Veterinary Research Communications, 23 (1999) 115^121 # 1999 Kluwer Academic Publishers. Printed in the Netherlands

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1 Veterinary Research Communications, 23 (1999) 115^121 # 1999 Kluwer Academic Publishers. Printed in the Netherlands Short Communication A Preliminary Study on the E ects of Dietary Energy and MelatoninontheEx Vivo Production of Progesterone and Prostaglandin F 2a by the Corpora Lutea and Endometrial Tissue of Ewes J.A. Abecia*, J.M. Lozano and F. Forcada Departamento de Produccio n Animal y Ciencia de los Alimentos, Facultad de Veterinaria, Universidad de Zaragoza, Miguel Servet 177, Zaragoza, Spain *Correspondence Abecia, J.A., Lozano, J.M. and Forcada, F., A preliminary study on the e ects of dietary energy and melatonin on the ex vivo production of progesterone and prostaglandin F 2a by the corpora lutea and endometrial tissue of ewes. Veterinary Research Communications, 23(2), 115^121 Keywords: corpus luteum, endometrium, melatonin, nutrition, progesterone, prostaglandin, sheep Abbreviations: H, high-intake group; L, low-intake group; PG, prostaglandin INTRODUCTION Seasonality in sheep is mediated by photoperiod (Yeates, 1949), which is conveyed to the reproductive neuroendocrine axis by melatonin (Bittman et al., 1983). Administration of melatonin has been widely used as a means of advancing the breeding season (Koumitzis et al., 1989; McMillan and Sealey, 1989; Haresign et al., 1990), but an increased lambing percentage has also been reported following such treatment (Haresign et al., 1990; Chemineau et al., 1991). The higher proli cacy observed in melatonintreated ewes could be mediated through a higher rate of embryonic survival, either by an improvement in luteal function or by a reduction in the antiluteolytic mechanisms. Several studies have demonstrated that melatonin stimulates luteal progesterone production, either in vivo or in vitro (Durotoye et al., 1985; Webley and Luck, 1986; Webley et al., 1988), but little is known of the e ect of melatonin on the signals involved in the maternal recognition of pregnancy, such as prostaglandin (PG) F 2a production. Some e ect of nutrition on the response to melatonin treatments has been observed (Robinson et al., 1991). In particular, in Mediterranean breeds, an increase in ovulation rate was found to occur in undernourished ewes and in those with low body condition scores, but not in well-nourished ewes (Forcada et al., 1995; Rondön et al., 1996). 115

2 116 The aim of this experiment, which formed part of a larger study of the e ect of nutrition on embryonic survival rate, was to study the e ect of melatonin on luteal function and prostaglandin secretion by the uterus, and its interaction with nutrition. An in vitro model was used to determine progesterone production by luteal tissue and PGF 2a by endometrial tissue, incubated with or without melatonin. MATERIALS AND METHODS The study was conducted at the experimental farm of the University of Zaragoza, Spain (latitude 'N), which meets the requirements of the European Union for Scienti c Procedure Establishments. In mid-november, 30 adult Rasa Aragonesa ewes were allocated randomly by body condition score to two initially similar groups, weighed and synchronized in oestrus using intravaginal progestagen pessaries ( uorogestone acetate, Intervet S.A., Salamanca, Spain). From then on, the animals were fed to provide 1.5 times (high, H; n = 15) or 0.5 times (low, L; n = 15) the energy requirements for live weight maintenance. The pelleted diet consisted of barley (79%), soya bean (15%) and a mineral supplement (6%). From when the pessaries were inserted until slaughter, the ewes were given daily either 0.55 kg of pellets and 0.80 kg of barley straw (H; 12.4 MJ metabolizable energy (ME)) or 0.10 kg of pellets and 0.5 kg of barley straw (L; 4.1 MJ ME). From 32 to 80 h after withdrawal of the pessaries, the ewes were mated and oestrus was checked every 8 h. Seven ewes (2 H and 5 L) that did not show behaviour characteristic of oestrus were removed from the experiment. Seven H and 5 L ewes were weighed and euthanized (Euta-Lender, Normon, Spain) 9 days after mating. Their uterine horns were ushed after slaughter using Foley catheters, and the embryos so recovered were counted. Corpora lutea were dissected from the ovarian stroma to determine progesterone secretion in vitro (Abecia et al., 1995). Brie y, each corpus luteum was nely chopped, suspended in 1 ml of culture medium (M-199) and incubated in duplicate (c. 0.5 mg per culture) for 2 h at 378C, with or without melatonin (10 ng/ml). After incubation and centrifugation, the supernatant was collected and the progesterone content was analysed. Because of the small amounts of luteal tissue available, no attempt was made to determine the protein content of these samples, the results being expressed as ng of progesterone/mg of tissue per h. Progesterone determinations were performed using solid-phase RIA kits based on antibody-coated tubes, 125 I-labelled progesterone, and rabbit antiserum (biomerieux, Marcy L'Etoile, France). The assay sensitivity was 0.05 ng/ml of progesterone. The intra-assay and inter-assay coe cients of variation were 7.6% and 9.1%, respectively. Endometrial tissue (approximately 100 mg) was collected, washed in TCM-199 and placed into individual wells of a 24-well tissue culture plate containing 500 ml of cell culture medium per well, with or without melatonin (10 ng/ml). The tissue was incubated for 24 h at 378C in an atmosphere of 5% CO 2. The cell culture medium was described previously by Thibodeaux and colleagues (1994), and consisted of Ham's F-

3 medium supplemented with antibiotics, l -glutamine (0.29 mg/ml), insulin (5 mg/ ml), transferrin (5 mg/ml) and selenium (5 ng/ml). There were two replicates for each tissue sample collected. After culture, the medium was collected and stored at ^208C until the PGF 2a content was assayed, using PGF 2a enzyme-immunoassay kits (Cayman Chemical Co., Ann Arbor, MI, USA). The samples were all assayed in a single assay with an intra-assay coe cient of variation of 8.4%. All the materials employed for tissue cultures were purchased from Sigma Chemical Co., St Louis, MO, USA. The protein content of the endometrial samples was measured by a protein assay kit (Sigma) based on the method of Lowry and colleagues (1951), using bovine serum albumin as the standard. The remaining ewes were weighed and euthanized on day 15 (6 H and 5 L ewes) and all the above procedures were repeated. The main e ects of food intake on production of PGF 2a and secretion of progesterone by luteal tissue were compared by analysis of variance. Hormonal secretion in vitro, with or without melatonin in the culture medium, was compared using Student's t-test. Ovulation and pregnancy rates were compared using w 2 tests. Because pregnant and non-pregnant ewes expressed di erent rates of PGF 2a secretion (p50.01), especially on day 15 ( vs ng PGF 2a /mg protein, for pregnant and non-pregnant ewes, respectively), and in order to distinguish between the nutritional and pregnancy e ects, only pregnant ewes were included in the initial statistical analysis (6 H and 4 L ewes on day 9 and 6 H and 2 L ewes on day 15). Since only 2 L ewes were pregnant on day 15, it was not considered appropriate to conduct statistical tests to compare the secretion of progesterone and PGF 2a on day 15. RESULTS L ewes declined signi cantly in live weight and body condition during the study ( to kg and to , respectively), while H ewes showed little or no change ( to kg and to , respectively). The nutritional treatments had no e ect on ovulation rate (H, ; L, corpora lutea). Only 2 ewes (1 H and 1 L) were not pregnant on day 9, while more H ewes than L ewes were pregnant on day 15 (H, 100%; L, 40%; p = 0.06). Neither the plane of nutrition nor the inclusion of melatonin in the culture medium modi ed progesterone production in vitro on day 9 of pregnancy (Figure 1). On day 15, the corpora lutea from L ewes tended to secrete more progesterone in the presence of melatonin than in its absence. There was no e ect from either nutrition or melatonin on PGF 2a production on day 9. On day 15 of pregnancy, the endometrial tissue from the 2 L ewes considered secreted more PGF 2a in vitro than that from the H ewes. However, when these samples were incubated with melatonin, tissue from L ewes secreted less in the presence of melatonin than in its absence (Figure 2).

4 118 Figure 1. Mean (+SEM) progesterone secretion in vitro (ng/mg tissue per h) by endometrial tissue, with (M+) or without melatonin (M^) in the culture medium (10 ng/ml), on days 9 and 15 of pregnancy, in ewes fed 1.56 (H; n = 6 on day 9 and n = 6 on day 15) and 0.56 (L; n = 4 on day 9 and n = 2 on day 15) maintenance requirements DISCUSSION Melatonin appeared to a ect PGF 2a secretion in vitro only in undernourished ewes on day 15 of pregnancy, when the signals of maternal recognition of pregnancy, the most critical stage for survival of the conceptus, are being established. However, this nding re ects the data from only 2 animals and must be regarded as provisional. These ewes had a lower pregnancy rate and higher secretion of PGF 2a, and this apparent reduction in endometrial luteolytic hormone suggests that melatonin treatment might improve embryonic survival in undernourished ewes through a reduction of the luteolytic signals. There was also evidence of an e ect of melatonin on luteal function in the L ewes, as measured by luteal progesterone secretion in vitro.

5 119 Figure 2. Mean (+SEM) prostaglandin F 2a secretion in vitro (ng/mg protein) by endometrial tissue, with (M+) or without melatonin (M^) in the culture medium (10 ng/ml), on days 9 and 15 of pregnancy, in ewes fed 1.56 H; n = 6 on day 9 and n = 6 on day 15) and 0.56 (L; n = 4 on day 9 and n = 2 on day 15) maintenance requirements Gimeno and colleagues (1980) reported that melatonin blocks in vitro generation of PGF 2a by the uterus, and it has been found that melatonin can modify PG biosynthesis in the hypothalamus (Bojanowska and Forsling, 1997). However, there have been reports of the presence of melatonin receptors in the endometrium. On the other hand, an e ect of melatonin on progesterone secretion by the corpus luteum has been reported in sheep (Durotoye et al., 1997), cattle (Webley and Luck, 1986) and humans (Webley and Luck, 1986; Webley et al., 1988) and the presence of melatonin receptors has been demonstrated in granulosa cells (Yie et al., 1995). The absence of a clear in vitro e ect of melatonin on progesterone production in the present experiment might be due to the shorter duration of the culture (2 h) than in previous studies (1^4 days).

6 120 Treatment of sheep with melatonin has resulted in increased proli cacy (Haresign et al., 1990; Chemineau et al., 1991; Durotoye et al., 1991), possibly by increasing the ovulation rate (Wigzell et al., 1986; Forcada et al., 1995; Rondön et al., 1996) or by improving embryonic survival (Durotoye et al., 1985), as suggested by Durotoye and colleagues (1997). The results of the present study suggest that, in Mediterranean sheep breeds, there may be an interaction between nutrition and melatonin. These results are consistent with the ndings of Forcada and colleagues (1995), who reported an increase in the ovulation rate in melatonin-treated ewes and especially in those with a low rather than a high plane of nutrition. Similarly, Rondo n and colleagues (1996) concluded that melatonin treatment could improve the ovulation rate in ewes with a moderately low body condition score at the beginning of the breeding season. It is concluded that melatonin does not modify the secretion of progesterone or PGF 2a in vitro 9 days after mating, regardless of the immediate plane of nutrition. On day 15, the addition of melatonin to the culture medium appeared to reduce the secretion of PGF 2a but only in two ewes with a low energy intake; these ewes also exhibited a higher embryonic mortality. However, further studies are required to con rm these results as they only represent data from two undernourished animals. ACKNOWLEDGEMENTS This study was supported by grant AGF from CICYT (Spain). REFERENCES Abecia, J.A., Rhind, S.M., Bramley, T.A. and McMillen, S.M., Steroid production and LH receptor concentrations of ovarian follicles and corpora lutea and associated rates of ova wastage in ewes given high and low levels of food intake before and after mating. Animal Science, 61, 57^62 Bittman, E.L., Karsch, F.J. and Hopkins, J.W., Role of the pineal gland in ovine photoperiodism: regulation of seasonal breeding and negative feedback e ects of estradiol upon luteinizing hormone secretion. Endocrinology, 113, 329^336 Bojanowska, E. and Forsling, L., The e ects of melatonin on vasopressin secretion in vivo: interactions with acetylcholine and prostaglandins. Brain Research Bulletin, 42, 457^461 Chemineau, P., Vandaele, E., Brice, G. and Jardon, C., Utilisation des implants de me latonine pour l'ame lioration des performances de reproduction chez la brebis. Recueil de Me decine Vëterinaire, 167, 227^239 Durotoye, L.A., Argo, C.M., McNeil, M.E., Graham, N.B. and Rodway, R.G., Early lambing and increased lambing percentage with slow-release hydrogel melatonin implants. Journal of Reproduction and Fertility, Abstract Series, 1, 83 Durotoye, L.A., Rajkumar, R., Argo, C.M., Nowak, R., Webley, G.E., McNeil, M.E., Graham, N.B. and Rodway, R.G., E ect of constant-release melatonin implants on the onset of oestrous activity and on reproductive performance in the ewe. Animal Production, 52, 489^497 Durotoye, L.A., Webley, G.E. and Rodway, R.G., Stimulation of the production of progesterone by the corpus luteum of the ewe by the perfusion of melatonin in vivo and by treatment of granulosa cells with melatonin in vitro. Research in Veterinary Science, 62, 87^91 Forcada, F., Zarazaga, L. and Abecia, J.A., E ect of exogenous melatonin and plane of nutrition after weaning on oestrous activity, endocrine status and ovulation rate in Salz ewes lambing in the seasonal anoestrus. Theriogenology, 43, 1179^1193

7 121 Gimeno, N.F., Landa, A., Sterin-Speziale, N., Cardinali, D.P. and Gimeno, A.L., Melatonin blocks in vitro generation of prostaglandin by the uterus and hypothalamus. European Journal of Pharmacology, 62, 309^317 Haresign, W., Peters, A.R. and Staples, L.D., The e ect of melatonin implants on breeding activity and litter size in commercial sheep ocks in the UK. Animal Production, 50, 111^121 Koumitzis, S.A., Belibasaki, S. and Doney, J.M., Melatonin advances and condenses the onset of seasonal breeding in Greek dairy ewes. Animal Production, 48, 399^405 Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J., Protein measurements with the Folin phenol reagent. Journal of Biological Chemistry, 193, 265^275 McMillan, L.H. and Sealey, R.C., Do melatonin implants in uence the breeding season in Coopworth ewes? Proceedings of the New Zealand Society of Animal Production, 49, 43^46 Robinson, J.J., Wigzell, S., Aitken, R.P., Wallace, J.M., Ireland, S. and Robertson, I.S., The modifying e ects of melatonin, ram exposure and plane of nutrition on the onset of ovarian activity, ovulation rate and the endocrine status of ewes. Animal Reproduction Science, 26, 73^91 Rondön, Z., Forcada, F., Zarazaga, L., Abecia, J.A. and Lozano, J.M., Oestrous activity, ovulation rate and plasma melatonin concentrations in Rasa Aragonesa ewes maintained at two di erent and constant body condition score levels and implanted or reimplanted with melatonin. Animal Reproduction Science, 41, 225^236 Thibodeaux, J.K., Broussard, J.R., Godke, R.A. and Hansel, W., Stimulation of progesterone production in bovine luteal cells by co-incubation with bovine blastocyte-stage embryos or trophoblastic vesicles. Journal of Reproduction and Fertility, 101, 657^662 Webley, G.E. and Luck, M.R., Melatonin directly stimulates the secretion of progesterone by human and bovine granulosa cells in vitro. Journal of Reproduction and Fertility, 78, 711^717 Webley, G.E., Luck, M.R. and Hearn, J.P., Stimulation of progesterone secretion by cultured human granulosa cells with melatonin and catecholamines. Journal of Reproduction and Fertility, 84, 669^677 Wigzell, S., Robinson, J.J., Aitken, R.P. and McKelvey, W.A.C., The e ect of oral administration of melatonin at two times of the year on ovarian activity in ewes. Animal Production, 42, 448^449 Yeates, N.T.M., The breeding season of the sheep with particular reference to its modi cation by arti cial means using light. Journal of Agricultural Science, 39, 1^43 Yie, S.M., Niles, L.P. and Younglai, E.V., Melatonin receptors on human granulosa cell membranes. Journal of Clinical Endocrinology and Metabolism, 80, 1747^1749 (Accepted: 27 January 1999)