The effect of condensed tannins in Lotus corniculatus upon reproductive efficiency and wool production in ewes during autumn

Animal Feed Science and Technology 92 (2001) 185±202

The effect of condensed tannins in Lotus corniculatus upon reproductive ef®ciency and wool production in ewes during autumn B.R. Mina,c, J.M. Fernandezb, T.N. Barrya,*, W.C. McNabbc, P.D. Kempd a

Institute of Veterinary, Animal and Biomedical Science, Massey University, Palmerston North, New Zealand b Department of Animal Science, Louisiana State University, Baton Rouge, LA 70803-4210, USA c Nutrition and Behaviour Group, Agricultural Research, Grasslands Research Centre, Palmerston North, New Zealand d Institute of Natural Resources, Massey University, Palmerston North, New Zealand Received 25 May 2000; received in revised form 3 May 2001; accepted 16 May 2001

Abstract A grazing experiment, conducted for 81 days (from 13 February to 4 May) in the late summer/ autumn of 1999 at Massey University, Palmerston North, New Zealand, compared the reproductive ef®ciency and wool growth of ewes grazing Lotus corniculatus (birdsfoot trefoil) or perennial ryegrass (Lolium perenne)/white clover (Trifolium repens) pasture (referred to as pasture). Half the ewes grazing L. corniculatus were given twice daily oral polyethylene glycol (PEG: molecular weight 3500) supplementation to inactivate the condensed tannins (CTs) in lotus. A rotational grazing system with 225 mixed age ewes (53:2  3:78 kg per ewe) was used, with 75 ewes per treatment. The effect of forage species and PEG supplementation upon voluntary feed intake (VFI), concentration of plasma metabolites, reproductive ef®ciency, wool production and wool length was measured during three synchronised oestrous cycles. The ewes were restricted to maintenance feeding for the ®rst 12 days of each oestrous cycle and then increased to ad libitum for the 5 days prior to and including ovulation. Lotus contained 18 g total CT/kg dry matter in the feed offered. There were only trace amounts of total CT in pasture. In vitro organic matter digestibility (OMD) of the diet selected was higher for lotus (0.82 versus 0.74) than for pasture, whilst the two forages contained a similar nitrogen concentration. Mean ovulation rates (ORs) at cycle 3 for CT-acting and PEG sheep grazing lotus and for sheep grazing pasture were, respectively, 1.79, 1.58 and 1.48, with corresponding lambing percentages being 1.69, 1.39 and 1.22. Fecundity (number of corpora lutea (CL) per ewe ovulating) at cycle 3 *

Corresponding author. Tel.: ‡64-6-350-5799; fax: ‡64-6-350-5636. E-mail address: [email protected] (T.N. Barry). 0377-8401/01/$ ± see front matter # 2001 Elsevier Science B.V. All rights reserved. PII: S 0 3 7 7 - 8 4 0 1 ( 0 1 ) 0 0 2 5 8 - 9

186

B.R. Min et al. / Animal Feed Science and Technology 92 (2001) 185±202

ovulation and lambs born per ewe lambing were greater for ewes grazing lotus than pasture (P < 0:01), and for CT-acting than for PEG sheep grazing lotus (P < 0:05). Increases in fecundity were due to a lower proportion of single ovulations and a greater proportion of multiple ovulations. CT increased reproductive ef®ciency by increasing fecundity and by reducing embryonic loss. Rumen ammonia and blood plasma urea concentrations were lower for ewes grazing lotus than pasture (P < 0:01), and were generally lower for CT-acting than for PEG sheep grazing lotus. The nutritional treatments had little effect on plasma ammonia concentration. Organic matter intake (OMI) (P < 0:05), clean wool production (P < 0:01) and staple length (P < 0:001) were signi®cantly higher for ewes grazing lotus than for pasture. Liveweight gain (LWG) was low, and lower for sheep grazing lotus than pasture. There was no difference in OMI and LWG due to CT in lotus, but wool length (P < 0:01) and clean wool production (P ˆ 0:07) were higher for CT-acting than for PEG supplemented ewes (CT not acting). It was concluded that grazing lotus during mating increased the ef®ciency of reproduction and clean wool production, with a component due to the action of CT. Possible mechanisms for CT increasing reproductive ef®ciency are discussed. # 2001 Elsevier Science B.V. All rights reserved. Keywords: Condensed tannins; Lotus corniculatus; Reproduction; Wool growth

1. Introduction Condensed tannins (CTs) are polyphenolic compounds which are widespread in the plant kingdom and are found in a number of legumes including Lotus corniculatus, L. pedunculatus, sainfoin (Onobrychis viciifolia) and sericea lespedeza (Lespedeza cuneata; Terrill et al., 1992; Jackson et al., 1996). They occur in only trace amounts in the leaves of perennial ryegrass and white clover. The CT precipitate dietary proteins in the rumen and the CT:protein complex is insoluble at pH 3.5±7.0, but is soluble and releases protein at pH < 3:5 (Jones and Mangan, 1977). High concentrations of CT in L. pedunculatus (over 55 g CT/kg DM) have been shown to reduce feed intake and nutritive value in sheep (Barry and Forss, 1983; Barry and Manley, 1986). However, CT in L. corniculatus (30±35 g/kg DM) have reduced protein solubility and degradation in the rumen (Min et al., 2000), increased the absorption of essential amino acids (EAA) from the small intestine by 62% (Waghorn et al., 1987a) and increased the ¯ow of cysteine to body synthetic reactions (Wang et al., 1994). Action of CT in L. corniculatus increased wool growth (12%) during summer and increased milk protein secretion (14%) from ewes in mid and late lactation during spring (Wang et al., 1996a,b; Min et al., 1998). Nutrition is one of the most signi®cant factors in¯uencing wool growth and ovulation rate (OR) in sheep. Wool growth has been long known to respond to increasing protein absorption (Reis, 1979), whilst the proportion of multiple ovulations in ewes has also been shown to increase with increasing protein absorption (Cruickshank et al., 1988; Smith, 1991). However, studies in sheep and cattle (Blanchard et al., 1990; Elrod and Butler, 1993; O'Callaghan and Boland, 1999) have not shown consistent effects of high protein diets on reproductive rate. High intake of dietary soluble protein or rumen degradable protein may negatively effect reproduction via direct effects on the uterine environment where toxic by-products of nitrogen metabolism, such as ammonia/ammonium ions from

B.R. Min et al. / Animal Feed Science and Technology 92 (2001) 185±202

187

the rumen may impair sperm, ova or early embryo survival (Blanchard et al., 1990; Elrod and Butler, 1993). This effect may be mediated by changes in uterine pH (Elrod and Butler, 1993). As action of CT in L. corniculatus simultaneously reduces protein degradation to ammonia in the rumen and increases EAA absorption from the small intestine, it is possible that they could increase both reproductive rate and wool growth in grazing sheep. The objectives of the present investigation were to measure effects of feeding L. corniculatus upon reproductive ef®ciency and wool production in grazing ewes during autumn, and to determine the proportion of any response that was due to its CT content. Perennial ryegrass/white clover dominant pasture, containing only trace amounts of CT, was also fed as a control diet. 2. Materials and methods 2.1. Experimental design A grazing trial involving 225 mixed age Romney ewes, including 122 rising 2-year-old mated for the ®rst time, was conducted at Massey University, Palmerston North, NZ, from 13 February to 4 May 1999 (81 days). The experiment involved ewes grazing two types of forage (L. corniculatus v. perennial ryegrass/white clover pasture), with half the ewes grazing L. corniculatus receiving twice daily oral supplementation with polyethylene glycol (PEG; MW 3500; PEG-supplemented group), giving three treatment groups each of 75 ewes. The PEG binds with CT, preventing the CT from binding with protein (Jones and Mangan, 1977; Barry and Manley, 1986). Effects of CT can be quanti®ed by comparing unsupplemented ewes (CT-acting) with ewes given PEG (CT-inactivated). The experiment was conducted over three oestrous cycles, with oestrus being synchronised in each cycle for all ewes. Reproductive ef®ciency was measured as OR in three synchronised oestrous cycles using laparoscopy and as lambs born/ewe, data are expressed in terms of ewes cycling per ewes mated and fecundity (number ovulations per ewe ovulating). Wool production was determined by shearing the ewes at the beginning and end of the experiment (from 12 February to 4 May 1999). 2.2. Forages Pure vegetative L. corniculatus (birdsfoot trefoil; cv. Grasslands Goldie) and pasture were grazed in breaks by the ewes, with each break lasting 3 or 4 days. Measurement of herbage mass before and after grazing and collection of samples of feed on offer (cut to soil level) and diet selected (using sheep ®stulated in the oesophagus; OF) were performed as described by Min et al. (1998). Pre- and post-grazing herbage mass were determined weekly, immediately before and after grazing, by cutting eight random quadrats (0.125 m2) per paddock to ground level and drying at 908C for 17 h. A further eight samples per paddock were cut to ground level, pooled, and stored at 208C for nutritive value analysis of feed on offer. The diet selected was determined using six OF Romney sheep, which allowed sampling for organic matter digestibility (OMD), total nitrogen (N)

188

B.R. Min et al. / Animal Feed Science and Technology 92 (2001) 185±202

and CT. Samples were stored analysis.

208C, and then freeze±dried and ground for chemical

2.3. Grazing management Total grazing days were calculated according to the method described by Min et al. (1998). All 225 ewes were randomly allocated to treatment groups, which were balanced for age of ewe, and grazed on either pasture (75 ewes) or lotus (150 ewes) during the three ovulation cycles. Half the ewes grazing lotus (n ˆ 75 ewes per group) were supplemented orally with PEG. During ovulation cycles 1±3 the feed allowance was kept at maintenance (1.5 kg DM per ewe) for the ®rst 12 days of each cycle, and then increased to ad libitum allowance (ca. 2.2 kg DM per ewe) for the 5 days prior to and including ovulation. Day of ovulation was de®ned as day zero. After ovulation, the feed allowance was reduced to maintenance again. The rationale for this was that a minimum of 5 days of increased protein supply immediately prior to ovulation was required to increase ovulation rate in sheep (Smith et al., 1983; Stewart and Oldham, 1986). Ad libitum feeding over this period was designed to give the action of CT in L. corniculatus maximum opportunity for increasing EAA supply over this critical period, whilst restricted feeding ensured that all ewes on both forages were kept close to the maintenance level of energy intake at all other times. At the end of oestrous cycle 3, the ewes continued grazing either lotus or pasture for a further 27 days in order to measure voluntary feed intake (VFI), with and without PEG supplementation. The groups were then joined and grazed on pasture until lambing. 2.4. Animals Mean initial liveweight (LW) was 52.4 kg (S.D. 3.96) for rising 2-year-old ewes and 53.9 kg (S.D. 3.60) for ewes aged 3 years or older. At the start of the experiment, all ewes were weighed, tagged and drenched with anthelmintic (Ivomec; Merck, Sharp & Dohme, NZ Ltd.) to control internal parasites, and treated for external parasites (Wipeout; Coopers Animal Health, NZ). The animals were weighed at fortnightly intervals, and feed supply adjusted if needed to keep the ewes at maintenance. All ewes were shorn before the experiment commenced (10 February) and again at the conclusion of the grazing experiment (4 May). The dose of PEG was calculated on the basis of estimated daily VFI and the CT content in L. corniculatus, with the objective of administering 1.8 g PEG/g CT, this being the minimum amount to bind all the CT and prevent binding with soluble protein (Barry and Forss, 1983). The PEG (71 g per day during maintenance and 110 g per day during ad libitum feeding) was administered as a 50% w/v solution, given daily as two equal doses at 08.00 and 16.00 h. VFI was measured using slow release chromium capsules (Cr2O3 matrix, Nufarm, Auckland, NZ) according to the method described by Parker et al. (1989) and Min et al. (1998). Fifteen ewes per treatment group were used to estimate VFI. Three rumen-®stulated Romney sheep were grazed on each forage for 27 days to measure the Cr release rate of capsules suspended in the rumen. Measurements started on day 5 after chromium capsule insertion, and proceeded at 3-day intervals until day 27.

B.R. Min et al. / Animal Feed Science and Technology 92 (2001) 185±202

189

2.5. Synchronisation of oestrus and determination of ovulation rate Ovulation was synchronised using controlled release intravaginal devices (CIDR; type G; Carter Holt Harvey; containing 0.3 g progesterone). They were inserted for 12 days of the ®rst ovulatory cycle and were inserted for 8 days of the second and the third cycles. Ewes were mated with vasectomized teaser rams ®tted with tupping crayons (four rams per group; during 8 days) at the ®rst and the second ovulatory cycles and then entire rams ®tted with tupping crayons (®ve rams per group; during 25 days) were mated with ewes at the third cycle. Ovulation rate was determined by counting corpora lutea (CL) using laparoscopy (Kelly and Allison, 1976) ca. 7 days after oestrus started. A total of three laparoscopy measurements were made (8 March, cycle 1; 24 March, cycles 2 and 9 April, cycle 3) following the initial synchronisation. Pregnancy was diagnosed by ultrasound scanning approximately 50 days after mating (cycle 3) and the number of foetuses per pregnant ewe was recorded. Lambing records were also collected. 2.6. Plasma samples Blood samples (7 ml) were taken from the jugular vein of 60 sheep (20 per treatment group) grazing each forage on day-8 (maintenance feed) and on day 1 (ad libitum) before ovulation during each synchronised oestrous cycle. Samples taken during the ad libitum feeding were analysed for ammonia concentration, whilst samples taken from the same sheep during maintenance and ad libitum feeding were analysed for urea concentration. The blood samples were collected into vacutainers with Na-EDTA (Becton Dickinson, USA) as an anticoagulant and placed on ice. Sample preparation for both ammonia and urea were performed using the methods described by Min et al. (1998). All vacutainers were held on ice and centrifuged (3200 g for 20 min at 48C) to obtain blood plasma for ammonia and urea analysis. Plasma samples for ammonia (1.5 ml) were mixed with 0.5 ml of 30% trichloroacetic acid (TCA; w/w) in a pre-weighed plastic centrifuge tube, mixed well and centrifuged at 3200 g (48C) for 20 min. The supernatant was ®ltered into a labelled 4 ml vial with disposable millipore syringe ®lter paper (20 mm) and then stored at 858C until analysed. Samples of rumen ¯uid were withdrawn by stomach tube from 10 sheep per treatment during the maintenance and ad libitum parts of cycle 3 (31 March and 7 April) and at the end of the maintenance (30 April) when the sheep were grazing at maintenance. Ammonia concentration in rumen ¯uid was determined as described by Min et al. (1998). 2.7. Laboratory analyses 2.7.1. Forage and faeces Samples of feed offered and diet selected were stored at 208C, freeze±dried, and ground to pass through a 1 mm diameter sieve for laboratory analysis. Acetone/waterextractable, protein-bound and ®bre-bound CT fractions in forages and OF extrusa were determined using a butanol±HCl colorimetric procedure (Terrill et al., 1992). All CT

190

B.R. Min et al. / Animal Feed Science and Technology 92 (2001) 185±202

concentrations were determined using CT extracted from L. pedunculatus as a reference standard (Jackson et al., 1996). Total N was determined by the Kjeldahl method, OM by ashing samples for 16 h at 5508C and in vitro OMD by the enzymatic method of Roughan and Holland (1977). Chromium in faeces was determined by the method of Costigan and Ellis (1987). As extractable and protein-bound CT would be dissolved in the initial in vitro extraction steps, but are known to be indigestible in vivo (Terrill et al., 1994), extractable and protein-bound CT (% OM) values were deducted from all in vitro OM digestibilities. 2.7.2. Wool samples Fleeces were weighed at shearing to determine greasy ¯eece weight, with samples of 200±300 g being taken from both the left and right mid-side areas for laboratory analyses. A standard greasy wool washing procedure and measurements of staple length (cm) and clean wool yield were made using the methods described by Min et al. (1998). 2.7.3. Plasma samples Plasma ammonia concentration was determined at the Nutritional Laboratory, Agricultural Research, Grassland, Palmerston North, New Zealand, using a ¯ow injection analyser (FIA; FIAstar 5010 Analyser, Sweden). Plasma ammonia was determined using the method described by Hara and Matsumoto (1994) and Lopez-Fernandez et al. (1995). Plasma urea concentration was determined with an enzymatic ultraviolet colourimetric assay that utilises glutamate dehydrogenase and NADH, using an auto-analyser (COBAS FARA, Basel, Switzerland) as described by Min et al. (1998). 2.8. Calculation of data and statistical analyses Data were analysed using general linear model (GLM) procedures of statistical analysis system package (SAS, 1985) for a completely randomised block design experiment to test the effects of PEG supplementation (within lotus forage), forage types (lotus versus pasture) and age of ewes (2 versus 3 years and older; reproductive rate only). Data for liveweight gain (LWG) wool production, VFI, rumen and plasma metabolites were analysed using the GLM procedures of the SAS statistical package (SAS, 1985), with the variables ®tted being forage type and PEG supplementation. Data for chemical composition of forage and forage mass were analysed using the GLM procedures of the SAS statistical package (SAS, 1985), with the variables ®tted being forage type. Ovulation data were analysed as recorded and also in terms of ewes ovulating/ewes mated and fecundity (CL per ewe ovulating). Lambing data were analysed as recorded and also as ewes lambing/ewes mated and as lambs born/ewe lambing. The fecundity and lambing percentage of ewes giving birth are presented in the form of a percentage of ewes in the group having multiple (one, two or more ovulations). For statistical analysis the data were transformed using a logistic regression model (SAS, 1995) and treatment effects were established using the Chi-square procedure (Tables 4 and 5) as described by Smith (1985), with the factors ®tted being forage type, PEG supplementation and age of ewes.

B.R. Min et al. / Animal Feed Science and Technology 92 (2001) 185±202

191

3. Results 3.1. Forages Both pasture and lotus were in a vegetative state throughout the experiment. Pre- and post-grazing herbage mass were generally higher for pasture forage than for lotus and were higher during ad libitum than maintenance feeding (Table 1). 3.2. Chemical composition Total CT concentration in the lotus was 18 g/kg DM for feed offered and 10 and 7 g/kg DM for OF extrusa representing the diet selected at the beginning and end of grazing each break (Table 2). Only trace amounts of total CT were detected in pasture. Most CT in the lotus on offer was readily extractable (49.4%) and protein-bound (47.2%) with a much smaller amount being ®bre-bound (3.4%; Table 2). In the diet selected (OF extrusa), a much lower component was readily extractable (3±8%), with the largest component being protein-bound (82±84%). For both forages, in vitro OMD and total N concentration were higher for the diet selected than for feed on offer. In vitro OMD was signi®cantly higher for lotus (P < 0:01) than for pasture in both feed on offer and late OF extrusa. Lotus contained similar concentrations of N in both feed on offer and early OF extrusa compared with pasture, but was higher for late OF extrusa. 3.3. Rumen and plasma metabolites Rumen ammonia concentrations were signi®cantly lower for ewes grazing lotus than for ewes grazing pasture (P < 0:01; Table 3). Action of CT in lotus decreased rumen ammonia concentration during both maintenance (autumn) and ad libitum (end of summer) feeding (P < 0:01) compared to PEG-supplemented animals (Table 3). There was a signi®cantly lower blood plasma ammonia concentration for ewes grazing lotus than for pasture during ovulatory cycle 1 (P < 0:01), but thereafter there were Table 1 Pre-grazing and post-grazing forage mass (tonnes of dry matter/ha) of L. corniculatus (cv. Grasslands Goldie) and perennial ryegrass/white clover pasture (pasture) Lotus

Number of measurements Maintenance intake perioda Ad libitum periodb a

Pasture

S.E.M.

Pre-grazing

Post-grazing

Pre-grazing

Post-grazing

8 2.04 2.26

8 0.93 1.44

5 3.02 3.10

5 1.12 1.54

0.202 0.125

Fed for the initial 12 days in each cycle (12±24 February, 1±11 and 18±28 March) before ad libitum feeding started. b Fed for the final 5 days in each cycle (25±28 February, 12±17 March and 29 March to 2 April) with day of ovulation: day 0.

192

Diet selected (OF extrusa)a

Feed offered Pasture

Number of observations OM (g/kg DM) In vitro OMD (% OM) Unajusted Adjustedc Total N (g/kg OM) Condensed tannin (g/kg DM) Extractable CT Protein bound CT Fibre bound CT Total CT % Bound CTc a

12 914

Lotus

12 915

61.1 61.0 27.1

72.2 70.5 27.3

0.2 0.8 1.2 2.2 91

8.7 8.3 0.6 17.6 51

P value

S.E.M.

Earlyb

Lateb

Pasture

Lotus

P value

Pasture

Lotus

P value

S.E.M.

6 840

6 821

NS

6 798

6 836

*

11.01

NS

6.87

***

1.37 1.36 1.50

76.0 75.8 44.6

84.3 83.4 45.1

0.07 0.09 NS

0.34 0.74 0.17 0.84 ±

0.0 2.5 0.7 3.2 100

0.8 8.1 0.8 9.7 92

±

***

NS *** *** ** ***

±

***

NS **

±

72.2 72.1 31.1

82.7 82.1 44.4

**

0.0 0.8 1.1 1.9 100

0.2 5.8 1.1 7.1 97

±

** **

***

NS **

±

OF: oesophageal fistulae. Early: samples taken at the start of grazing each break; late: samples taken at the end of grazing each break. * (P < 0:05): level of statistical significance. ** (P < 0:01): level of statistical significance. *** (P < 0:0001): level of statistical significance. c After deducting extractable and protein-bound CT, as described in laboratory analysis. % bound CT ˆ ((protein bound ‡ fibre bound CT)/total CT). b

2.12 2.13 2.36 0.31 0.64 0.15 0.77 ±

B.R. Min et al. / Animal Feed Science and Technology 92 (2001) 185±202

Table 2 Organic matter (OM), total N and condensed tannin (CT) contents and in vitro OM digestibility of feed offered and diet selected by sheep grazing L. corniculatus and perennial ryegrass/white clover pasture (pasture)

Group

Rumen ammonia (mg/l) Number of observations Maintenance fed (end summer)a Maintenance fed (autumn)b Ad libitum feed (end summer)a Plasma ammonia (ad libitum; mg/l) Number of observations Cycle 1 Cycle 2 Cycle 3 Plasma urea (mM/l) Number of observations Maintenance feed Ovulatory cycles 1±3 Ad libitum feed Ovulatory cycles 1±3 a

Dietary treatment

S.E.M.

Lotus ‡ PEG (PEG sheep)

Lotus (CT-acting)

Pasture

10 130.6 318.2 268.9

10 119.7 255.8 168.1

10 336.8 (n ˆ 7) 334.3 268.5

13.22 17.02 13.44

10 29.2 103.7 92.0

10 28.5 95.3 83.0

10 47.6 (n ˆ 6) 98.1 83.7

20

20

20

Lotus vs. lotus ‡ PEG

NS

Lotus vs. pasture

***

**

**

***

***

2.79 4.61 4.20

NS NS P ˆ 0.1

NS NS

**

7.13

6.59

8.80

0.153

**

***

8.59

8.36

9.40

0.157

NS

***

Samples taken at the ovulatory cycle 3 (31 March and 7 April for ad libitum and maintenance fed, respectively). Samples taken at the end of grazing trial (30 April). ** (P < 0:01): level of statistical significance. *** (P < 0:0001): level of statistical significance. b

Contrasts

B.R. Min et al. / Animal Feed Science and Technology 92 (2001) 185±202

Table 3 Rumen concentration of ammonia and plasma concentrations of ammonia and urea in ewes grazing perennial ryegrass/white clover pasture and L. corniculatus, with or without twice-daily oral administration of PEG

193

194

B.R. Min et al. / Animal Feed Science and Technology 92 (2001) 185±202

Table 4 The effect of grazing ewes on L. corniculatus or perennial ryegrass/white clover pasture (pasture), and of supplementation with PEG, on mean ovulation rate (corpora lutea/ewe mated) and lambing (lambs born/ewe mated) Cycle

Dietary treatment Lotus ‡ PEG (PEG sheep)

Ovulation rate (OR) Number of ewes 75 First cycle 1.55 Second cycle 1.63 Third cycle 1.58 Ultra scan (lambs/ewe mated) 1.39 Lambing (lambs born/ewe mated) 1.39

S.E.M.

Lotus Pasture (CT-acting) 75 1.58 1.70 1.79 1.64 1.69

75 1.37 1.60 1.48 1.48 1.22

0.06 0.05 0.06 0.06 0.07

Contrasts Lotus vs. lotus ‡ PEG

Lotus vs. pasture

NS NS P ˆ 0.11

NS NS P ˆ 0.17 P ˆ 0.06

*

**

***

*

(P < 0:05): level of statistical significance. (P < 0:01): level of statistical significance. *** (P < 0:0001): level of statistical significance. **

no differences in plasma ammonia concentration between any of the nutritional treatments. Mean plasma urea concentration, averaged over all three cycles, was signi®cantly lower for ewes grazing lotus than pasture (P < 0:001) and was signi®cantly lower in CT-acting than PEG-supplemented sheep at maintenance (P < 0:01) but not at ad libitum feeding.

Fig. 1. The effect of grazing ewes on L. corniculatus or perennial ryegrass/white clover pasture (pasture), and of supplementation with PEG, on ewes ovulating or lambing per ewes mated. Ultra: ultra scan (ewes pregnant/ewe mated) at 50 days of pregnancy. (&) Pasture; (*) lotus CT-acting group; (*) lotus PEG-group; ( ) period of lotus feeding and PEG supplementation, up to 27 days of pregnancy.

B.R. Min et al. / Animal Feed Science and Technology 92 (2001) 185±202

195

3.4. Reproductive rate Effects of the nutritional treatments upon mean ovulation rate and lambing % are shown in Table 4, whilst ewes ovulating or lambing (% ewes mated) and fecundity are shown in Fig. 1 and Table 5, respectively. Ewes ovulating/ewes mated was 95% in cycle 1, 98% in cycle 2 and 94% in cycle 3, and was not affected by forage type, PEG supplementation or age of ewe (Fig. 1; 2 versus 3 years and older). Ewes pregnant/ewes mated at 50 days of pregnancy, as determined by ultrasound scanning, was greater for ewes that had grazed lotus than pasture (P ˆ 0:06) and was greater for CT-acting ewes grazing lotus than for PEG supplemented ewes (P < 0:05; and Fig. 1). Ewes lambing/ewes mated (Fig. 1) was greater for ewes that had Table 5 The effect of grazing ewes on L. corniculatus or perennial ryegrass/white clover pasture (pasture), and of supplementation with PEG, on fecundity at ovulation (corpora lutea/ewe cycling) and at lambing (lambs born/ ewe lambing) L. corniculatus

S.E.M.

Fecundity 1 Number of ewes 1st cycle Lotus ‡ PEG Lotus (CT-acting) Pasture 2nd cycle Lotus ‡ PEG Lotus (CT-acting) Pasture 3rd cycle Lotus ‡ PEG Lotus (CT-acting) Pasture Lambing (%) All ewes Lotus ‡ PEG Lotus (CT-acting) Pasture 2 years old Lotus ‡ PEG Lotus (CT-acting) Pasture 3 years and older Lotus ‡ PEG Lotus (CT-acting) Pasture *

Lotus vs. pasture

Lotus vs. lotus ‡ PEG

2

3±4

40.9 35.0 60.7

54.9 61.3 35.7

4.2 3.8 3.6

0.32

***

NS

34.3 27.4 47.2

64.4 72.6 47.1

1.4 0.0 5.7

0.33

**

NS

33.8 19.5 40.8

61.8 75.6 54.3

4.4 4.9 4.9

0.36

***

*

38.7 24.4 45.5

56.5 74.4 48.5

4.8 1.2 6.1

0.35

**

P ˆ 0.06

37.8 23.5 58.3

56.8 74.5 36.1

5.4 2.0 5.6

0.57

**

NS

40.0 25.8 30.0

56.0 74.2 63.3

4.0 0.0 6.7

0.47

**

P ˆ 0.14

75

(P < 0:05): level of statistical significance. (P < 0:01): level of statistical significance. *** (P < 0:0001): level of statistical significance. **

Contrasts

196

B.R. Min et al. / Animal Feed Science and Technology 92 (2001) 185±202

grazed lotus (90.2%) than pasture (75.9%; P < 0:001), and was greater for CT-acting ewes that had grazed lotus (96.5%) than for PEG-supplemented ewes (83.8%; P < 0:01), but was not affected by age of ewe (2 versus 3 years and older). Fecundity of ewes grazing lotus was greater than that of ewes grazing pasture during cycles 1, 2 and 3 (P < 0:01). During cycle 3, fecundity of CT-acting ewes grazing lotus was greater than that of PEG-supplemented ewes (CT not acting; P < 0:05; Table 5), with this trend being evident but not statistically signi®cant in earlier cycles. Fecundity at lambing was greater for ewes grazing lotus than for pasture (P < 0:01; Table 5), and was greater for CT-acting ewes grazing lotus than for PEG-supplemented ewes (P ˆ 0:06). The increased fecundity of ewes grazing lotus is indicated by fewer ewes having only one CL and giving birth to one lamb, and to more ewes having multiple ovulations and giving birth to two or more lambs. These effects were apparent in both nutritional treatment groups, but the effect was of greater magnitude in CT-acting than PEG supplemented ewes. The response in fecundity to grazing lotus was not affected by age of ewe. 3.5. Voluntary feed intake, liveweight gain, wool production and wool processing characteristics VFI was higher for ewes grazing lotus than for pasture (P < 0:05) but there was no difference in VFI between the CT-acting and PEG-supplemented groups (Table 6). LWG was low, as expected from the experimental design, but was higher in sheep grazing pasture than lotus (P < 0:01). Clean ¯eece weight (P < 0:01) and staple length (cm; P < 0:001) were signi®cantly higher in ewes grazing lotus than those on pasture. Action of CT in sheep Table 6 OMI, LWG, wool production and wool processing characteristics of sheep grazing L. corniculatus and perennial ryegrass/white clover pasture (pasture), with or without PEG supplementation Group

OMIa (number of observations) (kg per ewe per day) LWG (number of observations) Mean value (g per day) Wool weight (number of observations) Greasy fleece (kg) Clean fleece (kg) Efficiency of wool production (number of observations) (g clean wool/kg OMI) Staple length (cm) a

Dietary treatment

S.E.M.

Lotus ‡ PEG (PEG sheep)

Lotus Pasture (CT-acting)

15 1.48 74 16.3 74

15 1.40 75 22.3 75

15 1.23 75 43.2 75

2.00 1.61 74

2.09 1.71 75

14.05 5.97

14.90 6.23

Contrasts Lotus vs. Lotus lotus ‡ PEG vs. pasture

0.067

NS

*

6.88

NS

**

1.76 1.41 75

0.037 0.036

NS P ˆ 0.07

**

13.96 5.78

0.341 0.104

**

P ˆ 0.08

NS

**

***

OMI measured using a in vitro OMD (CT adjusted; diet selected from early OF extrusa; Table 2). (P < 0:05): level of statistical significance. ** (P < 0:01): level of statistical significance. *** (P < 0:0001): level of statistical significance. *

B.R. Min et al. / Animal Feed Science and Technology 92 (2001) 185±202

197

grazing lotus increased wool length (P < 0:01) and tended to increased clean ¯eece weight (P ˆ 0:07) and ef®ciency of wool production (gram clean wool/kg OMI; P ˆ 0:08). 4. Discussion The principal objective of the present study was to determine if feeding lotus during autumn effected the reproductive rate and wool production in ewes and to determine the proportion of any response that was due to CT. The most signi®cant ®ndings in this study were that light ewes (mean LW 53 kg) grazing lotus rather than pasture for three oestrus cycles during autumn increased both lambing percentage (21%) and wool production (18%). The increase in reproductive rate was due to increases in ewes lambing/ewes mated and to increases in fecundity at both ovulation and birth. Responses to PEG supplementation showed that approximately 50% of the effect of lotus on reproductive rate was due to its CT content. The CT in the lotus reduced rumen ammonia and blood plasma urea concentration in the present experiment, indicating reduced ruminal protein breakdown, and increased EAA absorption (Waghorn et al., 1987a,b, 1990). The reduced rumen ammonia and plasma urea concentration in CT-acting compared to PEG supplemented sheep grazing lotus at maintenance feeding is consistent with other ®ndings (Min et al., 1998, 1999). A high OR is the ®rst step in achieving a high lambing percentage. A 10% increase in OR typically causes 6.9% more lambs born per ewe lambing and 5.7% more lambs docked per ewe mated (Geenty, 1997). Results gained in the present experiment can be compared with results obtained in other similar grazing trials (Table 7). Averaged over three experiments, Table 7 The effect of grazing ewes on L. corniculatus or perennial ryegrass/white clover (pasture), and of supplemention with polyethylen glycol, on VFI, maximum ovulation rate (OR), clean fleece weight and staple length

Min et al. (1999) Luque et al. (2000) Current experiment Min et al. (1999) Luque et al. (2000) Current experiment Min et al. (1999) Luque et al. (2000) Current experiment Min et al. (1999) Luque et al. (2000) Current experiment a

Pasture

Lotus

PEG

‡PEG

(Maximum OR) 1.33 1.46 1.45 1.66 1.48 1.66 (Clean fleece weight) 1.12 1.31 1.54 1.69 1.41 1.61 (Staple length) 4.10 4.27 6.72 6.89 5.78 5.97 (VFI) 1.91 1.85 1.78 1.86 1.23 1.48

Calculated as …OR lotus … PEG†

CT-acting

Response to lotus feeding (%)a

1.76 1.64 1.79

32.2 13.1 20.9

1.35 1.74 1.71

20.5 12.33 21.30

4.37 6.99 6.23

7.0 4.0 7.8

1.70 1.96 1.40

11 10.1 13.8

OR pasture … PEG††=…OR pasture … PEG††  100.

198

B.R. Min et al. / Animal Feed Science and Technology 92 (2001) 185±202

relative to ewes grazed on pasture, grazing lotus increased OR by an average of 22%, with the response being present in all years (Table 7). Magnitude of the response was related to the duration of lotus feeding, with maximum response occurring after feeding for 2±3 oestrus cycles (5±7 weeks). As judged by response to PEG supplementation, approximately 50% of the increase in OR due to lotus feeding could be explained by action of CT in the present experiment and in the work of Min et al. (1999), whereas none of the OR response could be explained by CT in the experiment of Luque et al. (2000). Possible reasons for this differences are that the lighter ewes used in the present experiment and by Min et al. (1999) were more responsive to the additional protein absorption caused by the action of CT than were the heavier ewes used by Luque et al. (2000), and that the lighter ewes gained a small amount of weight during the experiments whilst the heavier ewes lost weight (Table 7). This is similar to the result obtained by Knight (1980) who found that over the liveweight range of 39±49 kg, the advantage of supplementation with lupin grain (21.4% crude protein per DM) decreased with increasing ewe liveweight. It has been deduced that the increase in OR due to action of CT in Min et al. (1999) was due to increased plasma concentration of EAA and especially the branched chain amino acids (BCAA). Previous research has reported that the strongest correlation was found between OR and the plasma concentration of BCAA (r ˆ 0:95) and EAA (r ˆ 0:61) (Waghorn, 1986; Waghorn et al., 1990). This has been con®rmed by the ®ndings that intravenous infusion of a BCAA mixture (33.1 g total BCAA per day ewe) for 5 days before luteolysis (late stages of the oestrous cycle), produced an increase in OR (2.4 versus 1.5; Downing and Scaramuzzi, 1991; Downing et al., 1995). The critical period for protein supplementation to increase OR is therefore the last 5 days before ovulation (Stewart and Oldham, 1986) which is why the feeding level was restricted to maintenance for the ®rst 12 days of each cycle and increased to ad libitum for the last 5 days in this study. An additional possibility is that the high intake of dietary soluble protein may affect reproduction via direct effects on the uterine environment where toxic by-products of nitrogen metabolism, such as ammonia/ammonium ions from the rumen may impair sperm, ova or early embryo survival. This effect may be mediated by changes in uterine pH (Elrod and Butler, 1993; Kaur and Arora, 1995). Negative correlations between high protein intake and fertility have been reported by Hewett (1974) and Lotthammer (1974). Furthermore, reduced fertility and embryonic loss can occur when dietary urea is in excess in ewes (McEvoy et al., 1997) or when rumen degradable protein is increased in cattle (Blanchard et al., 1990). Ruminal ¯uid and blood plasma urea and ammonia±N levels are dependent on many factors, including the level and degradability of dietary protein and non-protein N sources in the animal's diet (NRC, 1985; Fernandez et al., 1997). It is free, nonionized (NH3) form of ammonia that is readily absorbed across the rumen epithelium and into the portal and systemic circulation that can result in hyperammonemia (Visek, 1968). Recently, it has been reported that overfeeding (ad libitum) can decrease the survival rate of embryos in ewes (Fahey et al., 1998; O'Callaghan and Boland, 1999) which may be attributed to their effect on the follicle or oocyte development. This may have affected the results in the present experiment, as it is clear from Fig. 1 that differences in embryonic mortality occurred between treatments by 50 days of pregnancy, when scanning was undertaken. Lowest embryonic losses occurred in the CT acting group, which grazed lotus for approximately half of this 50 days

B.R. Min et al. / Animal Feed Science and Technology 92 (2001) 185±202

199

period. The reason for this is unclear, as plasma ammonia concentration was not lower for this group during the time of ad libitumn feeding at cycles 2 and 3 ovulation; in future experiments of this type blood samples for plasma urea and ammonia need to be taken during the ®rst 50 days of pregnancy. Mean VFI in Table 7 was not greatly affected by the nutritional treatments applied, whilst a small liveweight loss occurred in the experiment of Luque et al. (2000) that did not differ between the nutritional treatments. Overall, mean clean wool weight was greater in sheep grazing lotus (18%) than pasture. As there were responses to PEG supplementation in two of the four experiments, it seems that action of CT contributed to the increase in wool growth in some cases. As VFI was similar for sheep grazing two forages, it seems that the increases in reproductive ef®ciency and wool production (Table 7) from feeding lotus were due to improved utilisation of ingested nutrients. Field observations from previous studies have shown that supplements of 1±3 g per day of methionine bypassing the rumen increased wool growth by 10±40% (Dove and Robards, 1974). Infusion of methionine and cysteine into the abomasum has been shown to be effective in stimulating wool growth and nitrogen retention in sheep fed poor roughage diets (Black and Reis, 1979; Reis, 1979; Revell et al., 1999). More recently, studies with L. pedunculatus (McNabb et al., 1993) and with L. corniculatus (Wang et al., 1994) showed that the action of CT increased the irreversible loss rate (ILR) of cystine from blood plasma, mainly due to reducing the loss of SAA (30%) in the rumen. Part of the increase in clean ¯eece weight (15%) from feeding lotus in the present experiment could be due to CT increasing the absorption of SAA and also that of all other EAA. This is similar to the result obtained by Min et al. (1998, 1999) who found CT in lotus increased the ef®ciency of wool production (gram wool produced/kg OMI). Lotus feeding in the present experiment also increased length of the wool grown. Feeding the CT-containing legume L. corniculatus to grazing ewes can be used to increase both reproduction and wool production in grazing ewes. Action of CT resulted in more lambs born and more wool produced without affecting VFI, showing improved utilisation of ingested nutrients, with the most likely mechanism being increased plasma concentration and supply of BCAA for reproduction and SAA and EAA for wool growth. From an agronomic point of view, lotus grows best on acid soils in dry areas, such as the east coast of New Zealand, where ewe liveweights are also likely to be lowest. Thus it may ®nd application in these areas, especially if only 5±7 weeks of grazing during autumn are required in order to increase reproductive ef®ciency. Acknowledgements This study was supported by a grant from Wools of New Zealand. The authors wish to thank G.S. Purchas, B. Parlane, R. Scho®eld, A. Luque and P. Back, who assisted with data collection. The skilled assistance from the Wool and Nutrition Laboratory staff, Massey University and J.S. Peters, Agricultural Research, is greatly acknowledged. P.C.H. Morel and D.J. Garrick are thanked for advice with statistical analysis and T.G. Harvey is thanked for advice on grazing management. Scholarship support to B.R. Min from Wools of New Zealand is greatly acknowledged.

200

B.R. Min et al. / Animal Feed Science and Technology 92 (2001) 185±202

References Barry, T.N., Forss, D.A., 1983. The condensed tannin content of vegetative Lotus pedunculatus, its regulation by fertilizer application, and effect upon protein solubility. J. Sci. Food Agric. 34, 1047±1056. Barry, T.N., Manley, T.R., 1986. free condensed tannin and lignin in Lotus sp. and their possible consequences in ruminant nutrition. J. Sci. Food Agric. 37, 248±254. Black, J.L., Reis, P.J., 1979. Speculation on the control of nutrient partition between wool growth and other body functions. In: Black, J.L., Reis, P.J. (Eds.), Physiological and Environmental Limitations to Wool Growth, University of New England Publishing Unit, New South Wales, Armidale, pp. 269±294. Blanchard, T., Ferguson, J., Love, L., Takeda, T., Henderson, B., Hastler, J., Chalupa, W., 1990. Effect of dietary crude protein type on fertilisation and embryo quality in dairy cattle. Am. J. Vet. Res. 51, 905±908. Costigan, P., Ellis, K.J., 1987. Analysis of faecal chromium from controlled release devices. NZ J. Tech. 3, 89±92. Cruickshank, G.J., Smith, J.F., Fraser, D.G., 1988. The influence of abomasal infusion of protein or energy on ovulation rate in ewes. Proc. NZ Soc. Anim. Prod. 48, 77±79. Dove, H., Robards, G.E., 1974. Effect of abomasal infusions of methionine, casein, and starch plus methionine on the wool production of merino wethers fed on lucerne or wheaten chaff. Aust. J. Agric. Res. 25, 945±956. Downing, J.A., Scaramuzzi, R.J., 1991. Nutrient effects on ovulation rate, ovarian function and the secretion of gonadotrophic and metabolic hormones. J. Rep. Fert. (Suppl. 43), 209±227. Downing, J.A., Joss, J., Scaramuzzi, R.J., 1995. A mixture of the branched chain amino acids leucine, isoleucine and valine increases ovulation rate in ewes when infused during the late luteal phase of the oestrus cycle: an effect that may be mediated by insulin. J. Endo. 145, 315±323. Elrod, C.C., Butler, W.R., 1993. Reduction of fertility and alteration of uterine pH in heifers fed excess ruminally degradable protein. J. Anim. Sci. 71, 694±701. Fahey, J., Boland, M.P., O'callaghan, D., 1998. Effects of dietary urea on embryo development in superovulated donor ewes and on embryo survival following transfer in recipient ewes. Proc. Br. Soc. Anim. Sci., 182. Fernandez, J.M., Sahlu, T., Lu, C.D., Ivey, D., Potchoiba, M.J., 1997. Production and metabolic aspects of nonprotein nitrogen incorporation in lactation rations of dairy goats. Small Rumin. Res. 26, 105±117. Geenty, K.G., 1997. Mating and early pregnancy. A Guide to Lambing Percentage for Farmers and Advisors. Wools of New Zealand and the New Zealand Meat Producers Board, pp. 33±46. Hara, H., Matsumoto, S., 1994. Continuous flow system for the accurate determination of low concentrations of ammonium ions using a gas-permeable polytetrafluoroethylene tube decontaminator and all ammonia gassensing membrane electrode. Analyst 119, 1839±1842. Hewett, C., 1974. On the causes and effects of variations in the blood profile of Swedish dairy cattle. Acta Vet. Scandi. 15 (Suppl. 50), 1. Jackson, F.S., McNabb, W., Barry, T.N., Foo, Y.L., Peters, J.S., 1996. The condensed tannin content of a range of subtropical and temperate forages and the reactivity of condensed tannin with ribulose-1,5-bis-phosphate carboxylase (Rubisco) protein. J. Sci. Food Agric. 72, 483±492. Jones, W.T., Mangan, J.L., 1977. Complexes of the condensed tannins of sainfoin (Onobrychis viciifolia scop.) with fraction 1 leaf protein and with submaxillary mucoprotein, and their reversal by polyethylene glycol and pH. J. Sci. Food Agric. 28, 126±136. Kaur, H., Arora, S.P., 1995. Dietary effects on ruminant livestock reproduction with particular reference to protein. Nut. Res. Rev. 8, 121±136. Kelly, R.W., Allison, A.J., 1976. Measurements of ovulation rates by laparoscopy and effects on reproductive performance. Proc NZ Soc. Anim. Prod. 36, 240±246. Knight, T.W., 1980. Effects of diet and liveweight on ovulation rates in Romney ewes. Proc. NZ Soc. Anim. Prod., 38±39. Lopez-Fernandez, J.M., Rios, A., Valcarcel, M., 1995. Assessment of quality of flow injection methods used in food analysis. A Rev. Anal. 120, 2393±2400. Lotthammer, K.H., 1974. Haifge Futterungs fehler als ursache der herdenslerilitat (common feeding errors as causes of herd infertility). Colleguim Vet. 38±42. Luque, A., Barry, T.N., McNabb, W.C., Kemp, P.D., McDonald, M.F., 2000. The effect of grazing Lotus corniculatus during late summer±autumn on reproductive efficiency and wool production in ewes. Aust. J. Agric. Res. 51, 385±391.

B.R. Min et al. / Animal Feed Science and Technology 92 (2001) 185±202

201

McEvoy, T.G., Robinson, J.J., Aitken, R.P., Findlay, P.A., Robertson, I.S., 1997. Dietary excess of urea influence the viability and metabolism of preimplantation sheep embryos and may affect fetal growth among survivors. Anim. Rep. Sci. 47, 71±90. McNabb, W.C., Waghorn, G.C., Barry, T.N., Shelton, I.D., 1993. The effect of condensed tannins in Lotus pedunculatus on the digestion and metabolism of methionine, cysteine and inorganic sulphur in sheep. Br. J. Nutr. 70, 647±661. Min, B.R., Barry, T.N., McNabb, W.C., Kemp, P.D., 1998. The effect of condensed tannins on the production of wool and on its processing characteristics in sheep grazing Lotus corniculatus. Aust. J. Agric. Res. 49, 597±605. Min, B.R., McNabb, W.C., Barry, T.N., Kemp, P.D., Waghorn, G.C., McDonald, M.F., 1999. The effect of condensed tannins in Lotus cornioculatus upon reproductive efficiency and wool production in sheep during late summer and autumn. J. Agric. Sci. Cam. 132, 323±334. Min, B.R., McNabb, W.C., Peters, J.S., Barry, T.N., 2000. Solubilization and degradation of protein from white clover (Trifolium repens) and Lotus corniculatus by rumen microorganisms and the effect of condensed tannins on these processes. J. Agric. Sci. Cam. 134, 305±317. NRC, 1985. Ruminant nitrogen usage. National Academy of Sciences. National Academic Press, Washington, DC, p. 138. O'Callaghan, D., Boland, M.P., 1999. Nutritional effects on ovulation, embryo developemnt and the establishment of pregnancy in ruminants. Anim. Sci. 68, 299±314. Parker, W.J., McCutcheon, S.N., Carr, D.H., 1989. Effects of herbage type and level of intake on the release of chromic oxide from intraruminal controlled release capsules in sheep. NZ J. Agric. Res. 32, 537±546. Reis, P. J., 1979. Effects of amino acids on the growth and properties of wool. In: Black, J.L., Reis, J. (Eds.), Physiological and Environmental Limitations to Wool Growth Reis, University of New England Publishing Unit, Armidale, New South Wales, pp. 223±242. Revell, D.K., Baker, S.K., Purser, D.B., 1999. Nitrogen and sulfur mobilised from body tissue can be used for wool growth. Aust. J. Agric. Res. 50, 101±108. Roughan, P.G., Holland, R., 1977. Predicting in vitro digestibilities of herbages by exhaustive enzymic hydrolysis of cell walls. J. Sci. Food Agric. 28, 1057±1064. Statistical Analysis System, 1985. User's Guide: Statistics, Version 5, SAS Institute, North Carolina, USA. Statistical Analysis System, 1995. Logistic Regression Examples Using the SAS System, 1st Edition. Version 6, SAS Institute, North Carolina, USA. Smith, J.F., 1985. Protein, energy and ovulation rate. In: Land, R.B., Robinson, D.W. (Eds.), Genetics of Reproduction in sheep. Butterworths, London, pp. 349±359. Smith, J.F., 1991. A review of recent developments on the effect of nutrition on ovulation rate (the flushing effect) with particular reference to research at Ruakura. Proc. NZ Soc. Anim. Prod. 51, 15±23. Smith, J.F., Jagusch, K.T., Farquhar, P.A., 1983. The effects of the duration and timing of flushing on ovulation rate in ewes. Proc. NZ Soc. Anim. Prod. 43, 13±16. Stewart, R., Oldham, C.M., 1986. Feeding lupins to ewes for four days during the luteal phase can increase ovulation rate. Proc. Aust. Soc. Anim. Prod. 16, 367±369. Terrill, T.H., Rowan, A.M., Douglas, G.B., Barry, T.N., 1992. Determination of extractable and bound condensed tannin concentrations in forage plants, protein concentrate meals and cereal grains. J. Sci. Food Agric. 58, 321±329. Terrill, T.H., Waghorn, G.C., Woolley, D.J., McNabb, W.C., Barry, T.N., 1994. Assay and digestion of 14 Clabelled condensed tannins in the gastrointestinal tract of sheep. Br. J. Nut. 72, 273±467. Visek, W.J., 1968. Some aspects of ammonia toxicity in animal cells. J. Dairy Sci. 51, 286±295. Waghorn, G.C., 1986. The effect of different protein/energy intakes on nutritional and physiological parameters in young sheep. Proc. NZ. Soc. Anim. Prod. 46, 31±35. Waghorn, G.C., Ulyatt, M.J., John, A., Fisher, M.T., 1987a. The effect of condensed tannins on the site of digestion of amino acids and other nutrients in sheep fed on Lotus corniculatus L. Br. J. Nut. 57, 115±126. Waghorn, G.C., John, A., Jones, W.T., Shelton, I.D., 1987b. Nutritive value of Lotus corniculatus L. containing low and medium concentrations of condensed tannins for sheep. Proc. NZ Soc. Anim. Prod. 47, 25±30. Waghorn, G.C., Smith, J.F., Ulyatt, M.J., 1990. Effect of protein and energy intake on digestion and nitrogen metabolism in wethers and on ovulation in ewes. Anim. Prod. 51, 291±300.

202

B.R. Min et al. / Animal Feed Science and Technology 92 (2001) 185±202

Wang, Y., Waghorn, G.C., Barry, T.N., Shelton, I.D., 1994. The effect of condensed tannins in Lotus corniculatus upon plasma metabolism of methionine, cysteine and inorganic sulphate by sheep. Br. J. Nut. 72, 923±935. Wang, Y., Douglas, G.B., Waghorn, G.C., Barry, T.N., Foote, A.G., 1996a. Effect of condensed tannins in Lotus corniculatus upon lactation performance in ewes. J. Agric. Sci., Cam. 126, 353±362. Wang, Y., Douglas, G.B., Waghorn, G.C., Barry, T.N., Foote, A.G., Purchas, R.W., 1996b. Effect of condensed tannins upon the performance of lambs grazing Lotus corniculatus and lucerne (Medicago sativa). J. Agric. Sci. Cam. 126, 87±98.