Enhanced reproductive performance of ewes given a sustained-release multi-trace element/vitamin ruminal bolus

Small Ruminant Research 39 (2001) 25±30 Enhanced reproductive performance of ewes given a sustained-release multi-trace element/vitamin ruminal bolus R.G. Hemingway *, J.J. Parkins, N.S. Ritchie Department of Veterinary Clinical Studies, University of Glasgow, Veterinary School, Bearsden, Glasgow, UK Accepted 7 May 2000 Abstract A sustained-release multi-trace element/vitamin bolus has been devised for administration to ewes in advance of mating. Each bolus contained 5.3 g Cu, 50 mg Se, 90 mg Co, 100 mg I, 3.2 g Mn and 4.7 g Zn with 268 10 3 IU Vitamin A, 54 10 3 IU Vitamin D and 800 IU Vitamin E. About 50% of each nutrient is released during the rst 6 weeks. Thereafter the release rate slows and over the remaining life of the bolus (about 6 months extending to lambing time) the daily release rates are about 15 mg Cu, 0.1 mg Se, 0.2 mg Co, 0.3 mg I, 9.4 mg Mn and 13 mg Zn with 775 IU Vitamin A, 156 IU Vitamin D and 2 IU Vitamin E. In one study the proportion of twins born to ewes given the bolus was very signi cantly (P < 0:001) higher than for the untreated ewes and greater than for those given a copper injection or copper oxide needles. In a second study the proportion of ewes given a bolus and having twin lambs was signi cantly (P < 0:01) greater than for untreated ewes and signi cantly (P < 0:05) fewer ewes were non-pregnant. In a third study, giving a bolus to shearling ewes tended (P ˆ 0:06) to result in fewer non-pregnant animals than for those untreated. Blood copper and Vitamin B 12 concentrations and glutathione peroxidase activities were above normal values throughout. # 2001 Elsevier Science B.V. All rights reserved. Keywords: Ewes; Reproductive performance; Trace element/vitamin bolus supplementation; Blood copper; Blood glutathione peroxidase 1. Introduction The critical role of trace elements in the fertility of ewes has been reviewed by Hidiroglou (1979). Both copper (Howell, 1968) and selenium (Hartley and Grant, 1961) inadequacies impair conception by reduced ova implantation, early embryonic loss and foetal death. Segerson and Ganapathy (1981) recorded * Corresponding author. Tel.: 44-1360-770044; fax: 44-1360-770711. signi cantly more fertilised ova, signi cantly more uterine contractions and signi cantly more ewes with large numbers of sperm attached to the zona pellucida due to improved sperm mobility when ewes of marginal blood selenium status (0.05 mg/ml) were given injections of 10 mg Se and 136 IU Vitamin E before mating. Godwin et al. (1970) reported signi cant increases in lambing percentage when ewes with an initial low blood Se concentration (0.026 mg/ml) were given oral supplements of 5 or 25 mg Se as sodium selenite or as a selenium/iron powdered metal bolus 1 month before lambing. Hill et al. (1969) have described a signi cant copper/selenium 0921-4488/01/$ ± see front matter # 2001 Elsevier Science B.V. All rights reserved. PII: S 0921-4488(00)00175-9

26 R.G. Hemingway et al. / Small Ruminant Research 39 (2001) 25±30 supplementation interaction resulting in an increased proportion of twins born where inadequacies (described as `marginal') of both elements occurred. Scales (1974) recorded that in three of four trials where Merino ewes were given orally 5 mg Se as sodium selenate approximately 17 days before mating the proportion of barren ewes was reduced by 17%. Cobalt insuf ciency, by its detrimental effects on appetite and eventual body condition score, results in reductions in both oestrus activity and multiple ovulation. In an experimental situation where cobalt inadequacy was described as `marginal' because of absence of clinical signs, Fisher and MacPherson (1991) recorded that dietary cobalt supplementation from 16 days before mating resulted in fewer lamb losses due to abortion and probable reabsorption between scanning in mid-pregnancy and birth and signi cantly fewer neonatal lamb deaths. Egan (1972) recorded signi cant increases in lambing percentage when ewes consuming herbage of marginal manganese and zinc status (about 45 mg Mn and about 20 mg Zn/kg dry matter) were additionally given orally 45 mg Mn and 20 mg Zn per day. It is not possible to ensure appropriate and individually uniform consumption of trace elements and vitamins by groups of sheep and cattle at grass from mineral mixtures, molassed liquids or high density feedblocks (Hemingway, 1998). Earlier communications by Lawson et al. (1990) have demonstrated that two trace element/vitamin boluses (`All-Trace', Agrimin Ltd. DN20 0SP UK) gave substantial sustained responses in glutathione peroxidase activity () in beef cows together with a signi cant increase in conception rate and reduction in calving spread (Allan et al., 1993). Signi cant sustained responses in blood copper concentration have been reported in both growing cattle and beef cows at grass by Parkins et al. (1994). Smaller sized boluses (55 mm length 18 mm diameter) have been demonstrated to give sustained responses in blood copper concentrations and activities in studies with both suckling beef calves at grazing and housed dairy-bred calves for over 4 months (Hemingway et al., 1997). Two boluses are required as a treatment since nutrient release is controlled by a combination of solution and mutual erosion in the reticulum. The principle and construction of the multiple trace element/vitamin bolus system for calves (Hemingway et al., 1997) has been modi ed for use as a single bolus system suitable for ewes (`Small-Trace', Agrimin Ltd. DN20 0SP) and has now been evaluated in studies with three ocks of upland ewes to assess the effect of such supplementation on the number of lambs born per ewe 2. Materials and methods 2.1. Bolus construction and release rate evaluations The bolus is composed of copper oxide powder, sodium selenite, cobalt sulphate, potassium iodide, manganese sulphate, zinc oxide and zinc sulphate with Vitamins A, D 3 and E. The 30 g active matrix contains (mg) Cu 5300, Se 50, Co 90, I 100, Mn 3250, Zn 4700 with (IU) Vitamins A 268,000, D 3 54,000 and E 800. There is a small base weight within each bolus to increase the density. An additional weight is temporarily attached with a water-soluble xative to the upper ( at) exposed end of the otherwise completely polymer-coated cylinder. This further increases the initial density and increases the rate of movement of the bolus to the reticulum immediately following administration. It rapidly separates from the bolus and then acts as a `grinder'. Evaluations of bolus matrix release rates have been made in adult rumen-cannulated cows and slaughtered cast ewes (Ritchie et al., 1997) and in growing sheep (Hemingway et al., 1997). About half (15 g) of the active matrix weight (30 g) is released in the rst 6 weeks (about 2700 mg Cu, 26 mg Se and 46 mg Co) and with proportional amounts of the other contained nutrients. Thereafter, the daily release rate reduces and the mean amounts eroded each day from 6 weeks to an estimated 7 months are (mg) Cu 15, Se 0.14, Co 0.24, I 0.28, Mn 9.2, Zn 13.3 with Vitamins A 762 IU, D 3 163 IU and E 2.3 IU. This would cover the period from tupping to lambing in ewes and ensures a useful elevated release during the initial mating period of 6±8 weeks. Retention of the boluses by the sheep is good and all the boluses (with the separated grinders), have been recovered from the reticulum at slaughter (Hemingway et al., 1997; Ritchie et al., 1997). The liver copper concentrations of the sheep re ected the loss of matrix from the boluses recovered after different intervals extending to 16 weeks.

R.G. Hemingway et al. / Small Ruminant Research 39 (2001) 25±30 27 2.2. Studies with ocks of upland ewes Studies with a single `Small-Trace' bolus were conducted in three separate upland ocks in situations where one or more trace element inadequacies (copper, selenium and/or cobalt) were known to be present and where various forms of individual supplementation were regularly advised as either injections, oral solutions or by other means. In each study, blood samples were obtained as a routine diagnostic metabolic pro le procedure in view of the previous clinical histories, initially on the day that `Small-Trace' and other various supplements were given (in November, about 10 days before the introduction of the rams) and again in late pregnancy in March (when the ewes were scanned to determine pregnancy status). These were taken on each occasion from a random 10 ewes in each larger group in each study and analysed for copper, activity and Vitamin B 12. Copper was determined by atomic absorption spectroscopy and activity (Units/ml red cells at 378C) by a modi cation of the method of Paglia and Valentine (1967) using a Ransel kit (Randox Laboratories). Vitamin B 12 was assessed by a modi cation of the method of Taylor and Greer (1982). The numbers of lambs born per treatment group was examined by chi-squared analysis and the mean assessments of blood parameters were evaluated by analysis of variance. 2.2.1. Study 1 This was conducted on the same farm where previous studies (Parkins et al., 1994; Hemingway et al., 1997) had demonstrated sustained incremental responses in blood Cu concentrations and activities in beef cows and calves, respectively, following supplementation with two larger-sized `All- Trace' boluses. A ock of 120 Blackface ewes were randomly allocated to four treatment groups each of 30 ewes. Group 1 ewes were given a single `Small- Trace' bolus (B); Group 2 ewes were given a copper injection (I) (`Coppaclear', providing 12.5 mg Cu as copper heptonate, Crown Veterinary Pharmaceuticals Ltd.); Group 3 ewes were given a capsule containing copper oxide needles (N) (`Copporal' containing 4 g CuO and providing 3.4 g Cu, Beecham Animal Health Ltd.) and Group 4 ewes were untreated (U). The groups of ewes grazed together and were with Border Leicester rams for 8 weeks. 2.2.2. Study 2 This was undertaken on a farm where both copper and selenium inadequacies were found occasionally in both ewes and beef cows. Blackface ewes were randomly allocated to two dietary treatments. Ninety six ewes were given a single `Small-Trace' bolus (B) and 118 ewes were unsupplemented (U). Both groups grazed together. Bluefaced Leicester rams were with the ewes from 10 days later for a period of 8 weeks. 2.2.3. Study 3 This study was conducted with Swaledale shearling ewes. The younger ewes on this farm had a history of inadequate reproductive performance. The overall management and general nutrition were good and trace element inadequacies were suspected from previous studies but supplementation had generally been from mid-pregnancy onwards. One group of 40 ewes was given a single `Small-Trace' bolus (B) and a further comparable group of 40 ewes was untreated (U). Additionally, 2 weeks earlier all the ewes had been given an oral drench which supplied 10 mg Co, 5 mg Se, 500,000 IU Vitamin A, 12,000 IU Vitamin D 3, 50 mg Vitamin B 1 and 750 mg Vitamin B 12. Rams were with the ewes for three complete oestrus cycles. 3. Results The normal range for blood copper concentrations for sheep is 9±15 mmol/l (Suttle, 1994) and lower values of 3.0±4.5 mmol/l are commonly found in clinically affected sheep when accompanied by reduced liver copper contents (Whitelaw et al., 1979). Anderson et al. (1979) have described blood activities (U/ml red cells at 378C) and their equivalent plasma selenium concentrations for sheep as de cient, <18 U and <0.05 mg Se/ml; low/ marginal, 30 U and <0.075 mg Se/ml; marginal, 40 U and <0.11 mg Se/ml and adequate, >40 U and >0.11 mg Se/ml. In these studies there were no problems in the administration of the boluses to the ewes. The ewes

28 R.G. Hemingway et al. / Small Ruminant Research 39 (2001) 25±30 remained under veterinary control and all, including the untreated ewes, were in good health throughout with normal blood values and no inter-current disease. In the event, all Vitamin B 12 analyses from all the sampled ewes on both occasions were adequate and above 250 ng/l (and with the majority >1000 ng/l) and, in the interests of brevity of presentation, are not further discussed here. 3.1. Study 1 Very signi cantly (chi-squared ˆ 13:3, P < 0:001) more twins were born to the ewes given the bolus and to those given the copper injection (chi-squared ˆ 4:8, P < 0:05) than were born to the untreated ewes (Table 1). The increase in the proportion of ewes given the copper oxide needles and having twins approached signi cance (chi-squared ˆ 3:15, P ˆ 0:07). There were also more twins born to the ewes given a bolus than to those given copper oxide needles (chi-squared ˆ 3:93, P < 0:05). The overall lambing percentages (lambs born per 100 ewes mated) were 160 (ruminal bolus), 140 (copper injection), 130 (copper oxide needles) and 100 (untreated). Mean blood copper concentrations and activities for all groups on both sampling occasions were always well above the critical levels (9 mmol Cu/l and 18 U/ml red cells) and both would be considered as fully adequate. Only the ruminal bolus treatment signi cantly (P < 0:001) increased mean activity. Table 2 Study 2: the number of lambs born, the mean concentrations of copper (mmol/l) and the mean glutathione peroxidase activities (, units/ml red cells) in the blood of the ewes 3.2. Study 2 Bolus Untreated S.E.D. P Twins 54 42 ± ± Singles 38 63 ± ± Non-pregnant 4 13 ± ± Blood Cu November 14.8 13.3 0.96 NS a March 15.1 10.3 1.59 <0.05 November 42 47 9.9 NS March 180 64 21.6 <0.001 Signi cantly (chi-squared ˆ 6:86, P < 0:01) more twin lambs were born to the ewes given the bolus than to the untreated ewes. There were also signi cantly (chi-squared ˆ 3:99, P < 0:05) more barren ewes that were untreated. The overall lambing percentages were 152 for the bolus-supplemented ewes and 124 for the untreated ewes. Mean blood copper concentrations were adequate throughout but by March a signi cant difference between mean values had developed with the concentration for the untreated ewes (10.3 mmol/l) approaching the lower limit of the normal range. Mean blood activity was markedly and signi cantly enhanced by the bolus treatment (Table 2). Table 1 Study 1: the number of lambs born, the mean concentrations of copper (mmol/l) and the mean glutathione peroxidase activities ( units/ ml red cells) in the blood of the ewes Bolus Injection Needles Untreated S.E.D. P (B) (I) (N) (U) Twins 19 13 11 5 ± ± Singles 10 16 17 23 ± ± Non-pregnant 1 1 2 2 ± ± Blood Cu November 13.2 13.0 15.5 14.1 0.94 NS a March 15.2 13.1 11.1 13.5 1.89 NS November 122 96 113 105 18.4 NS March 188 121 98 120 8.5 B > I; N; U < 0:001

R.G. Hemingway et al. / Small Ruminant Research 39 (2001) 25±30 29 Table 3 Study 3: the number of lambs born, the mean concentrations of copper (mmol/l) and the mean glutathione peroxidase activities (, units/ml red cells) in the blood of the ewes 3.3. Study 3 Bolus Untreated S.E.D. P Twins 9 9 ± ± Singles 22 14 ± ± Non-pregnant 9 17 ± ± Blood Cu November 13.4 12.1 1.20 NS a March 16.9 15.8 0.46 NS November 87 84 9.7 NS March 170 130 15.9 <0.05 Nine sets of twins were born to the 40 ewes in each group. Nine of the 40 ewes given the bolus were found to be non-pregnant compared with 17 of the 40 untreated ewes. This difference failed to reach signi cance at P < 0:05 (chi-squared 3.84 required), but with a chi-squared value of 3.65 was signi cant at P ˆ 0:06. The overall lambing percentages were 100 for the ewes given a bolus and 80 for the untreated ewes. Mean blood copper concentrations and activities were always fully adequate (Table 3). 4. Discussion and conclusions The overall mean blood copper concentration for the three studies combined (30 ewes given the bolus and 30 untreated ewes) was signi cantly (P < 0:01) higher in March than that for the untreated ewes (Table 4). Mean blood activities were also signi cantly greater (P < 0:001) in March as a result of bolus treatment. It is recognised that neither blood copper concentrations nor activities would be considered as inadequate at any stage for any of the three studies. In Study 1, the signi cantly higher lambing percentage of the ewes given the bolus compared to those given the copper injection or the copper oxide needles above that for the untreated ewes must be due to the inclusion of some other trace element (than copper) or vitamin in the bolus. This was considered most likely Table 4 Studies 1±3 combined: mean blood copper (mmol/l) and mean activities (units/ml red cells) for 30 ewes per each combined group Bolus Untreated Pooled S.E.D. Copper November 13.8 13.2 0.51 NS a March 15.7 13.2 0.84 <0.01 Pooled S.E.D. 0.98 0.98 ± ± P NS NS ± ± November 84 79 7.7 NS March 179 105 9.4 < 0.001 Pooled S.E.D. 12.2 12.2 ± ± P <0.001 <0.01 to be attributed to the selenium content. Mudd and Mackie (1973) reported that a combined intra-muscular injection of 6 mg selenium with 272 IU Vitamin E given 4 weeks before mating to hill ewes at ve sites in Scotland signi cantly (P < 0:001) increased the proportion of ewes with multiple births from 10.1 to 29.6%. Mean initial blood selenium concentrations were normal (0.09 S.D., 0.025 mg/l). Earlier in New Zealand, Hartley and Grant (1961), Hartley (1963) and Andrews et al. (1968) gave a similar injection to ewes before mating and variously reported increases in lambing percentage from 74 to 118, from 25 to 90 and from 80 to 120. The ewes were in fair to good condition and the difference was due to both failure to conceive and early embryonic death at 3±4 weeks post conception. In Study 2, as a result of two mechanisms, the lambing percentage of the ewes given the bolus was 152 compared with 124% for the untreated ewes. The bolus treatment increased the number of twins born and at the same time reduced the proportion of barren ewes. In Study 3, the bolus treatment markedly, but just failing signi cance (P ˆ 0:06), reduced the number of non-pregnant shearling ewes. The contrasting lambing percentages were 80 (untreated) and 100 (bolus treatment). The bolus treatment resulted in a signi cantly higher mean activity in March than that for the untreated ewes (Table 3). This tendency to an improved lambing performance was found in spite of the fact that all the ewes in Study 3 P

30 R.G. Hemingway et al. / Small Ruminant Research 39 (2001) 25±30 had been given an oral drench containing cobalt, selenium and Vitamins A, B and D about 1 month before mating. It is concluded that the multi-trace element/vitamin bolus given to ewes before mating signi cantly increased the lambing percentage of upland ewes from the existing situation where 80±120 lambs were born to 100 ewes mated although blood copper and Vitamin B 12 concentrations and activities for the untreated ewes were considerably higher than the minimum of accepted normal ranges throughout. Acknowledgements The Cu and determinations were undertaken by C. Cameron. Vitamin B 12 determinations were undertaken at the Veterinary Investigation Centres at Penrith and the Scottish Agricultural College at Edinburgh. These studies would not have been possible without the full interest and co-operation of the farmers (Messrs A. Bain, J. Fraser and D. Lawton) and their veterinary surgeons (Messrs A. McInness, M. Shanks and M. Colston). References Allan, C.L., Hemingway, R.G., Parkins, J.J., 1993. Improved reproductive performance in cattle dosed with trace element/ vitamin boluses. Vet. Rec. 132, 463±464. Anderson, P.H., Berrett, S., Patterson, D.S.P., 1979. The biological selenium status of livestock in Britain as indicated by sheep erythrocyte glutathione peroxidase activity. Vet. Rec. 104, 235± 238. Andrews, E.D., Hartley, W.J., Grant, A.B., 1968. Seleniumresponsive diseases of animals in New Zealand. N.Z. Vet. J. 16, 3±17. Egan, A.R., 1972. Reproduction responses to supplementary zinc and manganese in grazing Dorset Horn ewes. Aust. J. Exp. Agric. Anim. Husb. 12, 131±135. Fisher, G.E.J., MacPherson, A., 1991. The effect of cobalt de ciency in the pregnant ewe on reproductive performance and lamb viability. Res. Vet. Sci. 50, 319±327. Godwin, K.O., Kuchel, R.E., Buckley, R.A., 1970. The effect of selenium on infertility in ewes grazing improved pastures. Aust. J. Exp. Anim. Husb. 10, 672±678. Hartley, W.J., 1963. Selenium and ewe fertility. Proc. N. Z. Soc. Anim. Prod. 23, 20±27. Hartley, E.D., Grant, A.B., 1961. A review of selenium responsive diseases in New Zealand livestock. Fed. Proc. 20, 679±688. Hemingway, R.G., 1998. Supplying trace elements to cattle. Feedmix 6 (4), 8±10. Hemingway, R.G., Parkins, J.J., Ritchie, N.S., 1997. Copper and glutathione peroxidase () responses in lambs given a sustained-release rumen bolus. Proc. Nutr. Soc. 56, 305A. Hidiroglou, M., 1979. Trace element de ciencies and fertility in ruminants: a review. J. Dairy Sci. 62, 1195±1206. Hill, M.K., Walker, S.D., Taylor, A.G., 1969. Effects of `marginal' de ciencies of copper and selenium on growth and productivity in sheep. N.Z. J. Agric. Res. 12, 261±270. Howell, J.McC., 1968. The effect of experimental copper de ciency on growth, reproduction and haemopoiesis in the sheep. Vet. Rec. 83, 226±227. Lawson, D.C., Ritchie, N.S., Parkins, J.J., Hemingway, R.G., Gresham, H.R., 1990. Use of a sustained-release bolus for enhancing the selenium status of cattle. Vet. Rec. 127, 67±68. Mudd, A.J., Mackie, I.L., 1973. The in uence of Vitamin E and selenium on ewe proli cacy. Vet. Rec. 93, 197±199. Paglia, D.C., Valentine, W.N., 1967. Studies on the quantitative and qualitative characteristics of erythrocyte glutathione peroxidase. J. Lab. Clin. Med. 70, 158±169. Parkins, J.J., Hemingway, R.G., Lawson, D.C., Ritchie, N.S., 1994. The effectiveness of copper oxide powder as a component of a sustained-release multi-trace element and vitamin rumen bolus system for cattle. Br. Vet. J. 150, 547±553. Ritchie, N.S., Hemingway, R.G., Parkins, J.J., 1997. A sustainedrelease multi-trace element/vitamin rumen bolus for ewes. Proc. Br. Soc. Anim. Sci. 154. Scales, G.H., 1974. Reproductive performance of Merino ewes dosed with selenium prior to mating. Proc. N. Z. Soc. Anim. Prod. 34, 103±113. Segerson, E.C., Ganapathy, S.N., 1981. Fertilisation of ova in selenium/vitamin E-treated ewes maintained on two planes of nutrition. J. Anim. Sci. 51, 386±394. Suttle, N.F., 1994. Meeting the copper requirements of ruminants. In: Garnesworthy, P.C., Cole, D.J.A. (Eds.), Recent Advances in Animal Nutrition. Nottingham University Press, Nottingham, UK, pp. 173±188. Taylor, C.N., Greer, J.C., 1982. Further studies of radio-assay techniques for the estimation of Vitamin B 12 in ovine serum. Medium 15, 15±18. Whitelaw, A., Armstrong, R.H., Evans, C.C., Fawcett, A.R., 1979. A study of the effects of copper de ciency in Scottish Blackface lambs on improved hill pasture. Vet. Rec. 445±460.