O Zúñiga

Variation in the ability of melatonin implants inserted at two different times after the winter solstice to restore reproductive activity in reduced seasonality ewes

The purpose of the present study was to assess the effects on reproductive activity of melatonin implants inserted immediately after the winter solstice compared with the classical implantation around the spring equinox. The experiment was conducted at latitude 41° N in the absence of rams. Twenty-one ovariectomised, oestradiol- treated Rasa Aragonesa ewes received (M1, n = 7, 15 January; M2, n = 7, 15 March) or not (C, n = 7) a single implant containing 18 mg melatonin. Blood samples were collected twice weekly from 15 January (M1) and from 15 March (M2, C), and assayed for luteinizing hormone. Six M1 ewes resumed their reproductive activity by 20 April, 95 days after implant insertion, although the reproductive season induced only lasted for 45 days. Melatonin implants inserted on 15 March did not induce a resumption of reproductive activity (0 ewes; P < 0.05). It is concluded that, in the absence of males, the response of Mediterranean ewes to melatonin implants is better when implantation takes place immediately after the winter solstice than around the spring equinox. Two subsequent field experiments with the same breed and year were carried out. Animals were implanted (M) or not (C) with melatonin on 29 December (M, n = 156; C, n = 159) and on 20 March (M, n = 150; C, n = 128). Results confirmed the efficacy of the treatment with melatonin immediately after the winter solstice in ewes with a short seasonal anoestrous period. Implants induced a mean productivity increase of 0.30 (December) and 0.36 (March) lambs per treated ewe compared with those in the control groups.

The effect of melatonin implants on the response to the male effect and on the subsequent cyclicity of Rasa Aragonesa ewes implanted in April

Rasa Aragonesa ewes were used to evalutate whether treatment with melatonin implants in spring could modify: (i) the response to the male effect in terms of oestrous behaviour and ovulation rate; and (ii) the maintenance of sexual activity and ovulation rate at medium term, i.e. over the next 306 days. On 12 April, 42 ewes were divided into two groups, with (M; n = 21) or without (C; n = 21) a subcutaneous implant containing 18 mg melatonin. On 17 May (day 0), three aproned rams were introduced to each group to induce a ram effect. Ewes were observed for oestrus daily. The rams were removed 40 days later after which one aproned ram was introduced daily. Oestrous detection continued until 28 February, 306 days after the first male-female contact. The ovulation rate was determined by endoscopy in the first three cycles after ram introduction and in September-October and January. Progesterone was assayed from blood samples taken on 6 May, 10 and from day 0 to day 22 after ram introduction. Luteal activity before ram introduction was seen in 33% (M) and 29 (C)% of the ewes, respectively. Significantly more M ewes showed oestrous behaviour during the first 40 days after ram introduction (M: 100%; C: 62%; P < 0.01). Similar differences were observed for ewes anovulatory at ram introduction (M: 100%, C: 47%; P < 0.01). These differences were maintained over the three oestrous cycles in both groups. Treatment with melatonin implants was without detrimental effect on cyclic functions in the following breeding season, after seasonal anoestrus. Melatonin treatment significantly increased (P < 0.05) the mean ovulation rate of the first (1.62 +/- 0.11 versus 1.31 +/- 0.13), second (1.78 +/- 0.15 versus 1.36 +/- 0.15) and third cycles (M: 1.73 +/- 0.12 versus C: 1.27 +/- 0.14). There was a significant interaction between the effects of cyclicity at day 0 and melatonin treatment on the ovulation rate in the first cycle (P < 0.05). The mean ovulation rates of both groups were similar at the beginning (September) and middle (January) of the subsequent breeding season. Overall, the results confirmed that melatonin implants, combined with the ram effect, improved the reproductive parameters of reduced-seasonality ewes during spring mating, without impairing sexual activity or ovulation rate during the subsequent breeding season.

Repeated superovulation of high-prolificacy Rasa Aragonesa ewes before culling as an inexpensive way to obtain high-quality embryos.

The purpose of this study was to assess the ovulatory response and embryo production after repeated superovulation of selected high-prolificacy Rasa Aragonesa ewes at the end of their reproductive life. A total of 211 superovulatory treatments were performed during the breeding seasons of 3 consecutive years. Ewes were given the same gonadotrophin treatment up to three times at intervals of at least 50 days. They were synchronized with intravaginal progestagen sponges and treated with ovine follicle stimulating hormone (oFSH) equivalent to a total dose of 176 NIH-FSH-S1 units in eight decreasing doses administered at 12-h intervals from 72 h before sponge removal. Embryos were recovered by laparotomy 7 days after the onset of oestrus. There was no effect of year on the superovulatory response as measured by the percentages of ewes in oestrus (80%) or ovulating (67%). An average of four freezable embryos (compacted morulae and blastocysts) were recovered at each treatment from the 141 ovulating ewes, with no significant differences amongst years, although the process seemed to be less efficient after the third treatment than after the first or second ones (2.4 vs. 4.1 ( , 0.10) or 4.5 (P , 0.05) freezable embryos, respectively). This was primarily attributable to a lower ovulation rate (three fewer corpora lutea; P , 0.10). Results indicate that the methodology used in the present study could be an efficient and inexpensive way to obtain high-quality embryos from selected high-prolificacy animals before culling.

The role of nutrition in the regulation of LH secretion during anestrus by the serotoninergic and dopaminergic systems in Mediterranean ewes treated with melatonin

The steroid-dependent inhibition of LH secretion involves dopaminergic and serotoninergic systems but it is unclear how the plane of nutrition affects this inhibition during anestrus in melatonin treated ewes. Melatonin implants (18 mg) were inserted (Day 0) into ovariectomized, estradiol treated adult Rasa Aragonesa ewes on a high (H; n = 8) or low energy diet (L; n = 6) which were applied in early anestrus (Day 29-57) and late anestrus (Day 90-104). Cyproheptadine (0.1 mg/ kg), a serotoninergic (SHT2) receptor antagonist, was administered in early and late anestrus (Day 50 and 107) followed by pimozide (0.08 mg/kg), a dopaminergic2 receptor antagonist (Day 57 and 114). The H ewes had significantly higher LH concentrations (P < 0.05) before cyproheptadine treatment in early anestrus. The H and L ewes responded in a similar way to the antagonists in both early and late anestrus, except for L ewes who had a higher LH pulse amplitude after pimozide treatment in both periods (P < 0.05). During early anestrus, cyproheptadine tended to increase (P = 0.06) LH pulse frequency in L ewes and LH concentrations in H ewes. The LH secretion also increased in L ewes after pimozide administration during early anestrus (P < 0.05 for mean LH concentrations and LH pulse frequency and amplitude). However, pimozide dramatically increased LH secretion during late anestrus (Day 114) irrespective of the plane of nutrition (P = 0.06-0.08 for LH pulse frequency and P < 0.05 for LH concentrations and pulse amplitude). In melatonin treated Mediterranean ewes, the plane of nutrition appeared to modify the effect of dopaminergic and serotoninergic systems on the steroid-dependent inhibition of LH secretion throughout anestrus.

Regulation of LH secretion during seasonal anestrus by dopaminergic pathways in Rasa Aragonesa ewes treated or not with melatonin

The involvement of the dopaminergic system in the steroid-dependent inhibition of luteinizing hormone (LH) secretion during anestrus in ovariectomized, estradiol-implanted adult Rasa Aragonesa ewes was investigated in both ewes treated with melatonin on 8 March (n = 10) and in control (n = 8) ewes. Melatonin implants did not significantly increase LH secretion. However, treatment with pimozide induced a significant increase (P < 0.05) in LH pulse frequency in both groups during early anestrus. We conclude that, in the absence of males, the dopaminergic system is clearly involved in the inhibition of LH secretion during anestrus in both melatonin-treated and control ewes. Key words: Sheep, melatonin, pimozide

Effect of exogenous melatonin on in vivo and in vitro prostaglandin secretion in Rasa Aragonesa ewes

The effect of exogenous melatonin on prostaglandin secretion was measured on Rasa Aragonesa ewes. Fourteen ewes received an 18 mg melatonin implant (M+) on 10 April and were compared with 13 control animals (without implants M-). Twenty days later, intravaginal pessaries were inserted in all animals to induce a synchronized oestrus (day 0). On day 14, ewes were injected, i.v., with 0.5 IU oxytocin. Plasma 15-ketodihydro-PGF(2alpha) (PGFM) concentrations were measured to assess uterine secretory responsiveness to oxytocin. After euthanasia, pieces of endometrium were collected to determine progesterone content and PGE(2) and PGF(2alpha) secretion in vitro, in the presence or absence of either 20 microg/ml recombinant ovine interferon-tau (roIFNt) or 1 nmol/l oxytocin in the medium. Endometrial progesterone content was similar in the two treatments (M+: 50.25+/-17.34 ng/mg tissue, M-: 43.08+/-11.21 ng/mg tissue). M+ ewes that responded to oxytocin had significantly higher plasma PGFM concentrations between 10 and 80 min after oxytocin administration, a higher mean PGFM peak (P<0.001), higher plasma PGFM levels after the challenge (P<0.05) and higher plasma progesterone concentrations (P<0.01) than control ewes. In the in vitro experiment, M+ and M- control samples secreted similar amounts of PGE(2). The presence of roIFNtau and oxytocin only stimulated PGE(2) production (P<0.05) in M- tissues. Control M+ tissues secreted higher amounts of PGF(2alpha) (P=0.07) and PGF(2alpha) secretion was significantly (P<0.01) stimulated by roIFNtau. Oxytocin produced this effect only in M- samples (P<0.01). In conclusion, although previous studies have demonstrated a positive effect of melatonin on lamb production, PGF(2alpha) secretion is higher in vitro and the PGE(2):PGF(2alpha) ratio is unfavourable in response to IFNtau, which could affect embryo survival. Whether or not these mechanisms are similar in pregnant ewes remains to be elucidated.