REPSON SCIENCE ELSEVIER
Animal Reproduction Science 48 ( 1997) 209-2 18
Effect of level of dietary energy and protein on embryo survival and progesterone production on day eight of pregnancy in Rasa Aragonesa ewes J.A. Abecia *, J.M. Lozano, F. Forcada, L. Zarazaga Departamento de Produccio’n Animal y Ciencia de 10s Alimentos, Facultad de Veterinaria, Uniuersidad de Zuragoza, Miguel Seruet, 177, 500/3 Zaragoza, Spain
Accepted 27 February 1997
Abstract 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 = 91, 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 < O.Ol), 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
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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. 0 1997 Elsevier Science B.V. Keywords: Sheep-pregnancy; Feeding and nutrition; Embryology; Progesterone
1. Introduction The effects of energy on embryo survival in sheep have been widely reported in the literature (Brien et al., 1981; Parr et al., 1987; Rhind et al., 1989; Abecia et al., 1996). Reduced rates of embryo survival have been reported in both overfed (Parr et al., 1987) and underfed ewes @hind et al., 1989). There are few experiments designed to determine whether protein level exerts any effect on embryo survival. Van der Westhuysen (1971) fed a high protein supplement in early pregnancy but failed to improve rates of survival. Brien et al. (1981) observed reduced rates of pregnancy in ewes with lupin supplementation, and an increase of embryo mortality in vitro in ewes supplemented with urea was reported by Bishonga et al. (1994). Investigations into the role of progesterone in mediating these effects have produced contradictory results. An inverse relationship between nutrition in early pregnancy and peripheral plasma progesterone concentrations has been observed (Cumming et al., 1971; Parr et al., 1982; Williams and Cumming, 19821, and explained by differences in the rate of clearance of progesterone from the circulation (Parr et al., 1982). A reduction in peripheral plasma progesterone concentrations was observed in the experiment of Brien et al. (1981), although Bishonga et al. (1994) showed no effect on plasma progesterone concentration. The absence of a clear association between embryo survival, plane of energy and peripheral progesterone patterns of secretion led Rhind et al. (1989) to suggest that measurement of progesterone profiles in either the ovarian vein or the ovarian artery could elucidate the relationships between pregnancy rate and nutrition. This is supported by the results obtained by Parr et al. (1982) and Kleemann et al. (19941, which provide evidence that changes to the hormonal environment of the early embryo can influence subsequent development. Abecia et al. (1996) observed higher progesterone concentrations in the ovarian vein and endometrial tissue in pregnant ewes than in animals experiencing embryonic mortality. The consequences of an increment in dietary protein intake in the delivery of progesterone to the uterus remain to be elucidated. This experiment was designed to extend previous works @hind et al., 1989; Abecia et al., 1995; Abecia et al., 1996), which showed negative effects of undernutrition on embryo survival rates, but contradictory results in the effect of treatments on progesterone secretion. Thus, the effect of level of dietary energy and protein on embryo survival and development, and associated progesterone concentrations in both ovarian and uterine veins, was investigated in Rasa Aragonesa ewes. This study was carried out 8 days after mating to determine whether nutritional treatments could affect these reproductive parameters some days before the critical period of implantation.
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2. Materials and methods The experiment was conducted at the experimental farm of the University of Zaragoza (Spain), meeting the requirements of the Scientific Procedure and Breeding of Animals for use in Scientific Procedure Establishments of the European Union. In mid-October, 29 adult Rasa Aragonesa ewes with a mean (+_s.e.) live weight (kg) of 52.4 + 1.32 and a mean body condition score of 2.92 * 0.05 were housed in individual pens at 41”4O’N, under natural daylength conditions. Animals were divided into three groups, on the basis of live weight and body condition score. They were fed to provide 1.5 X maintenance requirements for live weight (M) (high group; H; n = 9); 0.5 x M (low group; L; n = 12) or 0.5 M plus a protein supplement (low + protein group; LP; n = 8). The pelleted diet used consisted of alfalfa hay (60%); barley (30%) and soya bean meal (10%) (17.5% CP). From day - 14 relative to a synchronized mating (day 0) using intravaginal progestagen pessaries (Intervet, Spain) animals were given 1.135 kg (H; 12.4 MJ metabolizable energy (ME)); 0.450 kg (L; 4.1 MJ ME) pellets and 0.450 kg pellets plus 0.1 kg fishmeal (30.5% CP) (LP; 4.1 MJ ME plus 7.44 g CP/MJ ME) per head per day. These feeding regimes were maintained throughout the experiment. From 32 to 80 h after pessary withdrawal, ewes were mated, and the oestrus was checked every 8 h. Twenty-four hours after pessary withdrawal (follicular phase), and on day 7 (luteal phase), blood samples were collected via jugular catheters for 8 h at 15 min intervals, and assayed for LH (follicular phase) and LH and progesterone (luteal phase). On day 8, ovulation rate was recorded by laparotomy in six ewes of each group, and by laparoscopy in the remaining ewes. Laparotomies were performed under general anaesthesia induced by sodium thiopental (Tiobarbital, Braun Medical, Spain). The number of corpora lutea on each ovary were recorded. The uterine horns were then flushed using Foley catheters, and the embryos counted and classified by morphological criteria (Winterberger-Tomes and Sevellec, 1987) using a stereomicroscope. If, after the first flushing, the number of embryos counted was not equal to the number of corpora lutea, a new flushing was performed to confirm embryo loss. After that, a blood sample was collected from both ovarian veins, from the largest uterine branch of the ovarian veins, and from the jugular vein. Plasma samples were analysed for progesterone. Animals were ovariectomized, and corpora lutea and follicles with a diameter higher than 2.5 mm were dissected from the ovarian stroma. The rate of secretion of progesterone from luteal tissues and oestradiol, and testosterone from follicles were analysed as described by Abecia et al. (1995). Briefly, luteal tissue was finely chopped and suspended in l-ml culture medium (M199, Sigma Chemical, St. Louis) without (three replicates) or with (three replicates) an excess of LH (1000 ng LH; NIADDKoLH-26), and incubated for 2 h at 37°C. After incubation and centrifugation, the supematant was collected and progesterone content was analysed. The follicles were incubated individually under the same conditions. The medium was analysed for testosterone and oestradiol concentrations. The incidence of pregnancy in the remaining experimental animals was determined using progesterone concentrations at 17 days after mating (concentrations higher than
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0.5 ng/ml were deemed pregnant). Pregnancy was confirmed by the absence of new corpora lutea at laparoscopy at 25 days after mating. Plasma LH concentrations were determined by radioimmunoassay (RIA) using the techniques of McNeilly et al. (1986). The sensitivity of the assay was 0.2 ng/ml and intra- and inter-assay coefficients of variation were 11.2% and 14.0%. Progesterone determinations and testosterone and oestradiol content of the follicle incubates were performed using solid-phase RJA kits based on antibody coated tubes, 1251-labelled hormones, and rabbit antiserum (bioMCrieux SA, Marcy L’Etolie, France). The assay sensitivities were 0.05 ng progesterone per ml, 0.2 ng testosterone per ml and 8 pg oestradiol per ml. Irma- and inter-assay coefficients of variation for the progesterone assay were 7.7% and 14.3%, respectively. Testosterone and oestradiol samples were run in single assays, with intra-assay coefficient of variation of 2.7% and 2.0%, respectively. Hormone pulses were defined according to Baird et al. (1981); a pulse was deemed to have occurred when two consecutive values were higher than the two preceding values, and when the value of the highest (pulse amplitude) exceeded the mean basal value by at least four times the coefficient of variation of the assay. The effect of plane of nutrition on live weight and body condition, plasma LH and progesterone concentrations, LH and progesterone pulse frequencies and amplitudes, and testosterone, oestradiol and progesterone secretion in vitro, were compared using analysis of variance. Ovulation and pregnancy rates and embryo stage of development were compared using x2 tests.
3. Results Both L and LP animals significantly declined in liveweight and body condition compared with H ewes during the period in which nutritional treatments were applied (P < 0.05); most of the reduction occurred before mating. H animals showed little or no change (Table 1). The interval between pessary withdrawal and mating (h) was similar in the three groups (H: 50.2 f 4.4; L:53.0 f 9.42; LP:55.2 f 9.0). Both energy and protein supplement increased the mean ovulation rate (2.22 + 0.16; 1.50 f 0.16 and 1.88 + 0.12;
Table 1 Live weight (LW; kg) and body condition (BC) score 14 days before mating (- 14). at mating (0) and7 days after mating (+ 7) in ewes fed 1.5X (H), 0.5 X (L) and 0.5 X maintenance requirements plus 7.44 g CP/MJ metabolizable
energy (LP)
Group
H (n = 9)
L(n=12)
LP (n = 8)
Significance
LW -14 LWO LW f7 BC -14 BCO BC +7
52.5 55.2 52.4 2.94 2.83 2.89
52.1 49.9 47.5 2.94 2.58 2.54
52.4 50.0 48.9 2.84 2.56 2.59
NS P < 0.05 P < 0.05 NS P
