.8° and 21.4°C, respectively, and mean rainfall was 600 mm. The particular farm was selected on the basis of results of another study,7 in which the farm was determined to have a high prevalence of NMD in lambs and low concentrations of Se in soil and pasture.

Pasture grasses included various species, which mainly included Lolium spp, Bromus spp, Festuca spp, and Trifolium spp. In Mediterranean climates, pastures dry out early. Feed originating from these pastures is likely low in vitamin E because vitamin content of pastures and forages declines as plants mature.8 Ewes were stocked on pasture at a density of 4 ewes/hectare. They received no dietary supplementation with minerals, salts, or other feeds for the duration of the experiment.

Experimental design and treatments—The experiment was conducted between May 2007 and January 2008. At the beginning of this period, days are longer, and natural breeding is more difficult than at other times. Ewes were assigned to control (n = 50) or treatment (50) groups by means of coin flips. Treatment consisted of SC administration of implants containing 18 mg of melatonina in the lateral aspect of the pinna of the right ear 6 weeks prior to the introduction of rams. Control ewes did not receive implants.

After treatment, ewes were housed together and were managed with the same feeding, husbandry, and reproductive practices. For mating, rams and ewes were kept together for 45 days at a ram-to-ewe ratio of 1:4. Most mating occurred in the 2 or 3 weeks following ram introduction.

Sample collection and processing—Blood samples from a jugular vein of ewes from both groups were collected into EDTA, lithium heparin, and serum separation tubesb at 6 points during the study period: just before treatment (day 0), at early breeding (day 50), at late breeding (day 100), at gestation (day 150), at early lambing (day 200), and at late lambing (day 250). Blood samples were stored at 4°C until analysis. Whole blood samples in EDTA tubes were evaluated for erythrocyte count, Hb, Hct, MCH, MCHC, MCV, total and differential leukocyte counts, platelet count, and MCF. Blood samples in lithium heparin tubes were used for determination of GSHPx activity. Serum was evaluated for concentrations of total protein, total and direct bilirubin, cholesterol, triglycerides, glucose, urea, creatinine, calcium, phosphorus, magnesium, sodium, and potassium and activities of ALT, AST, GGT, ALP, CK, and LDH.

Determinations of erythrocyte count, Hb, Hct, MCH, MCHC, MCV, and leukocyte and platelet counts were performed by use of an automated cell counter.c Differential WBC count was performed on stained blood films.d Mean corpuscular fragility was measured spectrophotometricallye with a commercial kit.f Blood GSHPx activity was assayed with a kit for kinetic determination of the enzyme.g In the assay, glutathione peroxidase catalyses the oxidation of glutathione, with cumene hydroperoxide as a substrate. In the presence of glutathione reductase and NADPH, the oxidized glutathione is immediately converted to the reduced form, with a concomitant oxidation of NADPH to NADP+ and consequent decrease in absorbance at 340 nm. Serum sodium and potassium concentrations were measured via flame photometry.h Other serum biochemical values were determined by means of commercial kitsi and a human clinical chemistry analyzer.j Serum Se concentration was measured via graphite furnace atomic absorption spectrometry, after standard extraction procedures.k

Surveillance of clinical and reproductive variables— An experienced team of observers monitored reproductive performance and health status of all sheep throughout the experiment. Pregnancy rate (number of pregnant ewes/number of ewes available to mate) and degree of prolificacy (number of lambs born alive/number of ewes lambing) were measured. Lambs born to ewes were carefully surveilled for signs of NMD such as stiff gait, hunched back, and an increase in heart or respiratory rate. Lambs were also evaluated for muscles that appeared hardened or in which palpation elicited signs of pain.

Statistical analysis—Assessment of changes in the variables evaluated was conducted by use of a repeated-measures ANOVA, with melatonin treatment as a between-subject factor and time as a within-subject factor. A C2 test was used to investigate differences in pregnancy rates and proportions of lambs with NMD between control and implanted groups. Degree of prolificacy was expressed on a per-ewe basis before analysis and mathematically transformed (arcsine square root), and differences between groups were evaluated by means of a Student t test. All analyses were performed with statistical software.9,l Differences between groups were considered significant at a value of P < 0.05.

Results

Ewes in the implanted and control groups were not significantly different at any time before or after treatment with respect to any hematologic variable, including erythrocyte count, Hb, Hct, MCH, MCHC, MCV, total and differential leukocyte counts, and platelet count. The same lack of significant differences between implanted and control ewes was evident for serum concentrations of calcium, phosphorus, magnesium, sodium, potassium, total protein, total and direct bilirubin, cholesterol, triglycerides, glucose, urea, and creatinine and serum activities of ALT, GGT, ALP, AST, CK, and LDH.

Mean values were calculated for serum Se concentration, blood GSHPx activity, and MCF (Table 1). Serum Se concentrations were not significantly different between the 2 treatment groups at any sampling point. The opposite was true for blood GSHPx activity, which differed between groups at late breeding (P = 0.008), gestation (P < 0.001), and early lambing (P = 0.04). For MCF, differences between treatment groups were significant (P = 0.04) only at early breeding.

Table 1—

Mean ± SD serum Se concentrations, MCF values, and blood GSHPx activities in ewes from an Se-deficient region that did (implanted group; n= 50) and did not (control group; 50) receive a melatonin implant (18g), as measured at various times during the reproductive cycle.

TimeGroupSe (ppm)*MCF (%)GSPHx (U/g of Hb)
Before treatmentControl0.020 ± 0.0080.42 ± 0.0146 ± 2
Implanted0.020 ± 0.0080.41 ± 0.0145 ± 2
Early breedingControl0.019 ± 0.0080.43 ± 0.01§45 ± 2
Implanted0.019 ± 0.0080.40 ± 0.0152 ± 3
Late breedingControl0.020 ± 0.0080.42 ± 0.0148 ± 2§
Implanted0.018 ± 0.0080.41 ± 0.0162 ± 3
GestationControl0.020 ± 0.0080.42 ± 0.0146 ± 3§
Implanted0.019 ± 0.0080.41 ± 0.0172 ± 5
Early lambingControl0.019 ± 0.0080.43 ± 0.0144 ± 2§
Implanted0.020 ± 0.0080.41 ± 0.0150 ± 1
Late lambingControl0.019 ± 0.0080.43 ± 0.0150 ± 2
Implanted0.019 ± 0.0080.42 ± 0.0148 ± 3

Serum concentrations of Se were deemed to be deficient, marginal, or adequate at < 0.03 ppm, 0.03 to 0.10 ppm, and > 0.10 ppm, respectively.

Reference range, 0.38% to 0.42%.

Blood activities of GSHPx were deemed to be deficient, marginal, or adequate at < 60 U/g of Hb, 60 to 120 U/g of Hb, and > 120 U/g of Hb, respectively.

Value is significantly (P < 0.05) different between treatment groups at indicated time.

Pregnancy rates, degrees of prolificacy, and prevalences of NMD in lambs of both treatment groups were compared (Table 2). No instances of abortion or stillbirth were recorded. Pregnancy rate and degree of prolificacy were not significantly different between groups. Only 1 case of NMD was reported among lambs born to implanted ewes; however, NMD was detected in 8 lambs born to control ewes, which represented a significant difference (P = 0.002). Mean blood activity of GSHPx in all 9 lambs with signs of NMD was 12.8 ± 1.5 U/g of Hb (reference range, 60 to 120 U/g of Hb), and mean serum activities of AST, CK, and LDH were 356 ± 34 U/L (reference range, 60 to 70 U/L), 8,111 ± 1,026 U/L (reference range, 100 to 200 U/L), and 632 ± 58 U/L (reference range, 400 to 500 U/L), respectively.

Table 2—

Lambing rate (lambs born/1 ewe available to mate), mean ± SD degree of prolificacy (lambs born/1 ewe lambing), and proportion of lambs with NMD in ewes from an Se-deficient region that did (implanted group; n= 50) and did not (control group; 50) receive a melatonin implant (18g).

VariableControl groupImplanted groupP value*
Pregnancy rate (%)58720.14
Prolificacy1.03 ± 0.031.16 ± 0.060.09
Lambs born with NMD (%)26.72.40.002

A value of P < 0.05 was considered significant.

Discussion

Among the several postulated effects of melatonin on animal physiology, the peptide is believed to adversely affect hemopoiesis.10,11 In the study reported here, hematologic values for erythrocytes, Hb, Hct, MCH, MCHC, and MCV were not significantly different between ewes that received melatonin implants and those that did not, and all values remained within respective ranges12 throughout the study period. These results indicated that erythrocytes did not undergo serious changes as a result of treatment with melatonin. In findings of another study,13 IV injection of 5 mg of melatonin/kg was administered also did not significantly alter blood cells counts.

Similar to results of another study14 of sheep with naturally high or low plasma concentrations of melatonin, no significant differences were detected in total and differential leukocyte counts between implanted and control ewes in our study. In contrast, results of other studies indicate that melatonin is able to modify production and release of leukocytes. In sheep, total numbers of circulating leukocytes and monocytes are lower when nocturnal elevation in melatonin is eliminated by constant exposure to light,15 whereas SC injection of melatonin increases numbers of circulating neutrophils and decreases numbers of circulating lymphocytes.16

Platelets contain melatonin.17 Consequently, melatonin has been used to treat animals with disorders characterized by low platelet counts18 to increase platelet mass.13 However, platelet counts in the ewes in the present study did not appear to be affected by melatonin treatment.

In the study reported here, treatment with melatonin did not yield changes in serum concentrations of calcium, phosphorus, magnesium, sodium, and potassium, compared with values for untreated ewes. In fact, serum concentrations of calcium, phosphorus, and magnesium were within the respective reference ranges for the Merino breed.19 At 100 to 200 days after administration of the melatonin implants, mean serum potassium concentration in implanted ewes appeared to be lower than that in control ewes, although the difference was not significant. Changes in potassium concentration in erythrocytes as a result of melatonin administration can occur after challenge with cytotoxic solutions20 because melatonin is able to decrease the loss of potassium from the cells. No changes in serum sodium concentration were associated with melatonin administration, although such an increase reportedly occurs in sheep.10

Values for serum activities of liver enzymes in sheep in the present study were always within respective reference ranges.19,21 These findings agree with those of another study22 involving dairy cattle. In that study, treatment with melatonin did not affect liver enzyme activity despite the protection that melatonin purportedly provides against ischemic and toxic insults to the liver mediated by free radicals,23,24 which would result in decreases in serum activities of ALP, GGT, and aminotransferases.23,25

Melatonin also modulates several aspects of metabolism,24 and its influence on serum concentrations of cholesterol, triglycerides, and glucose has been evaluated. Serum concentrations of cholesterol and triglycerides significantly increase after melatonin is administered to dairy goats26 and dairy cows.22 Researchers in those studies suggested that the changes could be related to blood insulin concentration. However, in the study reported here, no differences in serum concentrations of glucose, cholesterol, and triglycerides were detected between implanted and control ewes, likely because sheep were not in a negative energy balance during lactation or at the end of gestation.

Because the sheep in our study were presumably free of previous renal or muscle damage, it was not surprising that we did not detect a significant difference between implanted and control ewes with respect to serum creatinine concentration and muscle enzyme activity. By inhibiting excessive oxidative stress, melatonin is able to protect physiologic systems and organs, reducing the increases in serum or plasma creatinine concentration and activities of AST, CK, and LDH that typically follow renal and muscle damage.25,27–29

The effect of melatonin on the kinetics of electrolytes in various physiologic or pathologic conditions has been evaluated.10,30 After treatment with melatonin, changes in serum concentrations of sodium and magnesium occur, whereas serum concentrations of potassium and ionized calcium remain unchanged.10 In the present study, the serum concentration of potassium at 50 to 200 days after treatment with melatonin was lower than the value before treatment, but the difference was not significant, possibly because the sheep did not have any condition characterized by hemolysis. The fact that we evaluated total calcium rather than ionized calcium may have contributed to the lack of a significant difference between groups with respect to serum calcium concentration.

In Se-deficient regions, several disorders responsive to treatment with Se and vitamin E have been reported for livestock.6 In the present study, serum Se concentrations were not different between implanted and control ewes. All sheep had Se concentrations indicative of deficiency (ie, < 0.03 ppm of wet weight), in accordance with findings of another study6 carried out in the same area.

Erythrocytes are highly susceptible to oxidative stress. Damage mediated by oxygen free radicals and lipid peroxidation occurs as a result of an imbalance between the oxygen free radical–producing system and the oxygen free radical–scavenging system. Melatonin might protect erythrocytes against oxidative stress.31 In our study, evidence of a protective effect was apparent, given that a decrease in osmotic MCF was detected in implanted ewes 50 days after implant insertion, when melatonin concentrations are supposedly optimal.32

Melatonin not only exerts its protective action against oxygen free radicals through its direct scavenging activity but also via its stimulating effect on the production of antioxidant enzymes such as GSHPx.33 In ewes that received melatonin implants, blood GSHPx activity was marginal, and in control ewes, it was deficient, despite the fact that serum Se concentrations remained deficient in both groups.

Other researchers have reported that GSHPx activity in erythrocytes and the liver significantly increases after melatonin treatment.34–36 Melatonin injection in mice results in greater increases of hepatic GSHPx activity than does combined treatment with Se and vitamin E.37 In our study, the variations recorded in the activity of the Se-containing antioxidant enzyme could not be attributed to Se intake because serum Se concentrations were not significantly different between implanted and control ewes. The importance of this fact should not be undervalued because improvement was detected in blood GSHPx activity, presumably attributable to melatonin, from deficient activity (< 60 U/g of Hb) to marginal activity (60 to 120 U/g of Hb). Sheep in many regions of the world are so accustomed to low dietary intake of Se that no clinical signs of Se deficiency are expected when GSHPx activities are > 60 U/g of Hb.7 Differences in blood GSHPx activities in our study were not likely attributable to nutritional changes. Both groups were managed as 1 flock, and early maturation and dry out of herbage associated with regional climatic conditions would likely have yielded low vitamin E content.

Treatment with melatonin did not significantly improve the pregnancy rate or degree of prolificacy in the ewes in our study. This apparently failed reproductive effect may have been attributable to small sample size because melatonin administered in the nonovulatory period brings ewes into fertile estrus earlier than is typical.3 Data regarding blood GSHPx and serum muscle enzyme activities from lambs with musculoskeletal impairment confirmed the diagnosis of NMD. The significant difference in prevalences of clinical cases of Serelated disorders in lambs born to the 2 groups of ewes might be suggestive of a protective effect of melatonin, similar to that obtained via treatment with Se salts.6

The apparent stimulating action of melatonin that was detected in the present study could allow an earlier start to the breeding season in ewes. In addition, the increase in blood GSHPx activity could protect ewes and fetuses against oxidative damage during the first stage of gestation.

Abbreviations

ALT

Alanine aminotransferase

ALP

Alkaline phosphatase

AST

Aspartate aminotransferase

CK

Creatine kinase

GGT

γ-Glutamyl transferase

GSHPx

Glutathione peroxidase Hb Hemoglobin

LDH

Lactate dehydrogenase

MCF

Mean corpuscular fragility

MCH

Mean corpuscular hemoglobin

MCHC

Mean corpuscular hemoglobin concentration

MCV

Mean corpuscular volume

NADP+

Nicotinamide adenine dinucleotide phosphate

NADPH

Reduced form of nicotinamide adenine dinucleotide phosphate

NMD

Nutritional myodystrophy

Se

Selenium

a.

Melovine, Ceva Salud Animal, Barcelona, Spain.

b.

Eurotubo, Barcelona, Spain.

c.

Syxmex F-900, Roche, Barcelona, Spain.

d.

Diff Quick, QCA Laboratorios, Tarragona, Spain.

e.

UV 160 A, Shimazdu, Kyoto, Japan.

f.

Osmotic Brittleness, Aulabor Industries, Barcelona, Spain.

g.

Ransel, Randox Laboratories, Crumlin, Ireland.

h.

Nak II, Pacisa, Sevilla, Spain.

i.

RAL Laboratories, Madrid, Spain.

j.

Clima Plus, RAL Laboratories, Madrid, Spain.

k.

550 SE, PerkinElmer, Wellesley, Mass.

l.

G-Stat, Glaxo-Smithkline, Madrid, Spain.

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