ELSEVIER SCIENCE

Animal Reproduction Science 44 (19%) 21 I-221

Ovulation rate in ewes after single oral glucogenic dosage during a ram-induced follicular phase

R.M. Rodriguez Iglesias *, N.H. Ciccioli, H. Irazoqui, C. Giglioli Departamento de Agronomiu, Universidad National de1 SW, &WI-Bahia Blanca, Argentina

Accepted 2 1 February I996

Abstract

A 2-factor factorial array with three replicates (N = 280) was used to simultaneously assess the effects on ovulation rate of two alternative doses of medroxy-progesterone acetate (MPA) (10 or 60 mg), applied during a 6-day priming period, and the effect of a single dosage of a glucogenic formulation, administered immediately before ram exposure to groups of adult seasonally anovular Corriedale ewes. The glucogenic formulation contained 1,2,3-propanetriol (glycerol; 70% v/v), 1,2-propanediol (propylene glycol; 20% v/v) and distilled water (10% v/v). At sponge with- drawal, a single oral dose of 100 ml of this formulation or the same volume of distilled water was administered to treated and control groups, respectively, and ewes were immediately exposed to rams and hormonally-induced oestrous ewes. Data from an ancillary experiment (n = 10) showed significantly (P < 0.005) above normal plasma glucose levels in treated animals at 3 and 6 h after dosage. A significant interaction (P = 0.0006) between MPA priming doses and glucogenic supplementation was detected. Supplemented ewes, among those exposed to the lower dose of MPA, exhibited a higher (P = 0.0098) mean ovulation rate (1.56 k 0.076) than ewes that did not receive glucogenic treatment (1.31 f 0.060). In contrast, ovulation rate was significantly de- creased (P = 0.021) from 1.30 f 0.058 to 1.13 + 0.042 after glucogenic treatment in ewes that were primed with sponges containing 60 mg of MPA. Ewes exposed to 60 mg of MPA were marked by the rams at a significantly later (P < 0.00001) mean time (54.8 + 1.44 h) than ewes receiving 10 mg sponges (43.6 + 1.08 h). These results reveal the potential for modifying ovulation rate through short-term glucogenic manipulations, at least during the compressed follicular phase typical of ram-induced ovulations,

Keywords: Glucose; Medroxy-progesterone acetate; Ovulation rate; Ram effect; Supplementation

* Corresponding address. Department of Rangeland Ecology and Management, Texas A&M University, College Station, TX 77843-2126, USA.

0378-4320/96/$15.00 Copyright 0 1996 Elsevier Science B.V. AI1 rights reserved. PII SO378-4320(96)01564-3

212 R.M. Rodriguez Iglesias et al./Animal Reproduction Science 44 (1996) 2X1-221

1. Introduction

Nutritionally-induced changes in the reproductive rate of sheep have been observed since the start of the domestication process of the species. The so called ‘flushing’ has been, and still is, a common practice to increase sheep reproductive performance, particularly in very seasonal environments. A relatively rapid improvement in body condition, usually associated with a sustained positive energy balance and liveweight gain has been frequently observed to increase both ovulation and lambing rates (Coop, 1966). The relative importance of body reserves, the availability of oxidizable metabolic fuels and the role of various hormones in translating and determining the effects of an improved nutrition is still controversial. Short term high-protein supplementation proto- cols have been observed to alter various reproductive variables (Oldham and Lindsay, 1984). This opened the possibility of increasing ovulation rate at low cost through short term supplementation with high quality diets instead of prolonged periods of improved nutrition.

One of the common factors in many effective short term nutritional protocols seems to be the glucose entry rate associated with nutrient-rich diets (Teleni et al., 1984; Teleni et al., 1989a; Teleni et al., 1989b). Although the role of circulating glucose in triggering mechanisms of ovulation rate control in the ewe is not clear, a strong relationship has been observed between this oxidizable fuel and ovulation rate in short-term supplemen- tation experiments (Teleni et al., 1989b).

Detecting meaningful nutritionally-induced effects on ovulation rate requires large numbers of animals. Intravenous infusion of metabolic fuels has been used extensively to mimic the effects of certain nutrients. However, frequent manipulation, particularly repeated blood extraction, alters ovulation rate in experimental ewes (Adams et al., 1993). In searching for an animal model to test the effect of an enhanced plasma glucose level on ovulation rate, previously anovular ewes induced to ovulate by means of the ‘rams + oestrous ewes effect’ (Rodriguez Iglesias et al., 1991) presented various advantages other than a high efficiency to induce ovulation. As the follicular phase in those animals is ‘compressed’ and reduced to a total duration of less than 2 days, this study hypothesized that a single dose of a glucogenic preparation would be sufficient to elicit a glucose-associated response without altering the normal hormonal profile of the ewes through frequent manipulations. On the other hand, using previously anovular ewes circumvents the problem of possible carry-over effects from previous oestrous cycles, thus increasing the power of statistical tests for the same number of animals.

To reduce the likelihood of return to anoestrus in successfully induced ewes, progesterone priming is required to promote oestrus at the first induced ovulation. From previous unpublished experiments, it was determined that reduced doses of medroxy- progesterone acetate (MPA) were as effective as the commercial formulation (60 mg> in intravaginal sponges for eliciting oestrous behaviour in ram-induced ewes. However, potential effects of alternative dosages of MPA on ovulation rate have not been previously reported. Results from treating cyclic ewes suggested that lower doses of MPA applied in intravaginal sponges were associated with earlier and tighter oestrous synchronization after sponge removal but no information was available for ram-induced oestrus. Thus, the objectives of this study were: (1) to determine the effect of a single

R.M. Rodriguez Iglesias et al./Animal Reproduction Science 44 (1996) 211-221 213

dose of a glucogenic formulation on the ovulation rate of Corriedale ewes induced to ovulate in the middle of the seasonal anoestrus, and (2) to assess the effect of two contrasting doses of MPA on ovulation rate and interval to breeding.

2. Materials and methods

2.1. Animals and experimental procedure

Recently shorn adult (2.5-5.5-year-old) Corriedale ewes from the Argerich Experi- mental Station (latitude: 38”44’S), isolated (sight, sound, smell) from rams since the mating season of the previous spring, were used for the study. Most of these ewes had weaned one or two lambs in early July of the year of the experiment. Sexually experienced adult Corriedale rams were also available. To ensure the presence of oestrous ewes at the start of the experimental period, intact adult Corriedale ewes were brought into standing oestrus when required by intramuscular injections of 500 Fg of oestradiol propionate after a 6-day progesterone priming period.

On 23 November (late spring in southern latitudes), 301 ewes were screened for ovarian activity through mid-ventral laparoscopy performed under local anaesthesia and 280 ewes (93.0%) found acyclic (i.e. no corpora haemorrhagica, corpora lutea or corpora albicantia visible on the surface of the ovaries). These ewes were randomly allocated to 12 experimental units on the basis of age and live weight (54.8 _+ 6.7 kg; mean + s.d.) and then assigned to a 2 X 2 factorial array with three replicates. Factors included were: exposure to two alternative doses (10 or 60 mg> of MPA administered through intravaginal sponges during the priming period before ram exposure and oral administra- tion of either 100 ml of distilled water or an equal volume of a glucogenic supplement. The glucogenic formulation contained 1,2,3-propanetriol (glycerol; 70% v/v), 1,2-pro- panediol (propylene glycol; 20% v/v> and distilled water (10% v/v>. It was orally administered in a single dosage with the aid of an automatic drencher at sponge withdrawal, immediately before introducing the experimental ewes to rams and oestrous ewes. This glucogenic formulation, currently commercialized to treat the symptoms of pregnancy toxaemia in sheep, is reputed to increase plasma glucose levels for about 6-8 h and was chosen on the basis of previously proved effectiveness in reducing the mortality of ewes affected by ketosis.

Intravaginal sponges containing MPA (Gador Labs) were inserted and then removed from different replicates in such a way as to ensure a 6-day priming period before ram exposure in all groups. On 30 November at 09:OO h sponges were removed from ewes in the first replicate and after administration of the oral treatments, the ewes were immediately introduced to 15 crayon-equipped rams and 14 oestrous ewes in an 18 ha paddock. The second and third replicates were treated in an identical way and introduced to rams and oestrous ewes 2 and 4 days later, i.e. on 2 December and 4 December at 09:OO h, respectively, in similarly sized paddocks. Replication allowed testing for interaction and helped in maintaining the logistics and timing of the experimental protocol under control. Ewes detected in oestrus were identified at 12-h intervals from 12 to 120 h after introduction of rams to each replicate. Incidence of ovulation and

214 R.M. Rodriguez Iglesias et al./Animal Reproduction Science 44 (1996) 211-221

ovulation rate were assessed by mid-ventral laparoscopy performed under local anaes- thesia after 5 days of continuous ram exposure.

To avoid any interference of serial blood sampling in the experimental animals on which ovulation rate effects were being assessed (Adams et al., 1993), a different group of 180 ewes, selected from a flock of 380 animals on the basis of similar age and liveweight, was used to test the ability of the glucogenic formulation to increase plasma glucose levels of treated ewes. These ewes were allocated to 18 experimental units through stratified randomization (age and liveweight) and then assigned at random by pairs (treated and control groups) to 9 blood collection times: 0, 3, 6, 9, 12, 15, 18, 21 and 24 h after receiving the oral treatment with either distilled water or glucogenic formulation. Blood samples (5 ml), collected via jugular venipuncture, were centrifuged immediately after collection and kept refrigerated until assay for plasma glucose. Plasma samples were assayed for glucose using a commercial kit within 12 h after collection. The described procedure was adopted to minimize any possible effect of serial blood sampling of the same animals on plasma glucose levels and to keep the glucose temporal trend as close as possible to that expected for the experimental groups exposed to rams. Ewes selected for this purpose were chosen for closeness to average liveweight and body condition. Average liveweight of individual experimental units (n = 10) varied from 57.1 to 57.9 kg while liveweight variances varied between 2.1 and 3.0 kg2. Ewes in the experimental and auxiliary groups were part of the same flock and had been exposed to similar nutritional management before the start of the experiment. All animals grazed native pastures throughout the study. Experimental protocols and animal care followed standards set in the Consortium guide (Consortium for Developing a Guide for the Care and Use of Agricultural Animals in Agricultural Research and Teaching, 1988).

2.2, Statistical analyses

Ovulation and oestrus data were analyzed by maximum likelihood, fitting generalized linear models (McCullagh and Nelder, 1989) with binomial errors and logit link function. Ovulation rate was treated as a threshold character (Falconer, 1981) and analyzed using the same modelling procedure with multinomial error distribution and probit link function. The models contained terms for the main factors (doses of MPA and glucogenic supplementation) and first order interaction. Interval to breeding and plasma glucose data were subjected to analysis of variance. The REG package (Gilmour, 1985) was used for calculations.

3. Results

The temporal trend for the difference in plasma glucose between treated and control ewes in the auxiliary group of 180 ewes is depicted in Fig. 1. Mean plasma levels ( f standard deviation) in control groups varied from 0.59 f 0.09 to 0.62 f 0.06 g l- ’ . Significantly higher (P = 0.0037) plasma glucose levels were observed among ewes supplemented with the glucogenic formulation as soon as 3 h after treatment (Fig. 1).

R.M. Rodriguez Iglesias et al./Animal Reproduction Science 44 (1996) 211-221 215

-0.1 i , I I I I I I I I

0 3 6 9 12 15 18 21 24

Time after treatment (h)

Fig. 1. Differences in plasma glucose concentrations between treated and control groups (n = 10) from 0 to 24 h after single oral dosage. Length of the bars is proportional to two standard errors of the differences.

This difference was even more marked in the next blood sampling period (P = 0.0022) but declined after that and was no longer significant 9 h after the application of the oral dosage.

Thirteen ewes among the experimental animals exposed to rams and oestrous ewes lost their sponges before removal. Their data were excluded from further analyses.

Similar incidences of ovulation were observed in ewes exposed to either 10 (132 of 133, 99.2%) or 60 mg (128 of 134, 95.5%) of MF’A during the priming period. Incidence of ovulation was also similar in ewes receiving distilled water or glucogenic supplement at ram introduction (129 of 132,97.7% and 131 of 135,97.0%, respectively).

A significant interaction (P = 0.0006) between main effects was detected in ovula- tion rate data (Table 1). Supplemented ewes, among those exposed to the lower doses of MPA, exhibited a higher (P = 0.0098) mean ovulation rate (1.56) than ewes that did not receive glucogenic treatment (1.3 1). In contrast, ovulation rate was significantly de- creased (P = 0.0210) from 1.30 to 1.13 after glucogenic treatment in ewes that were primed with sponges containing 60 mg of MPA (Table 1).

The proportion of oestrous ewes marked by the rams within 120 h of exposure was high and not affected by supplementation nor by the different doses of MPA tested (Table 1). However, ewes exposed to a higher content of MPA during the priming period were marked by the rams at a significantly later (P < 0.00001) mean time (54.8 f 1.44 h) than ewes receiving sponges with a lower progestagen content (43.6 &. 1.08 h). A significant trend was also detected (P = 0.044) for ewes treated with the

216 R.M. Rodriguez lglesias et al./Animal Reproduction Science 44 (1996) 211-221

Table 1 Effects of MPA content and glucogenic supplementation on the incidence of oest~s and ovulation, ovulation rate and interval to breeding

MPA content Glucogenic Ovulating Ovulation rate Marked Interval to breeding of the supplement b ewes (%) ’ (mean f s.e.m.) ewes(%) (mean f s.e.m.1 sponges (mg) a

10 + 67 of 68 (98.5) 1.56 kO.076 63 of 67 (94.0) 40.6 f 1.69 10 65 of 65 (100.0) 1.31 f0.060 64 of 65 (98.5) 46.2rt 1.31 60 + 62 of 64 (96.9) 1.13 YcO.042 58 of 62 (93.5) 54.0 f 2.13 60 - 66 of 70 (94.3) 1.30 kO.058 61 of 66 (92.4) 55.5 f 1.97

a Intravaginal sponges inserted for 6 days. b See text for further explanation. ’ Thiin ewes lost sponges.

glucogenic formulation to exhibit an earlier onset of oestrous behaviour as compared with ewes that received distilled water (47.2 f 1.49 vs. 50.6 + 1.23 h). The relationship between interval to breeding and ovulation rate was also explored by means of calculating for each replicate the deviation relative to the average interval to breeding for the four groups treated in each replicate. These deviations were then plotted against

2’o1

;:’

1.2 - i

/’

1.0 I I -I -10 -5 0 5 10

Deviation from mean value of replicate (h)

Fig. 2. Ovulation rate of ewes primed with 10 (open symbols) or 60 (filled symbols) mg of MPA and given a dosage of glucogenic formulation (circles) or distilled water (squares) before ram exposure. Lines connect results from experimental units (n varying from 20 to 24) included in the same replicate.

R.M. Rodriguez Iglesias et al./Animal Reproduction Science 44 (1996) 211-221 217

the corresponding mean ovulation rates for each replicate (Fig. 2). As indicated in Fig. 2, ewes primed with 60 mg of MPA were detected in oestrus consistently later (about 10 h) than ewes primed with 10 mg of MPA. Less marked differences were observed between mean values for ewes receiving the glucogenic supplement as compared with the corresponding controls. On the other hand, it is clear that the trend for each replicate was to show interval to breeding and ovulation rate associated in a negative way for ewes treated with 10 mg of MPA but in a positive one for ewes primed with 60 mg of progestagen.

4. Discussion

The rationale of this study was to test the ability of a glucogenic formulation, reportedly capable of improving clinical symptoms of pregnancy toxaemia, for enhanc- ing ovulation rate by means of producing a short term exposure to elevated plasma glucose levels during a ram-induced follicular phase. As previously observed in this breed, and despite their prolonged anoestrous period in our latitude (over 200 days; Rodriguez Iglesias et al., 1993), ewes readily responded to the stimulus of sexual activity displayed by rams on hormonally-induced oestrous ewes. The proportion of ewes effectively induced to initiate cyclic activity during anoestrus was high and similar to what has been previously reported for this breed in this environment (Rodriguez Iglesias et al., 1991; Rodriguez Iglesias et al., 1992). The experiment showed that ovulation rates as high as those only observed during the peak of the breeding season for this breed (Irazoqui and Menvielle, 1982; Rodriguez Iglesias et al., 1993) can be reproduced in the middle of anoestrus under adequate circumstances. High ovulation rates have been observed in Corriedale ewes in the first induced ovulation after ‘ram + oestrous ewes’ exposure (Rodriguez Iglesias et al., 1991) but mean values over 1.4 are unusual, particularly when animals are stimulated under field as opposed to confined conditions.

The glucogenic supplement caused an above-normal plasma glucose level that reached a peak of 25% over controls in treated ewes after 6 h. Above-normal levels persisted for 6-8 h and then declined to control levels without any apparent sign of rebound (Fig. 1). Drastic changes in ovulation rate in the experimental groups were observed that can be associated with patterns of plasma glucose change in the auxiliary groups. We are not aware of any possible direct effect of any of the components of the glucogenic formulation on ovulation rate other than through promoting an increase in plasma glucose. In particular, the hypothesized effect of injected propylene glycol on ovulation rate (Lopez-Sebastian et al., 1992) has not revealed a clear cause and is probably unrelated to the effects observed in the present study. In our experiment, propylene glycol was orally administered and most probably completely transformed into glucogenic metabolites before leaving the rumen and entering the blood stream.

The observed interaction between glucogenic supplementation and priming progesta- gen dosage was an unexpected though consistent result in the three replicates (Fig. 2). Both metabolic fuels and synthetic progesterone have marked effects on other hormones and metabolites involved in or that exhibit concomitant variation with ovulation control

218 R.M. Rodriguez lglesias et al./Animal Reproduction Science 44 (19%) 211-221

mechanisms (Bronson and Manning, 1991; Schillo, 1992; Kriel et al., 1992; Goodman, 1994). This makes it difficult to assess the possible causes of this interaction.

A progestagen-induced growth hormone (GH) excess has been observed in other animal species following prolonged exposure to MPA and other synthetic progesterone analogs (Selman et al., 1991, 1994). These elevated GH plasma levels are usually accompanied by a concomitant rise in insulin and insulin-like growth factor-I (IGF-I) levels. IGF-I, in turn, has been hypothesized to be involved in the well-known nutritional effects on follicular development observed in ruminants (Downing and Scaramuzzi, 1991; Richards et al., 1991; Momriaux and Pisselet, 1992). However, elevated peripheral IGF-I levels do not seem to be a causal factor in nutritionally- or genetically-increased ovulation rate in sheep (Davis et al., 1990; Spicer et al., 1993; Downing et al., 1995). Local effects on ovarian function have also been suggested in which GH would play an inhibitory role in ovulation rate regulation (Downing et al., 1995). However, IGF-I local production in the ovine ovary seems to be limited (Spicer et al., 1993; Wathes et al., 19951, associated with luteal tissue, and apparently unrespon- sive to GH (Wathes et al., 1995).

An intravenous glucose infusion was effective in increasing basal IGF-I levels, decreasing GH plasma concentrations and restoring IGF-I response to a GH challenge in nutritionally restricted lambs (Kriel et al., 1992). In turn, deprivation of energy sub- strates, particularly glucose, inhibited luteinizing hormone (LH) secretion (Schillo, 1992; Funston et al., 1995) in sheep and prevented oestrus and CL formation in heifers (McClure et al., 1978) apparently acting at centres higher than the anterior pituitary (Funston et al., 1995). Thus, the involvement of specific metabolic fuels in nutritionally-induced changes in reproduction is clearly established. Discrete mecha- nisms remain to be identified.

We have observed before (Rodriguez Iglesias et al., unpublished results) that ewes treated with doses of MPA as low as 5 or 10 mg initiate oestrous activity significantly earlier than those treated with the conventional dose (60 mg) during the breeding season. A prolonged clearance time associated with higher levels of MPA circulating at the time of sponge withdrawal in ewes primed with higher doses may explain this difference. Thus, the known effect of sexual stimulation upon LH pulsatility (Martin et al., 1980) elicited by the sexual activity of rams and oestrous ewes would have been more or less delayed until most of the remaining MPA in circulation was metabolized. Also, the LH surge has been shown to be delayed when MPA has been applied in intravaginal sponges for priming purposes (Martin et al., 1986). For the two alternative doses tested in this study, the difference in clearance time was of approximately 10 h. On the other hand, the period during which plasma glucose was higher than normal or increasing, probably did not exceed 6 h for most ewes treated. Also, the period during which glucose was decreasing to normal levels was of about 3 to 6 h. The delay between the introduction of rams and the preovulatory surge of LH (that can be associated with the onset of the induced oestrus) varies between about 25 to 40 h among different breeds of sheep (Martin et al., 1986). Thus, ewes primed with 10 mg of MPA and supplemented with the glucogenic formulation were exposed to high or increasing levels of plasma glucose during the first hours of hormonal arousal elicited by the rams. In contrast, ewes primed with 60 mg of MPA were exposed to decreasing or back to normal levels of

R.M. Rodriguez Iglesias et al./Animal Reproduction Science 44 (1996) 211-221 219

plasma glucose due to the 10 h delay relative to ewes primed with the lower MPA dose. Non-supplemented ewes primed with 10 or 60 mg of MPA showed almost identical ovulation rates (Table 1, Fig. 2) which is indicative of a null effect on this variable of the priming doses per se.

Nutritionally-induced changes in ovulation rate in sheep seems to be mediated by the action of specific metabolic fuels, particularly glucose, that influence and are influenced by many reproductive hormones/factors. The process by which variations in metabolic fuels are detected and translated into signals that will ultimately affect follicle develop- ment remains to be established. Our results showed that this process may be completed within 6 to 8 h, at least in ewes that are experiencing a ‘compressed’ follicular phase induced by rams and cestrous ewes in the middle of the seasonal anoestrus. This conclusion, however, depends upon the validity of considering the observed interaction as an artifact of the effect of MPA doses on the interval between sponge removal and the onset of LH pulses in successfully stimulated ewes. A thorough testing of this hypothesis would require using groups of ewes with different degrees of delay after supplementation relative to the onset of the ‘ram + oestrous ewes’ exposure. Neverthe- less, it is clear that ovulation rate in ewes can be drastically modified by means of altering the circulating levels of glucose just preceding a ram-induced ovulation. This suggests the possibility of eliciting substantial increases in ovulation rate by means of implementing short-term cost-effective protocols (White and Bowman, 1987) based upon glucogenic supplementation.

Ewes utilized in our experiment were in good, to very good, body condition at mating. Additional work will be needed to investigate whether or not this is a requisite for a consistent response in ovulation rate. Cost efficiency will depend upon the possibility of improving ovulation rate in ewes in sub-optimal and even poor body condition at mating.

Acknowledgements

This work was partially supported by a grant from CONICET (Argentina). MPA was kindly provided by Laboratorios Dr. Gador & Cia (Buenos Aires, Argentina). The skillful assistance of Hector Larrea in recording oestrous data is acknowledged.

References

Adams, N.R.. Atkinson, S., Martin, G.B., Briegel, J.R., Boukhliq, R. and Sanders, M.R., 1993. Frequent blood sampling changes the plasma concentration of LH and FSH and ovulation rate in Merino ewes. J. Reprod. Fertil., 99: 689-694.

Bronson, F.H. and Manning, J.M., 1991. The energetic regulation of ovulation: a realistic role for body fat. Biol. Reprod., ti 945-950.

Consortium for Developing a Guide for the Care and Use of Agricultural Animals in AgriculturaI Research and Teaching, 1988. Guide for the care and use of agricultural animals in agricultural research and teaching. Urbana, IL.

Coop, I.E., 1966. Effect of flushing on reproductive performance of ewes. J. Agric. Sci. (Camb.), 67: 305-323.

220 RN. Rodriguez lglesias et al./Animal Reproduction Science 44 (19%) 211-221

Davis, S.R., Smith, 1.F. and Gluckman, P.D., 1990. Effects of growth hormone injections on ovulation rate in ewes. Reprod. Fertil. Dev., 2: 173-178.

Downing, J.A. and Scaramuzzi, R.J., 1991. Nutrient effects on ovulation rate, ovarian function and the secretion of gonadotrophic and metabolic hormones in sheep. J. Reprod. Fertil. Suppl., 43: 209-227.

Downing, J.A., Joss, J., Connell, P. and Scaramuzzi, R.J. 1995. Ovulation rate and the concentrations of gonadotrophic and metabolic hormones in ewes fed lupin grain. J. Reprod. Fertil., 103: 137-145.

Falconer, D.S., 1981. Introduction to quantitative genetics (2nd edn.). Longman, London. Funston, R.N., Roberts, A.J., Hixon, D.L., Hallford, D.M., Sanson, D.W. and Moss, G.E., 1995. Effect of

acute glucose antagonism on hypophyseal hormones and concentrations of insulin-like growth factor (IGF)-I and IGF-binding proteins in serum, anterior pituitary, and hypothalamus of ewes. Biol. Reprod., 52: 1179-l 186.

Gilmour, A.R., 1985. REG - A Generalised Linear Models Program. Miscellaneous Bulletin No. 1. Department of Agriculture, N.S.W., Australia.

Goodman, R.L., 1994. Neuroendocrine control of the ovine estrous cycle. In: E. Knobil and J.D. Neil1 (Editors), The physiology of Reproduction (2nd edn.). Raven Press, New York, pp. 659-709.

hazoqui, H. and Menvielle, E.E., 1982. Ovulation rate of Corriedale ewes at Bahia Blanca, Argentina. Anim. Prod., 35: 313-320.

Kriel, G.V., Bryant, M.J. and Lomax, M.A., 1992. Effect of dietary protein intake and intravenous glucose infusion on plasma concentrations of insulin-like growth factor-I in lambs. J. Endocrinol., 132: 195-199.

Lopez-Sebastian, A., Gomez-Bnmet, A., Lishmau, A.W., Johnson, S.K. aud Inskeep, E.K., 1992. Modification by propylene glycol of ovulation rate in ewes in response to a single injection of FSH. J. Reprod. Fertil., 99: 437-442.

Martin, G.B., Oldham, C.M. and Lindsay, D.R., 1980. Increased plasma LH levels in seasonally anovular Merino ewes following the introduction of rams. Anim. Reprod. Sci., 3: 125-132.

Martin, G.B., Oldham, C.M., Cogni6, Y. and Pierce, D.T., 1986. The physiological responses of anovulatory ewes to the introduction of rams - a review. Livestock Prod. Sci., 15: 219-247.

McClure, T.J., Nancarrow, C.D. and Radford, H.M., 1978. The effect of 2-deoxy-D-glucose on ovarian function of cattle. Aust. J. Biol. Sci., 31: 183-186.

McCullagh, P. and Nelder, J.A., 1989. Generalized Linear Models (2nd edn.). Chapman and Hall, New York. Monniaux, D. and Pi&et, C., 1992. Control of proliferation and differentiation of ovine granulosa cells by

insulin-like growth factor-I and follicle stimulating hormone in vitro. Biol. Reprod., 46: 109-l 19. Oldham, C.M. and Lindsay, D.R., 1984. The minimum period of intake of lupin grain required by ewes to

increase their ovulation rate when grazing dry summer pasture. In: D.R. Lindsay and D.T. Pearce @ditors), Reproduction in Sheep. Australian Academy of Science, Canberra, pp. 274-276.

Richards, M.W., Wetternan, R.P., Spicer, L.J. and Morgan, G.L., 1991. Nutritional anestrus in beef cows: effects of body condition and ovariectomy on serum luteinizing hormone and insulin-like growth factor-I. Biol. Reprod., 44: 961-966.

Rodriguez Iglesias. R.M., Ciccioli, N.H., Irazoqui, H. and Rodriguez, B.T., 1991. Importance of behavioural stimuli in ram-induced ovulation in seasonally anovular Corriedale ewes. Appl. Anim. Behav. Sci., 30: 323-332.

Rodriguez Iglesias, R.M., Irazoqui, H. and Ciccioli, N.H., 1992. Response of anovular Corriedale ewes to teasing in spring. Small Rumin. Res., 6: 317-322.

Rodriguez Iglesias, R.M., Irazoqui, H., Ciccioli, N.H. and Giglioli, C., 1993. Seasonality of oestrus and ovulation in Corriedale ewes: repeatabilities and phenotypic correlations. J. Agric. Sci. (Camb.), 120: 115-119.

Schillo, K.K., 1992. Effects of dietary energy on control of luteinizing hormone secretion in cattle and sheep. J. Anim. Sci., 70: 1271-1282.

Selman, P.J., Mol. J.A., Rutteman, G.R. and Rijnberk, A., 1991. Progestin and growth hormone excess in the dog. Acta Endocrinol. (Copenh.1, 125: 42-47.

Selman, P.J., Mol. J.A., Rutteman, G.R. and van Garderen, E., 1994. Progestin-induced growth hormone excess in the dog originates in the mammary gland. Endocrinology, 134: 287-292.

Spicer, L.J., Ham&an, J.P., Zavy, M.T. and Enright, W.J.. 1993. Relationship between ovulation rate and concentrations of insulin-like growth factor-l in plasma during the oestrous cycle in various genotypes of sheep. J. Reprod. Fertil., 97: 403-409.

R.M. Rodriguez. Iglesias et al./Animal Reproduction Science 44 (19%) 211-221 221

Teleni, E., Rowe, J.B. and Croker, K.P., 1984. Ovulation rate in ewes: the role of energy- yielding substrates. In: D.R. Lindsay and D.T. Pearce @ditors), Reproduction in Sheep. Australian Academy of Science, Canberra, pp. 277-278.

Teleni, E., King, W.R., Rowe, J.B. and McDowell, G.H., 1989a. Lupins and energy-yielding nutrients in ewes. I. Glucose and acetate biokinetics and metabolic hormones in sheep fed a supplement of lupin grains. Aust. J. Agric. Res., 40: 913-924.

Teleni, E., Rowe, J.B., Croker, K.P., Murray, P.J. and King, W.R., 1989b. Lupins and energy-yielding nutrients in ewes. II. Responses in ovulation rate in ewes to increased availability of glucose, acetate and amino acids. Reprod. Fertil. Dev., 1: 117-125.

Wathes, D.C., Perks, C.M., Davis, A.J. and Denning-Kendall, P.A., 1995. Regulation of insulin-like growth factor-I and progesterone synthesis by insulin and growth hormone in the ovine ovary. Biol. Reprod., 53: 882-889.

White, D.H. and Bowman, P.J., 1987. Economics of feeding energy-based supplements to grazing ewes before mating in order to increase the reproduction rate of a wool-producing flock. Aust. J. Exp. Agric., 27: 11-17.