Alargar la vida a los instrumentos rotatorios de NiTi con manipulación.

The coumestrol threshold of 25 mg/kg DM chosen for the regional risk assessment was based on the coumestrol levels reported in Chapter 8 and previous research by Smith et al. (1979). Coumestrol at approximately this level was shown in both cases to cause an effect on ewe reproductive

performance. However, it is probable that levels below this will also have an effect and so a true safe value has not yet been determined. The threshold and median predicted coumestrol level do, however, provide an indication of the differences between regions, with Blenheim likely to have

157 lower coumestrol levels in any given autumn than Lincoln, Lauder or Napier (Figures 6.12 & 6.13), and therefore less likely to have reduced reproductive performance. The risk assessment model also showed an effect of delaying the mating period on the prevailing coumestrol content of lucerne (Figures 6.10 & 6.11). It was predicted that the later into autumn that ewes are mated, the more likely they are to be consuming high coumestrol lucerne. The exception was Napier which tended to have a consistent median predicted coumestrol (19 and 34 mg/kg DM for four and six week old lucerne, respectively), and a constant likelihood of exceeding the 25 mg coumestrol/kg DM threshold (38 and 65% for four and six week old lucerne, respectively; Figure 6.17), throughout the mating period.

In Lauder, Lincoln and Blenheim, the lowest risk of heightened coumestrol values was for lambing dates between 15 July and 1 September (Figures 6.14 to 6.16). However, data from The Sheep and Beef Farm Survey (Beef and Lamb New Zealand, 2014) showed that across New Zealand only 2% of ewes lamb before 3 August and only 17% by the end of August. There was a regional difference. In Marlborough and Canterbury, lambing dates were spread out with approximately 20% of ewes lambed by the end of August, 40% by mid-September, 60% by the end of September, and 90% by the end of October. In contrast, in Otago and Southland there is a lot less spread with no lambing in August, approximately 30% of lambing completed by the end of September and 90% by the end of October.

The lambing date distributions (Figures 6.15 & 6.16) mean that in Canterbury and Marlborough there may be lee-way for farmers to reduce the fecundity impairing risk of coumestrol by mating ewes earlier in the season. However, the spread in lambing date distribution could also indicate a greater variation in suitable lambing date between areas within Canterbury and Marlborough. On the other hand in regions such as Central Otago (Lauder), ewes are mated in late-autumn when coumestrol is most at risk of being high (Figure 6.14), and it is unlikely to be viable to move outside of this norm and mate ewes earlier in the season. The best way to reduce the risk from coumestrol in lucerne across all regions would be to mate ewes on four week old regrowth rather than on six week old regrowth as the risk is substantially reduced. In addition, at the end of a period of high rainfall and/or high humidity lucerne allocated for mating ewes could be grazed off by non-mating livestock or cattle, which are unlikely to be affected by coumestrol in the lucerne. This would enable new

regrowth to utilise the improved soil moisture from this rainfall, and as long as no further substantial rainfall and few high humidity days occur in the crop, and it is grazed while still relatively young (i.e. three or four weeks), it should be safe for ewes to return to it.

158

Chapter 7

Experiment 12 Morphological response of ewes to coumestrol

Introduction

7.1

Experiment 12 investigated the morphological response of ewes exposed to coumestrol as part of Objective 4 which was to quantify animal responses to elevated coumestrol. Teat growth has

previously been reported in wethers grazing oestrogenic lucerne (Newton and Betts, 1968) but not in ewe lambs. This chapter reports a serendipitous incident (Experiment 12a) where ewe lambs grazing lucerne on a commercial sheep farm were observed to have premature mammary and teat

development. The farmers contacted Lincoln University about this observation and further investigation was undertaken as part of this PhD research. Specifically, this spontaneous on-farm event provided an opportunity to investigate the occurrence of mammary development in ewe lambs grazing an oestrogenic lucerne crop and to determine if these effects had consequences for

reproductive performance after removal from the crop. The null hypothesis was that there would be no difference in mammary development between ewe lambs on grass or lucerne.

Where there is uncertainty about whether a lucerne crop is sufficiently oestrogenic to cause

impairment of fecundity in breeding sheep it would be useful to have a simple means to monitor the crop for this propensity. For farmers the occurrence of mammary and/or teat development in female sheep may be an easily recognisable indicator of oestrogenic activity in forage crops. However, if there is a difference in sensitivity of sheep that is dependent on their age, it may be necessary to use immature females for this purpose. This chapter also includes a comparison of coumestrol responsiveness between ewe lambs and adult ewes that was designed to address this question (Experiment 12b). In the study, ewe lambs and adult ewes were injected with coumestrol for nine days and teat growth and mammary development were monitored in comparison with those of animals receiving control (saline) injections. The hypothesis under test was that ewe lambs are more responsive to the effects of an exogenous coumestrol than adult ewes. The null hypothesis was that ewe lambs were not more responsive than adult ewes to coumestrol.

Methodology

7.2

7.2.1

Experiment 12a: Ewe lambs exposed to an oestrogenic lucerne pasture (an

on-farm study)

In Experiment 12a sheep were cross-bred ewe lambs of predominantly Texel-East Friesian-

159 North Otago, New Zealand. They were born in spring 2014 and at weaning on 27 November 2014 any with a live weight below 32.5 kg were allocated to a lucerne crop (n = 36). Those with higher live weights were returned to a predominantly ryegrass/white clover pasture (n = 22). The average live weights were 25.1 ± 0.84 kg for sheep on lucerne and 34.7 ± 0.41 kg for the grass-based pasture. This allocation was a commercial management decision intended to provide a superior nutrient supply to the lighter lambs. Lambs assigned to lucerne were nine days younger (P < 0.001) than lambs assigned to grass. The average date of birth (± SEM) was 3 September 2014 ± 0.9 days and 12 September 2014 ± 1.6 days for grass- and lucerne-fed lambs, respectively.

Throughout the post-natal growth period the lambs received routine husbandry treatments for endo- and ecto-parasites, anti-clostridial vaccinations and vitamin supplements. Their fleeces were shorn on 2 March 2015. Live weight was measured with electronic scales at weaning (27 November 2014), pre-shearing (2 March 2015), on 12 March 2015, and at commencement of mating (20 April 2015). On 12 March and 16 April 2015, all ewe lambs were checked by palpation for protruding mammary glands. Measurements of teat length, teat width at the base and mammary gland diameter were recorded using a digital calliper for 22 of the lucerne-fed ewe lambs and for 10 of the grass-fed lambs selected randomly. For each lamb, data for right and left teat measurements were averaged to give a single value.

On 20 March 2015, sheep were removed from the lucerne crop and re-united with their grass-fed cohorts on the grass-based pasture. A vasectomised ram was placed with the ewe mob on 9 April 2015 and remained there for two weeks before being replaced with a crayon-harnessed entire ram from 20 April 2015. Crayon marking of the ewes was recorded and the number of fetuses present was determined by transabdominal ultrasound recording carried out by a commercial operator on 24 June 2015.

7.2.1.1

Lucerne coumestrol measurement

Plant samples were collected on 12 March 2015 from the grass-based pasture and the lucerne paddocks used in the grazing rotation and analysed by HPLC for coumestrol content using the methodology described in Sections 4.4 to 4.7.

7.2.1.2

Statistical analysis

Data in Experiment 12 (Chapter 7) were analysed in Minitab 17. For Experiment 12a two-sample t- tests were used to compare the two treatment groups for live weight at each weighing, teat width, teat length, and scanning rate. Regression models were used to account for the effects of diet, live weight, growth rate, birth rank and age on mammary presence, teat width and teat length. These models were built with terms through order two and stepwise regression (α to enter = 0.15). Ordinal

160 regression ((R (version 3.3.3) with ‘polr’ function from package ‘MASS’) was used to account for the effects of diet and live weight on the number of fetuses per ewe at scanning.