Effect of diet and GnRH administration on post-partum ovarian cyclicity in autumn-lambing ewes

Animal Reproduction Science 76 (2003) 67–79

Effect of diet and GnRH administration on post-partum ovarian cyclicity in autumn-lambing ewes L.M. Mitchell∗ , M.J. Ranilla, G. Quintans, M.E. King, F.E. Gebbie, J.J. Robinson SAC, Animal Biology Division, Craibstone Estate, Bucksburn, Aberdeen AB21 9YA, UK Received 1 May 2002; received in revised form 22 October 2002; accepted 8 November 2002

Abstract Using autumn-lambing ewes, this study investigated (i) the effects of diet on gonadotrophin secretion and responsiveness of the hypothalamic-pituitary-ovarian axis to exogenous GnRH during the early post-partum period; and (ii) whether ovulation prior to completion of uterine involution results in an increased incidence of aberrant ovarian cycles. Thirty-two ewes rearing 1.9 ± 0.12 lambs were equally allocated to two dietary treatments at lambing (22 October ±0.2 day). Diets comprised ad libitum hay and 1.5 kg per ewe per day of one of two concentrates (11.5 MJ ME, 195 g CP per kg) containing 300 g kg−1 cracked maize grain (M) or 300 g kg−1 sugar beet pellets (S). Half of the ewes on each diet (G) received 25 i.v. injections of 250 ng GnRH in 2 ml 0.9% saline at 2 h intervals from days 12–14 post-partum while remaining ewes (N) were monitored for the resumption of spontaneous ovarian cyclicity. Blood samples were obtained from all ewes throughout the study (lambing to 18 December) for measurement of circulating hormone concentrations and the uteri and ovaries of all ewes were examined via laparoscopy on day 21 post-partum. There were no effects of dietary treatment on ewe daily live weight loss, lamb daily live weight gain or the immediate post-partum increase in circulating FSH concentrations. Diet did not affect insulin concentrations or LH pulse frequency on day 12 post-partum but LH pulse amplitude was lower in ewes fed concentrate M compared to concentrate S (1.4 ± 0.10 versus 1.7 ± 0.12 ng ml−1 , respectively, P < 0.05) and this was associated with an increased interval to the resumption of spontaneous ovarian cycles (35 ± 3.1 versus 26 ± 2.1 day, respectively, P < 0.05). Administration of exogenous GnRH increased (P < 0.05) the proportion of ewes on both diets that ovulated within 20 days of parturition and advanced the onset of ovarian cyclicity in ewes fed concentrate M by 9.5 days (significance of interaction, P < 0.05). Four ewes, all of which ovulated before day 22 post-partum, had extended luteal activity while in remaining ewes, duration of the first luteal phase was inversely related to the time of first ovulation (r 2 = 0.16, P < 0.05). Results demonstrate that (i) the onset of ∗ Corresponding author. Tel.: +44-1224-711067; fax: +44-1224-711292. E-mail address: [email protected] (L.M. Mitchell).

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ovarian cyclicity is influenced by diet and can be advanced by administration of exogenous GnRH; and (ii) ovulation during the early post-partum period is associated with an increased incidence of extended luteal activity. © 2002 Elsevier Science B.V. All rights reserved. Keywords: Sheep-ovary; Post-partum period; Gonadotrophins; GnRH; Ovulation; Ovarian cyclicity

1. Introduction Post-partum anoestrus and anovulation allow the functional integrity of the reproductive system to be restored following pregnancy. In both cattle and sheep, high circulating oestrogen concentrations during pregnancy inhibit the pulsatile release of GnRH from the hypothalamus. This in turn, inhibits the synthesis, storage and release of FSH and LH from the anterior pituitary (Nett, 1987). The dramatic fall in circulating oestrogen concentrations at the time of parturition leads to an immediate post-partum increase in FSH secretion which initiates a wave of ovarian follicular growth (cattle: Beam and Butler, 1997; sheep: Mandiki et al., 1990). Follicle maturation and ovulation do not however, occur unless there is a concomitant increase in LH pulsatility. In cattle, the presence of the calf and suckling prevent restoration of LH pulsatility during the early post-partum period (Williams, 1990). While there is little evidence for suckling-induced inhibition of LH pulsatility in sheep (Schirar et al., 1990), most ewes lamb at the end of their natural breeding season when photoperiod is inhibitory. If calves are removed within 24 h of parturition (as in modern dairying systems), or ewes lamb within their natural breeding season, the early restoration of high frequency LH pulses can lead to ovulation of the first large follicle(s) formed (cattle: Beam and Butler, 1997; sheep Mitchell et al., 1998). This may occur at a time before uterine involution is fully completed with possible adverse consequences for fertility. In the absence of suckling-induced inhibition (cattle) or non-stimulatory photoperiod (sheep), the main factor influencing activity of the post-partum reproductive axis is nutrition. Energy supply affects both pulsatile LH secretion and ovarian responsiveness to gonadotrophin stimulation via alterations in the availability of glucose and (or) associated metabolic hormones, such as insulin (Schillo, 1992; Beam and Butler, 1999). Ruminants depend on hepatic gluconeogenesis from the rumen volatile fatty acid, propionate, to meet their glucose requirements. However, some feeds, such as maize grain, contain starch in a form that can partially escape rumen fermentation and supply glucose directly from the small intestine (Ørskov, 1986). The aim of the present study was to investigate the effect of diets containing maize grain or sugar beet pellets on post-partum gonadotrophin secretion and ovarian cyclicity in autumn-lambing ewes. These dietary ingredients were chosen because they contain similar levels of energy and protein but differ in their starch content (700 versus 65 g kg−1 DM, respectively), with potential consequences for neuroendocrine status and post-partum ovarian function (Monget and Martin, 1997). Exogenous GnRH was administered to half of the ewes on each dietary treatment to determine their responsiveness, and the effect of ovulation prior to completion of uterine involution on subsequent ovarian cyclicity. The research is

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of relevance to flocks of pedigree or dairying ewes that lamb within their natural breeding season, and to dairy cows where ovulation may occur during the early post-partum period. 2. Materials and methods 2.1. Animals and experimental design Experimental procedures were licensed under the UK Animals (Scientific Procedures) Act of 1986. Protocols and animal care were approved by the Animal Ethics Committee of the Scottish Agricultural College. The study was conducted at 57◦ N using 32 Mule (Bluefaced Leicester × Scottish Blackface) ewes selected from a larger population which established pregnancy following withdrawal of intra-vaginal progestagen sponges, i.m. injection of 600 IU Pregnant Mares Serum Gonadotrophin (Intervet, Cambridge, UK), and laparoscopic intra-uterine insemination during mid-anoestrus. Ewes were maintained at pasture until 7 October when they were housed, and fed hay and molasses ad libitum and up to 700 g d−1 of a standard ewe concentrate (11.5 MJ ME, 195 g CP per kg) during the last 4 weeks pre-partum. Selected ewes were equally allocated to four groups at lambing (mean ± S.E.M. date, 22 October ±0.2 day), and were group-housed, with their lambs, under natural photoperiod. Throughout the study (lambing to 18 December), ewes were fed 1.5 kg d−1 of one of two concentrates (16 ewes per diet) and had ad libitum access to hay. Concentrates were formulated to contain 11.5 MJ metabolisable energy and 195 g crude protein per kg and were identical in composition except that one contained 300 g kg−1 cracked maize grain (concentrate M) and the other, 300 g kg−1 sugar beet pellets (concentrate S). Remaining ingredients were bruised barley, soya bean meal, fishmeal and a mineral/vitamin premix. Half of the ewes on each diet were treated with exogenous GnRH to induce ovulation within 20 days of parturition (treatment G) while remaining ewes (treatment N) were monitored for the resumption of spontaneous ovarian cyclicity. 2.2. Measurements Ewe body condition score was assessed (Russel et al., 1969), and ewes and their lambs were weighed at 2-week intervals throughout the study. The protocol used for blood sampling and administration of exogenous GnRH is shown in Fig. 1. Blood samples were obtained from all ewes by jugular venepuncture (Vacutainer, Becton-Dickinson Limited, Coventry, UK) twice-daily (0800 and 1800 h) from parturition until day 11 post-partum, and three times per week (Monday, Wednesday, Friday) during the remainder of the study for measurement of circulating FSH and progesterone concentrations. All ewes (and their lambs) were temporarily housed in individual pens from days 12 to 14 post-partum. Ewes were fitted with an indwelling jugular catheter (Intraflon 2 i.v., Vycon, Ecouen, France) and blood samples were withdrawn at 15 min intervals (0900–1700 h on day 12) for measurement of pulsatile LH secretion. Administration of exogenous GnRH to ewes in groups MG and SG commenced 2 h after the final 15 min sample and comprised a series of 25 i.v. injections (250 ng GnRH (Sigma L7134) in 2 ml 0.9% saline) given via the

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Fig. 1. Protocol used for blood sampling and administration of exogenous GnRH to ewes fed concentrates containing maize grain (group MG) or sugar beet pellets (group SG). Procedures are shown relative to lambing (22 October ±0.2 day).

catheter at 2 h intervals. LH concentrations in blood samples obtained immediately prior to each GnRH injection were measured to determine the occurrence of an induced LH surge. The ovaries and uteri of all ewes were examined via laparoscopy (Boyd and Ducker, 1973) on day 21 post-partum. For this procedure, ewes were sedated by i.m. injection of 0.6 ml acepromazine maleate BP (ACP injection, 10 mg ml−1 , C-Vet Limited, Bury St Edmunds, UK) and local anaesthesia at trochar and cannula entry sites was achieved by s.c. injection of 10 ml lignocaine hydrochloride BP/adrenaline BP (Lignol, Arnolds Veterinary Products, Shrewbury, UK). 2.3. Radioimmunoassay Plasma was separated from blood samples by centrifugation and stored at −20 ◦ C prior to analysis. FSH, LH and progesterone concentrations, and insulin concentrations in five samples obtained at 2 h intervals between 0900 and 1700 h on day 12 post-partum were measured by radioimmunoassay. For each assay, at least three quality control samples were included with concentrations known to be low, intermediate and high in relation to the standard curve. These quality controls were used to determine the inter- and intra-assay coefficients of variation for each hormone. FSH concentrations were measured in duplicate 200 ␮l aliquots of plasma as described by Crowe et al. (1995). Mean concentrations in the low, medium and high quality controls were 0.13, 0.40 and 0.96 ng ml−1 , respectively. Inter- and intra-assay coefficients of variation (n = 30 assays) were 10.0 and 5.7%, respectively, and the minimum detection limit was 0.05 ng ml−1 . LH concentrations were measured in duplicate 50 ␮l aliquots of plasma using the modified method of Niswender et al. (1969). Mean concentrations in the low, medium and high quality controls were 0.80, 2.45 and 7.59 ng ml−1 , respectively. Inter- and intra-assay coefficients of variation (n = 41 assays) were 18.1 and 12.6%, respectively, and the minimum detection limit was 0.30 ng ml−1 . Progesterone concentrations were measured in duplicate 100 ␮l aliquots of plasma as described by McNeilly and Fraser (1987). Mean concentrations in the low, medium and high quality controls were 1.19, 2.80 and 6.00 ng ml−1 , respectively. Inter- and intra-assay coefficients of variation (n = 16 assays) were 14.0 and 10.3%, respectively, and the minimum detection limit was 0.60 ng ml−1 .

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Insulin concentrations were measured in duplicate 100 ␮l aliquots of plasma using the modified method of Starr et al. (1979). Mean concentrations in the low, medium and high quality controls were 6.95, 17.08 and 24.18 ␮lU ml−1 , respectively. Inter- and intra-assay coefficients of variation (n = 10 assays) were 13.2 and 9.6%, respectively, and the minimum detection limit was 2.73 ␮lU ml−1 . 2.4. Definitions Mean LH concentrations, and the frequency and amplitude of LH pulses on day 12 post-partum were determined by the use of algorithms (PULSAR, Merriam and Wachter, 1982). An LH surge was considered to have occurred in response to exogenous GnRH if LH concentrations were maintained above 4.5 ng ml−1 (the amplitude of the highest recorded LH pulse) in at least three samples obtained at 2 h intervals during the period of GnRH administration. The onset of the surge was defined as the time when LH concentrations first exceeded 4.5 ng ml−1 , and the end as the time when LH values returned to below 4.5 ng ml−1 . The onset of ovarian cyclicity was defined as the date of the first rise in progesterone concentrations to a value greater than 1 ng ml−1 that was sustained for at least two consecutive samples. Ovarian cycles were defined as ‘short’ if the duration of luteal activity (progesterone concentrations above 1 ng ml−1 ) was less than 10 days, and ‘extended’ if the duration of luteal activity was more than 20 days. 2.5. Statistical analyses Data pertaining to ewe body condition scores, and ewe and lamb live weights at parturition were analysed by ANOVA (Minitab 11.12, Minitab Inc., USA) with the number of lambs born included as a covariate. Daily live weight change for ewes and their lambs throughout the study was estimated as the slope of a linear regression line of weight on time for each animal. The immediate rate of post-partum increase in FSH concentrations was similarly estimated as the slope of a linear regression line drawn from parturition to the first maximum value obtained for each ewe. ANOVA was used to determine the effect of diet on ewe and lamb daily live weight change, FSH secretion, LH pulsatility and insulin concentrations on day 12 post-partum. The number of lambs reared was included as a covariate in the analyses. The effects of diet and administration of exogenous GnRH on the interval from parturition to the onset of ovarian cyclicity, and the duration of the first luteal phase were analysed using ANOVA and χ 2 . 3. Results Ewes reared between one and three lambs (overall mean ±S.E.M., 1.9±0.12) with those rearing singles being equally allocated to treatment groups. Daily concentrate allowances, divided between three feeds of 0.5 kg per ewe at 0730, 1230 and 1730 h, were completely consumed.

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3.1. Ewe body condition score and ewe and lamb live weight The overall mean ± S.E.M. body condition score of ewes at lambing was 2.8 ± 0.05 units (scale of 1–5; 1: emaciated, 5: obese) and their live weight was 72 ± 1.2 kg. Daily live weight losses throughout the study for ewes fed concentrates M and S (119 ± 15.8 versus 104 ± 12.2 g d−1 , respectively) were not significantly different. The overall mean ± S.E.M. birth weight of lambs reared by ewes fed concentrates M and S was 4.4 ± 0.13 kg, and their daily live weight gains throughout the study were 255 ± 13.6 versus 257 ± 13.1 g d−1 , respectively. 3.2. FSH concentrations Mean ± S.E.M. FSH concentrations during the early post-partum period for ewes fed concentrates M and S are shown in Fig. 2. There was no effect of dietary treatment on the rate of immediate post-partum increase in FSH concentrations (0.14 ± 0.086 versus 0.14 ± 0.016 ng ml−1 d−1 , respectively), the first peak value attained (1.02 ± 0.101 versus 0.93 ± 0.083 ng ml−1 , respectively) or the interval from parturition to the first peak value (8.3 ± 0.57 versus 7.3 ± 0.55 d, respectively). 3.3. Pulsatile LH secretion Diet did not affect LH pulse frequency on day 12 post-partum but LH pulse amplitude and mean concentration were significantly (P < 0.05) lower in ewes fed concentrate M compared to concentrate S (Table 1). Within diets, measures of pulsatile LH secretion did not differ for ewes subsequently treated with GnRH or monitored for the resumption of spontaneous ovarian cyclicity (data not shown).

Fig. 2. FSH concentrations during the early post-partum period in autumn-lambing ewes fed concentrates containing 300 g kg−1 maize grain (䊉) or 300 g kg−1 sugar beet pellets (䊊). Means ± S.E.M.

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Table 1 LH secretion on day 12 post-partum in autumn-lambing ewes fed concentrates containing 300 g kg−1 maize grain (M) or 300 g kg−1 sugar beet pellets (S). Means ± S.E.M.

No. ewes LH pulse frequency (h−1 ) LH pulse amplitude (ng ml−1 ) LH concentration (ng ml−1 )

Concentrate M

Concentrate S

Significance

16 0.25 ± 0.021 1.36 ± 0.095 0.80 ± 0.047

16 0.32 ± 0.028 1.72 ± 0.119 1.01 ± 0.078

ns P < 0.05 P < 0.05

3.4. Insulin concentrations Peripheral insulin concentrations showed little fluctuation during the 8 h period of measurement on day 12 post-partum (Fig. 3). Overall mean ± S.E.M. values for ewes fed concentrates M and S were not significantly different (15.3±1.25 versus 18.4±2.83 ␮lU ml−1 , respectively). 3.5. Response to exogenous GnRH Three ewes, one fed concentrate M and two fed concentrate S, experienced an LH surge during the period of GnRH administration (Table 2). All three ovulated as evidenced by the presence of luteal tissue on their ovaries and progesterone concentrations above 1 ng ml−1 at the time of laparoscopy. Early ovulations were recorded for a further four ewes following GnRH treatment but their pre-ovulatory LH surges did not occur within the period of measurement.

Fig. 3. Insulin concentrations on day 12 post-partum in autumn-lambing ewes fed concentrates containing 300 g kg−1 maize grain (䊉) or 300 g kg−1 sugar beet pellets (䊊). Means ± S.E.M.

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Table 2 Response to administration of exogenous GnRH in autumn-lambing ewes fed concentrates containing 300 g kg−1 maize grain (M) or 300 g kg−1 sugar beet pellets (S) Concentrate M No. ewes Treated with GnRH With a measured LH surge Ovulating within 20 days post-partum

Concentrate S

8 1 4

LH surge characteristics Interval, first GnRH injection to LH surge (h) LH surge duration (h) Peak LH concentration (ng ml−1 )

8 2 3 31 ± 5.0 9 ± 1.0 13.7 ± 1.85

16 8 28.8

3.6. Onset and pattern of ovarian cyclicity In contrast to ewes treated with exogenous GnRH, none of the ewes monitored for the resumption of spontaneous ovarian cyclicity had ovulated at the time of laparoscopy. Three ewes fed concentrate S were however, on the point of ovulation as evidenced by the presence of at least one large (>6 mm) ovarian follicle and an increase in peripheral progesterone concentrations in the subsequent blood sample. There was no effect of diet or GnRH administration on the mean±S.E.M. interval from parturition to the onset of ovarian cyclicity (Table 3) but the interaction between these factors was significant (P < 0.05). The resumption of spontaneous ovarian cyclicity occurred 9.5 days later in ewes fed concentrate M compared to concentrate S and was advanced by exogenous GnRH. For ewes on both diets, GnRH administration increased the proportion ovulating within 20 days of parturition (P < 0.05). The relationship between the time of onset of ovarian cyclicity and duration of the first luteal phase is shown in Fig. 4. Prolonged elevations in peripheral progesterone concentrations characteristic of persistent corpora lutea were apparent in four ewes (12.5%), all of which ovulated before day 22 post-partum. In each case, progesterone concentrations Table 3 Interval from parturition to the onset of ovarian cyclicity, and duration of the first luteal phase in autumn-lambing ewes fed concentrates containing 300 g kg−1 maize grain (M) or 300 g kg−1 sugar beet pellets (S) and treated with exogenous GnRH or monitored for spontaneous ovarian cycles Concentrate M

No. ewes Mean ± S.E.M. post-partum interval (day) Range (day) No. ewes, short (<10 days) first luteal phase No. ewes, long (>20 days) first luteal phase

Concentrate S

Significance

+GnRH

−GnRH

+ GnRH

−GnRH

Diet × GnRH

8 24.0 ± 3.23

8 35.3 ± 3.10

8 28.6 ± 3.85

8 25.8 ± 2.14

P < 0.05

15–39 0

25–44 1

16–43 1

21–37 0

2

0

1

1

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Fig. 4. Relationship between the interval from parturition to the onset of ovarian cyclicity and duration of the first luteal phase in autumn-lambing ewes.

remained elevated throughout the study. Three ewes (9.4%), all of which ovulated after day 33 post-partum, experienced an initial short cycle, while a further six late-ovulating ewes (18.8%) experienced a transient (one sample) increase in progesterone concentrations immediately prior to the first luteal phase. Duration of the first luteal phase was not affected by diet or GnRH administration but was inversely related to the time of onset of ovarian cyclicity (r 2 = 0.16, P < 0.05 for ewes without extended luteal activity). 4. Discussion In the present study, autumn-lambing ewes were fed one of two concentrates formulated to contain 30% cracked maize grain or 30% sugar beet pellets and the same levels of metabolisable energy and crude protein. Diet did not affect ewe daily live weight loss or lamb daily live weight gain during the first 8 weeks post-partum, implying that there were no major differences in ewe milk yield or composition in response to concentrate feeding. Maize grain has around 10 times the starch content of sugar beet pellets and the starch is in a form that can escape rumen fermentation and supply glucose directly from the small intestine (Ørskov, 1986), with potential stimulatory effects on metabolic hormones, such as insulin. Circulating insulin concentrations measured on day 12 post-partum were not however, increased in ewes fed the maize-containing compared to the sugar beet-containing concentrate and we can only, in retrospect, speculate as to the possible reasons for this. While maize-containing diets have been shown to increase glucose entry rate in non-pregnant, non-lactating sheep (Janes et al., 1985; Landau et al., 1992), no such advantage has been demonstrated in late-pregnant ewes (Landau et al., 1999) or lactating dairy cows (Crocker et al., 1998). The response of ruminants to dietary maize may therefore depend on their physiological status (Reynolds et al., 1997).

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During late-pregnancy and early lactation, rumen outflow rates are increased (Coffey et al., 1989) and this may be associated with an increase in the proportion of maize starch escaping rumen fermentation. Following digestion in the small intestine, uptake of glucose across the apical brush border requires the sodium-dependent glucose co-transporter (SGLT1), the activity of which is regulated by hexose availability (Hocquette and Hiroyuki, 2000). It takes several days for maize to pass through the rumen (Warner, 1981) and a further 4 days for sugars released in the small intestine to up-regulate the SGLT1 gene (Zhao et al., 1998). In the present study, experimental diets were introduced at lambing and it is possible therefore, that insufficient time had elapsed before measurement of circulating insulin concentrations for full expression of glucose co-transporter activity. It is also possible that circulating insulin concentrations were lower in ewes fed the maize-containing compared to the sugar beet containing concentrate during the immediate post-partum period as a consequence of their inability to absorb glucose from the small intestine at this time. In agreement with previous studies (Mandiki et al., 1990; Beam and Butler, 1997), ewes on both dietary treatments experienced an immediate post-partum increase in FSH secretion in response to the withdrawal of placental oestrogen. This would have stimulated a synchronous wave of ovarian follicular development with the fate of the follicles being determined by the restoration of LH pulsatility (Nett, 1987). Dietary treatment did not affect LH pulse frequency on day 12 post-partum but pulse amplitude was lower in ewes fed the maize-containing compared to the sugar beet-containing concentrate. Pituitary stores of LH are minimal around the time of parturition as GnRH secretion is suppressed during late-gestation and synthesis of the LH-␤ sub-unit depends on frequent GnRH pulses (Shupnik, 1996). Hypothalamic stores of GnRH and pituitary GnRH receptors are not however, compromised (Wise, 1990) and LH release during the early post-partum period is strongly correlated with pituitary LH content (Moss et al., 1980; Crowder et al., 1982). It is likely therefore, that the lower LH pulse amplitude in ewes fed the maize-containing compared to the sugar beet-containing concentrate represented a more gradual restoration of pituitary LH stores after parturition. This may have occurred because lower circulating insulin concentrations during the period of intestinal adaptation delayed the post-partum restoration of GnRH pulsatility (Schillo, 1992), and perhaps contributed to the delayed resumption of spontaneous ovarian cyclicity (Moss et al., 1980). GnRH was administered to half of the ewes on each diet from days 12–14 post-partum, approximately 2 weeks earlier than in previous studies (Fray et al., 1995) and at a time when pre-ovulatory sized follicles may have been present on the ovaries (Cahill and Mauleon, 1980; Al-Gubory and Martinet, 1986). The frequency of exogenous GnRH pulses (one per 2 h) was greater than that of endogenous pulses (as determined from LH pulse frequency) and would have stimulated LH synthesis and secretion as previously shown in nutritionally-restricted ewes (Kile et al., 1991). It is likely therefore, that exogenous GnRH prevented the delay in the onset of ovarian cyclicity in ewes fed the maize-containing concentrate by enhancing post-partum LH pulsatility. Exogenous GnRH did not advance the onset of ovarian cyclicity in ewes fed the sugar beet-containing concentrate as in these ewes, spontaneous cycles commenced at the earliest possible time (Schirar et al., 1989; Mitchell et al., 1998).

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Macroscopic uterine involution in the ewe is completed by day 17 post-partum (Rubianes and Ungerfeld, 1993) but elevated concentrations of 15-keto-13,14-dihydro-PGF2␣, a metabolite of PGF2␣, and uterine oxytocin receptors have been detected as late as day 35 post-partum (Fredriksson, 1985; Wallace et al., 1993). In the present study, four ewes, all of which ovulated before day 22 post-partum, experienced extended luteal activity. Destruction of the corpus luteum requires approximately eight discrete pulses of PGF2␣ at 6–8 h intervals and it seems probable that extended luteal activity occurred because of a disturbance in the mechanism that regulates these pulses (Zarco et al., 1984). Results suggest that the period between days 30 and 40 post-partum represents a time when high circulating concentrations of PGF2␣ associated with uterine involution are diminished but the pulsatile release pattern of PGF2␣ associated with regular ovarian cycles is not fully re-established. In conclusion, results demonstrate that (i) maize-containing diets, when introduced at parturition, did not enhance circulating insulin concentrations or stimulate the reproductive axis; and (ii) ovulation during the early post-partum period was associated with an increased incidence of extended luteal activity. These findings show that when ovulation occurs too early following parturition in the ewe there may be subsequent failure of luteolysis, preventing the establishment of regular ovarian cycles. A similar phenomenon has been observed in high yielding dairy cows (Royal et al., 2000) where the metabolic demands of lactation, and associated alterations in circulating steroid hormone concentrations, may exacerbate any inherent deficiencies in the luteolytic mechanism.

Acknowledgements The authors are grateful to the Scottish Executive Environment and Rural Affairs Department for financial support of this study and wish to thank G. MacLean, K. Mackie and S. Robertson for their technical assistance. Maria J. Ranilla was in receipt of a scholarship from the Ministerio de Educacion y Cultura, Spain. Hormone antibodies were obtained as gifts from Diagnostics Scotland and the National Hormone and Pituitary Program, Torrance, USA.

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