The Effect of Melatonin Dosage and Progesterone on Reproduction in Anestrous Ewes

Clay D. Carlson

Abstract

Subcutaneous synthetic melatonin implants were applied at different dosages and used in conjunction with progesterone to induce cyclicity in anestrous ewes. Sixty-nine Suffolk and Suffolk X Polypay females were randomized into one of four treatment groups. Treatments included: 1) 500mg, 2) 250mg, 3) 125mg, and 4) a non-treated control. Serum progesterone samples were extracted 45d post ram induction to determine either cyclicity or potential pregnancies in the ewes. Ultrasonography readings 60d postbreeding were used to indicate pregnancy, and all lambing data was collected to calculate actual lambing percentages. Analysis of variance and chi-square methodology were used to interpret the data.

The conjunctive effect of melatonin and progesterone substantially increased pregnancy rates in the 500mg treated ewes when compared to non-treated controls (p<.05). The general trend suggests melatonin implanted ewes, as a whole, were more likely to become pregnant than their non-implanted counterparts.

Introduction

One of the challenges facing the sheep industry is the seasonality of breeding. Ewes are typically anestrous in the spring after lambing and lactating, and remain so until late August or early September. By forcing the ewes to breed out of season and conceive during the long daylight months of the year (spring and summer), management practices and product supply could improve.

In species that are photoperiod entrained, information is relayed to the reproductive axis by the pineal gland through nocturnal secretion of the hormone melatonin (Matthews et al., 1993; Stellflug et al., 1994; Viguie et al., 1995). Previous studies have shown erogenous melatonin can mimic the natural elevation of melatonin associated with an extended dark phase (Slyter and Weiskircher, 1993). The elevated levels of melatonin stimulate the hypothalamus to secrete gonadatropin-releasing hormone (GnRH). GnRH begins a series of endocrine events resulting ultimately in follicular development at the level of the ovary (Hafez, 1993). Some out of season fertility has been achieved with success using PMSG (pregnant mare's serum gonadotropin) and progestogens, but native ovine hormones such as melatonin and progesterone are viewed with more acceptance by the FDA for future usage (Wheaton et al., 1990).

Preliminary research at CSU, Chico initially determined the efficacy of melatonin as a means of inducing cyclicity in the anestrous ewe. The use of the subcutaneous implant Regulin was found to be an effective method of hastening the onset of estrus in non-cyclic ewes (Patton et al., 1994). Daley et al. (1997) found that the use of subcutaneous Regulin implants resulted in higher pregnancy and lambing rates (p< .05) when accompanied with a progesterone implant (P4) 12d prior to the breeding season. With a limited supply of Regulin and no availability on the commercial market, Daley et al. (I 997) compared the remaining Regulin@ implants to a synthetic silicone melatonin implant formulated in the research lab at the University of California, Davis. Results indicated there was no significant difference between the two implants, therefore the silicone implant offers an alternative to the Regulin@ implant.

The objectives of this study (phase 4) are to determine at what dosage the synthetic subcutaneous implant will be effective. P4 will again be utilized to achieve a tight synchrony among the ewes during breeding.

Materials/Methods

Sixty-nine Suffolk and Suffolk X Polypay ewes were utilized to study the dosage effect of melatonin in the anestrous ovine female. Thirty-seven ewes from the Paul L. Byrne Teaching and Research Facility, California State University, Chico, California (Flock A), and 32 ewes from a local producer (Flock B) were implanted subcutaneously with a synthetic silicone melatonin implant in the left foreflank. Ewes were randomly assigned within flock to four treatment groups including: 1) 500mg implant, 2) 250mg implant, 3) 125mg implant, and 4) a non-treated control. The two groups of ewes were housed at two different locations in the northern Sacramento Valley in the spring of 1997. Management practices, feeding programs, genetic make-up, and age of the flocks were similar as they were grazed on irrigated legume/grass pastures in a farm flock scenario.

A 3.0mg P4 implant (1/2 of a Syncromate-B or SMB implant) was administered to all ewes, as a method of synchronization, 12d prior to the breeding season. On June 7 SMB (Norgestomet) implants were pulled, and rams equipped with marking harnesses were introduced to the four treatment groups. Marking harness color was changed every 21d and ewes were checked daily to record breeding dates. Males were removed from the ewes on August 1. Ewes were bled via jugular venipuncture 45d post introduction of therams to assess serum progesterone levels. P4 profiles were created from the samples using radioimmunoassay to indicate which ewes responded to treatment. Ewes were considered cyclic if P4 levels were higher than .5 ng/ml. Ultrasound readings were recorded 60d post breeding by a licensed veterinarian to determine pregnancy rates.

Data were analyzed for treatment effects by analysis of variance (Steel and Torrie, 1960) and chi-square analysis (Snedecor and Cochran, 1967).

Results and Discussion

Serum progesterone samples extracted 45d after introduction of the rams show no difference among the treatment groups in Flock A (Table 1.). Ewes were considered cyclic or potentially pregnant if P4 levels were greater than .5 ng/ml.

Table 1. SERUM PROGESTERONE LEVELS 45d POST RAM INDUCTION

Flock A

Treatment n ewes Mean P4 S.E.

However, differences did appear between groups according to ultrasonography determination of pregnancy recorded 60d post-breeding (Table 2.). Chi-square analysis indicated differences in expected pregnancy rates; 500mg and 125mg treatments were different from the control group in Flock A (p< .05). This apparent discrepancy between progesterone levels and ultrasound data may be the result of fetal reabsorption, possibly due to environmental heat stress or inaccurate ultrasonography interpretation.

No differences in ultrasound determined pregnancy were observed in Flock B which may be the result of limited data (Table 2.).

Table 2. PREGNANCY DETERMINATION 60d POST-BREEDING

Flock A, Flock B, Flock A & B

Overall Treatment Pregnant Open % Pregnant Open % pregnant %

Means that do not have common superscript differ (P<.05)

Overall ultrasonography percentages (Flock A and B combined) show the 500mg treatment had a higher pregnancy rate (p<.05) than the control; 78% and 40%, respectively (Table 2.). This suggests that the combination of exogenous melatonin at the 500mg dosage, accompanied with progesterone, in necessary in order to attain a possible pregnancy in the anestrous ewe.

No differences were detected in Flock A or B lambing percentages, number of lambs, or number of lambs/ewe lambed, nor were there differences when the flocks were combined (Tables 3., 4., and 5.). However, there appears a trend in days to lambing, with the control group approximately 10d longer than the treatments receiving melatonin along with progesterone, regardless of dosage (Table 5.).

Table 3. ACTUAL LAMBING PERCENTAGES

Flock A

ewes lambs/ewe

Treatment n ewes lambed % lambed n lambs lambed

Table 4. ACTUAL LAMBING PERCENTAGES

Flock B

ewes lambs/ewe

Treatment n ewes lambed % lambed n lambs lambed

Table 5. ACTUAL LAMBING PERCENTAGES

Flock A & B Combined

ewes lambs/ avg.d to

Treatment n ewes lambed % lambed n lambs ewe lambed lambing

It is interesting to note that there was an obvious decline in overall lambing percent when compared to ultrasound pregnancy determination for the 500mg treatment. This is especially noteworthy in Flock B, where 87.5% of the 500mg treatment were detected as pregnant, yet only 50% lambed. This is potentially the result of fetal reabsorption, due to heat stress. Ambient temperatures in the Sacramento Valley normally exceed 100 degrees Fahrenheit for extended periods in August and September.

Conclusion

Generally, the data suggests the synergistic effect of melatonin and progesterone induce cyclicity and pregnancy in the anestrous ewe. Specifically, these data indicate there is a difference in pregnancy rate of the 500mg melatonin dose, accompanied by progesterone, when compared to non-treated controls.

Literature Cited

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Patton, W.R., Daley, D.A., Daley, C.A., Bramble, T. 1994. Use of melatonin for induction of estrus in noncyclic ewes. Submitted.

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