Reproductive traits of Tunisian Queue Fine de l’Ouest ewes fed on wheat straw supplemented with concentrate and Acacia cyanophylla Lindl. foliage with and without polyethylene glycol (PEG)

M Rekik, N Lassoued*, H Ben Salem* and I Tounsi**

Ecole Nationale de Médecine Vétérinaire, 2020 Sidi Thabet, Tunisia
rekik.mourad@iresa.agrinet.tn
*INRA-Tunisie, Laboratoire des Productions Animales et Fourragères, Rue Hédi Karray, 2049 Ariana, Tunisia
**Ecole Supérieure d’Agriculture du Kef, 7100 Le Kef, Tunisia

Abstract

To the question do Acacia cyanophylla Lindl. (Acacia) tannins affect reproductive performance of local ewes?  This experiment was carried out on forty four multiparous and 4 nulliparous ewes of the Queue Fine de l’Ouest breed (mean initial live weight, 44.8 ± 5.78 kg). Ewes were divided into two equal groups balanced for age and live weight. For 66 days prior to synchronised mating and 24 days thereafter, ewes in C treatment received each on daily basis 0.3 kg of air-dried acacia foliage, 0.4 kg of concentrate and 1.0 kg of wheat straw. Ewes in treatment polyethylene glycol received the same diet as C ewes and they were each orally drenched every day with 20 g of polyethylene glycol (PEG 4000). In vivo diet digestibilities were measured, live weight and body condition score changes were recorded and reproductive performances were measured from oestrous behaviour, ovulation rate and litter size.

Total extractable phenols and tannins and condensed tannins in Acacia averaged 25.9 and 22.4 g tannic acid equivalent and 37.3 g leucocyanidin equivalent/Dry matter, respectively. Acacia supply had no significant effect (P > 0.05) on straw intake, dry matter and organic matter digestibility of diets. However, neutral detergent fibre and crude protein digestibilities increased significantly (P < 0.05) with polyethylene glycol administration. Plasma urea and creatinine concentrations were similar among animals (P > 0.05). Neither live weight nor body condition score changes were affected by the dietary treatments. live weight loss averaged or slightly exceeded 1 kg in polyethylene glycol and C treatments, respectively. However, at synchronised mating, body condition score within treatment group was significantly (P < 0.05) lower than values of body condition score a month earlier. At synchronised mating, all ewes in the polyethylene glycol group manifested oestrus in comparison to only 83.3% in the C treatment group (P < 0.05). Levels of ovulation rate were low and averaged 1.05 ± 0.12 and 1.0 ± 0 in ewes fed on C and polyethylene glycol diets, respectively. Lambing rates after mating period were 91.6 and 100% in respectively ewes of the C and polyethylene glycol treatment groups (P > 0.05) and all the sheep in both treatment groups that lambed invariably yielded singles. Results suggest that loss of the body condition score is causing most of the observed effects in the reproductive results.

Keywords: Acacia cyanophylla Lindl., body condition, ewes, polyethylene glycol (PEG), reproduction, tannins

Introduction

Reproduction is very prone to changes in nutritional supply (O’Callaghan and Boland 1999). In sheep, the level of feeding, energy intake and protein absorption are an important regulator of reproductive function (Smith 1991) from very short-term effects (few days) as reported by Stewart and Oldham (1986) to long-term effects (scale of months) that have an incidence on the reproductive rhythms of the animals and that could extend to the reproductive capacity of the next generation (Fowden et al 2006). However, the obtained responses show large differences between breeds (Rhind 1992) indicating probably variations in feeding strategies in terms of quantity and quality of the feed but also in the timing of supplementation. In semi-arid Tunisia where regular food supply is not guaranteed, sheep flock performances have become closely geared to nutritional supply and very often, farmers use large quantities of concentrate as an expensive resort to supplement animals on low quality roughages and pastures (Ben Salem et al 2000). For reproduction, such a strategy may not be efficient. Lassoued et al (2004) showed that for the low prolific Queue Fine de l’Ouest breed ewes, increasing the level of nutrition through a heavier use of concentrate did not improve ovulation rate but substantially depressed litter size as a result of higher reproductive losses.

Under these challenging conditions, alternative feeding strategies need to consider non-conventional foodstuffs. In this respect, multipurpose shrubs largely established in Tunisia to rehabilitate degraded rangelands (Ben Salem et al 2002a) should be included in the feeding plans. Acacia cyanophylla Lindl., a widespread shrub species in Tunisia and despite the low nutritive value of its foliage (Degen et al 1997; Ben Salem et al 2002b), produces a high quantity of biomass which is relatively rich in crude protein throughout the year (Ben Salem 1998). However, abundant tannins in this plant species form insoluble complexes with the proteins rendering them unavailable for the rumen microflora and consequently the host animal. The use of polyethylene glycol, a synthetic polymer, has proven to provide satisfactory results in deactivating acacia tannins (Ben Salem et al 1999) hence improving the efficiency of utilisation of this shrub by animals (Makkar 2003a). In this respect, Lassoued et al (2006) produced evidence that deactivation of condensed tannins in acacia with polyethylene glycol produced an increase in ovulation rate of goats as a result of increased protein availability. However, the effect of acacia feeding on reproductive performance of ewes is, to our knowledge, not yet investigated. Using Queue Fine de l’Ouest ewes prior to and during mating, fed a diet including air-dried acacia, this study assessed the effect of polyethylene glycol administration on the diet digestibility, blood metabolites and live weight as well as their reproductive traits namely oestrus occurrence, ovarian cyclic activity, ovulation rate and litter size.
 

Materials and methods

Study location

This study was carried out in the sheep research station of the Ecole Supérieure d’Agriculture du Kef in North-West, semi-arid Tunisia (latitude: 35°7’ North, longitude: 9° East, average annual rain fall: 370 mm).

Experimental feeds

Acacia foliage (leaves and twigs) was hand-harvested from a plantation in the Jebibina’s farm of the Pasture and Livestock Agency (Central Tunisia, average annual rainfall 390 mm). It was spread on plastic sheets to dry in a shade for 4-5 days, then packed in sacks and stored for later use. Wheat straw and concentrate (mixture of processed barley grain, soybean meal and a commercial mineral and vitamins supplement, 80: 17.5 : 2.5) were also used in this experiment. 

Animals and experimental design

Forty four multiparous and 4 nulliparous ewes of the Queue Fine de l’Ouest breed with a mean initial live weight of 44.8 ± 5.78 kg were used. Animals of this breed thrive in the North Western part of Tunisia showing a great adaptation to the degraded mountainous pastures of the region. The flocks are usually mated in spring for an out of season breeding and their inherent prolificacy is low averaging 120% (Lassoued et al 2004).  The ewes were randomly allocated to two treatment groups balanced for age and live weight. Ewes in control treatment (C) received each and on daily basis 0.3 kg of air-dried acacia foliage, 0.4 kg of concentrate and 1.0 kg of wheat straw. Ewes in treatment polyethylene glycol received the same diet as ewes in treatment C and they were each orally drenched every day with 20 g of polyethylene glycol (PEG 4000) dissolved in 40 ml of water (1 kg PEG : 2 L water).

During the 90 days trial (April 1^st until June 30^th) including 10 days of acclimatization to experimental conditions and dietary treatments, animals were individually penned in a barn covered on the top and open on the sides allowing a free circulation of air and continuous exposure of animals to natural light. Each animal had free access to clean water at all times. Concentrate was offered in two equal meals at 08:00 h and 16:00 h. Acacia was offered once daily at 09:00 h immediately after being drenched with polyethylene glycol. Straw was distributed in two equal meals at 11:00 h and 16:00 h allowing, prior to the morning meal, sufficient time for the animals to consume totally or most of the acacia. Feedstuffs were offered in separate troughs.

Management of reproduction

All ewes were naturally mated at a synchronised oestrus starting on the 5th June, i.e. 66 days after the onset of the application of the feeding treatments and for animals not conceiving at the induced oestrus, mating continued until 30 June. Oestrus was synchronised using intravaginal progesterone pessaries impregnated with 40 mg fluorogestone acetate and left in situ for 14 days. At pessaries removal and for the subsequent 6 days, all the ewes were allowed to stay out of the pens and group-fed the same diets as described above within small groups of 5-6 ewes each. A ram of the Queue Fine de l’Ouest breed was introduced in each group and throughout the mating period, rams were daily rotated between the groups. This was done to ensure that a ram failure did not affect reproductive outcome. The ewes were monitored for external signs of oestrous occurrence and the onset of oestrus in relation to the timing of pessaries removal and ram introduction was recorded. Thereafter and until the end of the mating period, the ewes were individually checked for oestrus twice daily (morning 07:00 - 08:00 h and afternoon 18:00 - 19:00 h). The ewes that exhibited heat were allowed to naturally mate twice at 12 hours interval.

Measurements

Throughout this experiment, the amount of feed offered and refusals were weighed daily to determine dry matter intake of all the feedstuffs. Samples of feed offered were taken every week, dried at 50 °C, ground using a 1 mm screen then stored until analysed. Blood samples for urea and creatinine determination were withdrawn at the start, half-way (day 48) through and at the end of the trial (day 87), always before morning feeding. Blood samples were recovered in heparinised vacutainer tubes from each animal. After centrifugation (3000 Rotations per minute, 15 min.), plasma was stored at –20 °C until assayed. Throughout the experimental period, the ewes were weighed on the 35^th day following the start of the nutritional treatments and one month later immediately prior to the start of the mating period. At each occasion, the ewes were weighed before the morning feeding. On the same occasions, the ewes body condition score was measured according to the method described by Russel et al (1969).

The total faecal collection technique was used to determine diet digestibility. Five ewes selected randomly from each group were fitted with harnesses and allowed to adapt to the new experimental conditions for 4 days before starting the 7-day faecal collection period on June 25^th. Along with the digestibility period, ewes received the same dietary treatments and monitored as above. The amount of feed offered and of individual refusals and faeces were weighed and samples were taken for later analyses and for determination of Dry matter contents using a ventilated oven at 80 °C. At the end of the faecal collection period, the samples of feed offered, refusals and faeces were pooled by animal and subsamples were dried at 50 °C, ground using 1 mm screen then stored pending analysis.

Cyclic ovarian activity and ovulation rate were determined using the laparoscopy method as described by Thimonier and Mauléon (1969) 40 days after the application of the nutritional treatment and 35 days later corresponding to day 9 after the synchronised oestrus. At lambing, litter size was recorded.

Laboratory analyses

Total extractable phenols, tannins and condensed tannins contents were determined in triplicate on 0.2 g samples of feed offered according to Makkar (2003b). Feed offered, refusals and faeces samples were analysed for organic matter and Kjeldahl crude protein according to AOAC (1984) and for neutral detergent fibre (Van Soest et al 1991). The α–amylase and sodium sulphite were not included in the extraction solution. Contents of all analysed components were expressed on dry matter basis. The total extractable phenols and tannins contents were expressed as tannic acid equivalent and condensed tannins were expressed as leucocyanidin equivalent.

Urea and creatinine concentration in plasma were determined using commercially available kits (Biomaghreb®, Tunis, Tunisia). The kits were based on bio-chemically established procedures and concentrations were measured photometrically at specific wavelengths. 

Statistical analysis

Data on feed intake, diet digestibility, and the concentrations of blood metabolites were compared using a Student’s t-test (SAS 1991). Data on live weight and body condition score were submitted to an analysis of variance (SAS 1991) with dietary treatment and the period of measurement as the main effects. The data on the proportions of ewes exhibiting oestrus or cycling ovarian activity as well as those lambing were analysed using a χ² test. The same test was also used for the analysis of ovulation rate and litter size. The Fisher Exact test was preferred when the number of measurements per cell was below 5.

Results

Nutritive value of experimental diets

Acacia was higher in crude protein than wheat straw and its total extractable phenols, tannins and condensed tannins are reported in Table 1.

The amount of acacia included in both diets was completely consumed by ewes. Therefore, these animals were consuming approximately 6 and 10 g/day of tannins (tannic acid equivalent) and condensed tannins (leucocyanidin equivalent), respectively. As can be seen from Table 2, these secondary compounds had no effect (P > 0.05) on straw intake, and apparent digestibility of diet dry matter and organic matter.. polyethylene glycol supplementation increased (P < 0.05) crude protein and neutral detergent fibre digestibility but did not affect (P > 0.05) plasma urea and creatinine concentrations.

Live weight and body condition score variation

Sheep in both treatment groups lost weight during the course of the application of the nutritional treatments (Table 3). Weight loss averaged or slightly exceeded 1 kg in the polyethylene glycol and the C treatments respectively and in neither case, it was statistically significant (P>0.05).

Most of the weight loss occurred during the first month after the application of the nutritional treatments and thereafter, live weight remained constant for sheep in both treatment groups.

The initial body condition score at the start of the experiment was not measured and only its variation during the second month after the application of the nutritional treatments is reported. Similarly to live weight, a reduction in the average body condition score was recorded for sheep receiving the two experimental diets (Table 3). Within experimental groups, body condition score after 65 days of the application of the nutritional treatment was significantly (P < 0.05) lower than values of body condition score a month earlier.

Effect of acacia on reproductive traits

After 40 days of the application of the nutritional treatments, few ewes (2 and 5 respectively in the C and polyethylene glycol groups) had at least a corpus luteum present on one of their ovaries (Table 4).

Nine days after removal of the sponges and the start of the mating period, all ewes in the polyethylene glycol treatment group had at least a corpus luteum on their ovaries whereas 87.5% of C ewes were cyclic (P = 0.08). Irrespectively of dietary treatment, all ewes shed only one ovum except one ewe in the C treatment that had twin ovulations. As can be seen from Table 4 at the synchronised mating, treatment differences in the proportions of ewes showing oestrus were significant (P < 0.05). The proportion of ewes detected in oestrus was higher in the polyethylene glycol than in the C treatment group. Ewes in both treatments exhibited oestrus during the first 3 days following pessaries removal with a tendency for sheep receiving polyethylene glycol to come to oestrus earlier than those fed the C diet (Figure 1).  

Lambing rates at the induced oestrus and after the mating period are shown in Table 4. No differences for lambing rate were recorded between the two treatment groups. All ewes in both the C and the polyethylene glycol treatments that lambed invariably yielded singles (Table 4).

Discussion

This study suggests that deactivation of tannins in Acacia cyanophylla Lindl. foliage by polyethylene glycol resulted in an improvement of energy and nitrogen values of experimental diets as indicated by the increased amount of digestible organic matter and digestible crude protein intakes. Such a beneficial effect of supplying polyethylene glycol on the feeding value of diets including acacia foliage was also reported by Ben Salem et al (2000).

polyethylene glycol supply in this study was not associated with an improvement of the end reproductive parameters namely lambing rate and litter size per lambing ewe. This result is unlikely to be due to insufficient protein supply because protein intake averaged 177 g protein/ewe/day in our experiment, well above the threshold intake level necessary to achieve an increase in ovulation rate (Smith 1985). In line with this, it is likely that polyethylene glycol provision increased the amount of protein absorbed by the ewes, hence amino acids availability at the gut level. However, the absence of any effect on ovulation rate possibly suggests that the protein input was not sudden to flush the ewes. Another possible explanation is the existence of secondary compounds, other than tannins. Nevertheless, published reports preclude the existence of such secondary compounds in Acacia cyanophylla Lindl. (Dynes and Schlink 2002) and, in Tunisia, Ben Salem (unpublished results) has failed to identify secondary compounds different from tannins (e.g. mimosine, saponins) in several accessions of Acacia cyanophylla Lindl. Alternatively, energy supply was insufficient to sustain the increase in ovulation rate as a result of increased protein availability. Such a hypothesis is forwarded because ewes in both the C and polyethylene glycol treatments loosed body condition but is not backed by the calculated energy provision of both diets. If the daily metabolisable energy maintenance requirements are estimated at 0.397 MJ ME/kg live weight^0.75 (Jarrige 1980), then ewes in the C and polyethylene glycol treatment groups were respectively receiving 1.67 and 1.69 of the daily metabolisable energy maintenance requirements. However, changes in live weight and in body condition score highlight the weakness of the currently used methods in estimating the energy requirements of the studied breed as previously signalled by Lassoued et al (2004).  It is most likely, in the case of this study, that the decline in body condition score over the period prior to and during mating has rendered ineffective the expected positive effect of the likely increased absorption of protein on ovulation rate in the   ewes (McWilliam et al 2004). Indeed, it is now well established from the male sheep model (Blache et al 2006) but also from dairy cows (Villa-Godoy et al 1990), that changes in the energy balance through several metabolic signals (glucose, insulin) can influence any of the three levels of the reproductive axis (hypothalamus, pituitary gland, gonads). Unfortunately, proper monitoring of these hormones and metabolites was not undertaken in our study.

Our results are in conflict with those by McWilliam et al (2004) who reported a significant improvement of the reproductive rate of Romney ewes with an increased absorption of protein as a result of poplar (Populus) supplementation and that were loosing weight and body condition score. These discrepancies are perhaps indicative of breed differences in the response of the reproductive mechanisms to several concomitant nutritional and metabolic signals. In the current study, live weight changes do not correlate with the possible relationship between body condition score loss and ovulation rate confirming previous conclusions (Smith et al 1983) that ovulation rate is a more sensitive indicator of nutritional and metabolic changes than live weight.

Beyond its nullifying effect of the increased protein availability on ovulation rate in the polyethylene glycol group, losses in body condition score provide the only explanation to the unusually observed decline of ovulation rate at mating and litter size at mating in both treatment groups. Working with the same experimental flock of Queue Fine de l’Ouest ewes as the one used in this study and during the same season of the year, Lassoued et al (2004) reported a mean ovulation rate varying from 1.17 to 1.26 according to the nutritional level and the obtained litter size in this study was well below the average figures commonly reported for the Queue Fine de l’Ouest breed (Rekik et al 2005).

The only positive effect on reproduction of deactivating acacia tannins by daily oral drenching with polyethylene glycol was on the proportion of ewes that were found cycling and those that exhibited oestrus at the synchronised mating. If the higher incidence of ovulating, polyethylene glycol-treated ewes could be related to their better long term nutritional status in terms of proteins, it remains difficult to explain why ewes in the polyethylene glycol treatment group exhibited oestrous behaviour earlier than their counterparts in the C group. There are no similar published results if we except findings by Melaku et al (2004) that supplementation of Menz ewes with the shrub Sesbania sesban 1198 had a negative influence on their reproduction by compromising manifestation of oestrus. The effect was related to the anti-nutritional factors in the studied plant. In our study, the experimental diets were maintained throughout the mating period and even though return to oestrus was not monitored, overall lambing rates preclude any real significant effect of the nutritional treatments, particularly acacia, on the capacity of ewes to reproduce.

Conclusions

• Supplementing Queue Fine de l’Ouest ewes fed wheat straw with concentrate and air-dried acacia foliage with and without polyethylene glycol resulted in very similar reproductive results being unusually low for ovulation rate and litter size.

  
  

• The mechanisms, whereby the likely increased absorption of protein in the ewes receiving polyethylene glycol should have caused an elevation of ovulation rate and consequently litter size, were perhaps blocked by other metabolic signals arising from a rapid decrease of the ewes’ body condition prior to and during mating.

Acknowledgements

This work was supported by the International Atomic Energy Agency (IAEA) and the Ministry of Scientific Research, Technology and Capacity Building of Tunisia under the framework of the technical cooperation project TUN 5/021.
 

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Received 27 July 2007; Accepted 3 August 2007; Published 1 November 2007

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