J.M. Lozano

Effect of level of dietary energy and protein on embryo survival and progesterone production on day eight of pregnancy in Rasa Aragonesa ewes

The aim of this experiment was to investigate the effects of dietary protein and energy on ovulation rate and embryo survival to day 8 of pregnancy, and the associated concentrations of progesterone in jugular, ovarian and uterine veins, in a Spanish breed of sheep. In mid-October, three groups of ewes were fed to provide 1.5 x (H; n = 9), 0.5 x (L; n = 12) or 0.5 x plus 7.44 g CP/MJ ME (LP; n = 8) energy requirements for maintenance of live weight from day -14 relative to a synchronized mating on day 0. A significant effect of nutrition on ovulation rate was observed (H: 2.22 +/- 0.16; L: 1.50 +/- 0.16; LP: 1.88 +/- 0.12 corpora lutea; P < 0.05). Mean LH and progesterone concentrations were affected by nutrition on day 7, L ewes showing the highest mean LH level (P < 0.01), while H ewes presented the lowest mean LH concentration and the highest mean plasma progesterone concentration (P < 0.01). Laparotomies were performed on six animals of each group on day 8 to determine the effect of nutrition on embryo development. A significantly higher percentage of embryos recovered from L and LP ewes presented an earlier stage of development (morulae or early blastocysts) (P < 0.001), while 100% embryos of H ewes were expanded blastocysts. The ratio expanded blastocysts/corpora lutea was significantly higher in H ewes (0.86) when compared with L and LP groups together (0.57; P < 0.05). Mean progesterone concentration in the ovarian vein was 800-fold higher than mean jugular venous levels with no differences between groups. Samples from ovarian veins contralateral to corpus luteum-bearing ovaries showed mean progesterone concentrations significantly lower than samples opposite to corpus luteum (ipsilateral: 1037.84 +/- 138.45; contralateral: 30.4 +/- 11.22 ng/ml; P < 0.001). Mean progesterone concentration in the uterine vein was approximately 30-fold higher than in jugular and similar in both uterine horns and treatments. No effect of nutrition on pregnancy rate was observed (H: 89%; L: 92%; LP: 100%). These results suggest that neither dietary energy nor protein are able to modify pregnancy rate or progesterone concentrations in ovarian and uterine veins eight days after mating. However, the delay in embryo development observed in the embryos collected from L and LP ewes may give rise to compromised embryo growth and development some days later.

Effect of undernutrition on the distribution of progesterone in the uterus of ewes during the luteal phase of the estrous cycle

The effect of undernutrition on ovarian and uterine venous progesterone concentrations and endometrial progesterone content on Days 5 and 10 of the estrous cycle were studied. Forty ewes were synchronized using progestagen pessaries. At pessary withdrawal, the ewes were fed diets to provide either 1.5 or 0.5 times the daily maintenance requirement (Group H, n = 20 and Group L, n = 20, respectively). Ewes fed the low nutrition diet (Group L) had higher mean peripheral progesterone concentrations than those fed the high plane diet (Group H; P < 0.05) but lower endometrial progesterone content on Day 5 (P < 0.05). Neither ovarian nor uterine venous levels were affected by nutrition on either Day 5 or 10. Progesterone concentrations in blood samples collected ipsilateral to ovaries bearing a corpus luteum (CL) were higher than in the contralateral samples (P < 0.001). It is concluded that undernutrition can produce a reduction of endometrial content of progesterone the first week after mating. Since no differences in ovarian venous concentrations were observed, it remains to be shown whether this variation is due to other variables, such as the population of endometrial progesterone receptors or other nonhormonal factors.

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.

Oestrous activity, ovulation rate and plasma melatonin concentrations in Rasa Aragonesa ewes maintained at two different and constant body condition score levels and implanted or reimplanted with melatonin

Forty-seven adult and non-pregnant Rasa Aragonesa ewes were used to study the effect of two constant body condition score levels on reproductive parameters in ewes implanted in early April or reimplanted 49 days later with melatonin. The duration of the experiment was 9 months (27 November 1992 – 3 September 1993), and was designed as a factorial study defined by two constant body condition (BC) scores, high (H; 2.75 or greater) or low (L; 2.50 or less) and the implant on 8 April (M) or reimplant (2M) with melatonin (18 mg, Melovine™). Oestrus was detected daily using vasectomized rams and ovulation rate was measured by laparoscopy 6 days after positive identification. Blood samples were collected twice weekly from each ewe and assayed for progesterone. In addition, samples were collected from 20 ewes (five in each treatment group) every 2 h and hourly for 24 h on Days 27 and 84 after first implantation, respectively, and assayed for melatonin. Onset of seasonal anoestrus was significantly influenced by the BC score (February 16 ± 8 and March 15 ± 5 for H and L groups; P < 0.01), but neither of the factors considered in the study influenced the interval between the first implantation (8 April) and the first detected oestrus (54 ± 7 days, 63 ± 8 days, 64 ± 10 days and 66 ± 5 days for HM, LM, H2M and L2M groups, respectively). Ovulation rate in the third cycle detected from 1 month after first implant insertion was significantly influenced by the BC score level (1.78 vs. 1.44 corpora lutea for H and L groups; P < 0.05). However, ovulation rate in the first and second cycles was slightly lower in the H group (1.46 vs. 1.64 and 1.48 vs. 1.56 corpora lutea, respectively), suggesting a positive effect in the short term of the melatonin implants in ewes with a moderately low BC score. No significant effect of the reimplant on ovulation rate was detected. Plasma melatonin concentrations on Day 84 were not different between groups of ewes receiving one or two implants. Furthermore, and except for nocturnal levels in the high BC score group, ewes that received one implant showed significantly higher melatonin concentrations on Day 84 than on Day 27 after implantation. It is concluded that (i) interval between melatonin implantation and first oestrus is not influenced by the BC score level; (ii) the reimplant does not improve reproductive parameters, although exogenous melatonin seems to overcome the positive effect of a high BC score in the early breeding season; (iii) one implant is adequate to maintain high plasma melatonin levels up to 3 months after implantation.

A preliminary report on the effect of dietary energy on prostaglandin f2α production in vitro, interferon-tau synthesis by the conceptus, endometrial progesterone concentration on days 9 and 15 of pregnancy and associated rates of embryo wastage in ewes

Two groups of ewes were fed to provide 1.70 x (high energy group; n = 15) or 0.56 x (low energy group; n = 15) energy requirements for maintenance of liveweight from 14 d before a synchronized mating in November until slaughter at 9 or 15 d after mating. We investigated the effects on interferon-tau (IFN tau) secretion by the conceptuses, prostaglandin F2 alpha (PG) production in vitro by endometrial tissue, and associated rates of embryo mortality, endometrial progesterone content and progesterone production by luteal tissue. No differences between groups in pregnancy rate were detected on Day 9 between the 2 groups. Proportionately (6/6 vs 2/5), there were more pregnant ewes in the high energy group on Day 15, although this difference did not reach significance (P = 0.06). The proportion of corpora lutea represented by embryos was significantly lower in undernourished ewes (P < 0.05). Secretion in vitro of PG was lower in the 2 pregnant ewes of the low energy group on Day 15, and it was accompanied by higher IFN tau secretion by conceptuses recovered from these ewes. However, the limited number of pregnant ewes recorded on Day 15 prevented any statistical comparison. Neither mean endometrial content of progesterone nor ovarian venous progesterone concentrations and production of progesterone by luteal were affected by nutrition. The provisional results of the present experiment indicate that undernutrition may induce a reduction in the rate of secretion of IFN tau and can therefore increase production of PG from the endometrium. This could initiate luteolysis. The lower pregnancy rates observed in underfed ewes could be mediated through this alteration in the signal of maternal recognition of pregnancy. However, these findings remain to be shown in further experiments including a larger number of animals, as they only represent data from 2 undernourished animals.

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 Effects on Reproductive Performance in the Short and Medium Term of the Combined Use of Exogenous Melatonin and Progestagen Pessaries in Ewes with a Short Seasonal Anoestrous Period

Three hundred and sixty-one adult Rasa Aragonesa ewes were used to evaluate the effect on their reproductive parameters of melatonin implants used in association with the classical synchronization treatment (progestagen plus pregnant mare serum gonadotrophin (PMSG)) in an accelerated breeding system (3 lambings in 2 years). The animals were divided into four groups on the basis of body condition score (H, ≥2.75; L, ≤2.5) and treatment (M) or no treatment (C) with a subcutaneous implant containing 18 mg of melatonin on 17 February 1997. Intravaginal pessaries containing 30 mg of fluorogestone acetate were inserted into all the ewes between 3 and 13 March and 500 IU PMSG was injected intramuscularly when the pessaries were withdrawn 14 days later. Fertile rams were introduced on 17 March. The pregnancy rate and litter size were unaffected by either melatonin treatment or body condition. The overall fertility at first plus second oestrus was between 76.4% (HC) and 82.8% (HM), and the litter size was between 1.58 (LM) and 1.82 (HC) lambs born per lambing. However, melatonin did seem to have a positive effect on the reproductive parameters of non-pregnant ewes at the second mating period 2 months later. Finally, no detrimental effect of melatonin implants on fertility and litter size was recorded in the medium term.

A preliminary study on the effects of dietary energy and melatonin on the ex vivo production of progesterone and prostaglandin F2alpha by the corpora lutea and endometrial tissue of ewes.

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) F2a 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). Veterinary Research Communications, 23 (1999) 115^121 # 1999 Kluwer Academic Publishers. Printed in the Netherlands

Effect of plane of protein after weaning on resumption of reproductive activity in Rasa Aragonesa ewes lambing in late spring

The effect of protein supplementation after weaning on the resumption of reproductive activity in a Spanish breed of ewes (Rasa Aragonesa) with a reduced seasonality was studied. Two equal groups of ewes were weaned in the first 2 weeks of July. From weaning to the end of the experiment both groups were fed identical energy-content rations but with different protein levels (high, Group H; low, Group L). The mean time between weaning and first detected estrus was 64 days. No differences between groups were found either in relation to this interval or in the ovulation rate in the first estrus. Ewes showing a multiple ovulation rate in the first cycle anticipated the onset of sexual activity after weaning. Significant differences in the ovulation rate were detected between the 2 groups from September to the end of the experiment (1.55 vs 1.05 corpora lutea for Groups H and L, respectively; P<0.001). In conclusion, the effect of a protein supplement after weaning in Rasa Aragonesa ewes lambing in late spring is reflected at mid-term in the early breeding season, with an evident rise of ovulation rate in the nutrient supplemented group.

Opioidergic and nutritional involvement in the control of luteinizing hormone secretion of postpartum Rasa Aragonesa ewes lambing in the mid-breeding season

The role of endogenous opioids and nutrition on the inhibition of luteinizing hormone (LH) secretion during the postpartum period was investigated in a Spanish breed of sheep lambing in the mid-late breeding season. Two groups of adult Rasa Aragonesa ewes housed in individual pens and lambing on 30 December were fed during the suckling period to provide maintenance requirements and the production of 1.1 (M; n = 8) or 0.55 (L; n = 8) kg of milk per day. On days 10, 20 and 30 after lambing, the effect of a treatment with the opiate receptor antagonist naloxone (1 mg/kg at four hourly intervals) on LH secretion was assessed in half of the ewes of each group, the remaining females receiving four saline injections. After weaning, animals were fed to provide requirements for maintenance of liveweight. Blood samples were collected twice a week from day 20 postpartum until the end of March, and assayed for progesterone and prolactin. Although underfed ewes showed significantly lower mean plasma concentrations during the control period on day 20 postpartum, nutrition did not seem to modify LH secretion before naloxone or saline injections. Moreover, no differences between nutritional groups in the response to naloxone injections on pattern of LH secretion were found. In fact, naloxone treatment induced an increase of mean LH concentrations on days 10, 20 and 30 postpartum (at least, P < 0.05), of LH pulse frequency on days 20 and 30 (P < 0.05), and of LH pulse amplitude on days 10 and 20 (P < 0.05). Underfed ewes during the postpartum period showed a slower decline in plasma prolactin levels, with significant differences on days 29, 36 and 39 after lambing (P < 0.05). Only 3 M ewes ovulated before the onset of the seasonal anoestrus period. It is concluded that endogenous opioids are involved in the inhibition of LH secretion during the early suckling period of a reduced seasonality breed of sheep without any influence of nutrition on the response to naloxone treatment; however, ewes underfed before weaning failed to reactivate their cyclicity prior to the onset of the seasonal anoestrus.