Accepted Manuscript

Effect of breed, plane of nutrition and age on growth, scrotal development, metabolite
concentrations and on systemic gonadotropin and testosterone concentrations
following a GnRH challenge in young dairy bulls

C.J. Byrne, S. Fair, A.M. English, C. Urh, H. Sauerwein, M.A. Crowe, P. Lonergan,
D.A. Kenny
PII: S0093-691X(17)30152-8
DOI: 10.1016/j.theriogenology.2017.04.002
Reference: THE 14053

To appear in: Theriogenology

Received Date: 11 November 2016

Revised Date: 8 March 2017
Accepted Date: 1 April 2017

Please cite this article as: Byrne CJ, Fair S, English AM, Urh C, Sauerwein H, Crowe MA, Lonergan
P, Kenny DA, Effect of breed, plane of nutrition and age on growth, scrotal development, metabolite
concentrations and on systemic gonadotropin and testosterone concentrations following a GnRH
challenge in young dairy bulls, Theriogenology (2017), doi: 10.1016/j.theriogenology.2017.04.002.

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 ACCEPTED MANUSCRIPT
1 Effect of breed, plane of nutrition and age on growth, scrotal development,

2 metabolite concentrations and on systemic gonadotropin and testosterone

3 concentrations following a GnRH challenge in young dairy bulls

4 C.J. Byrne1,2, S. Fair3, A.M. English1,3, C. Urh4, H. Sauerwein4, M.A. Crowe5, P.

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5 Lonergan2, D.A. Kenny1,6

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6 Animal and Bioscience Research Department, Teagasc, Dunsany, Co. Meath,

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7 Ireland. 2School of Agriculture and Food Science, University College Dublin, Belfield,

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8 Dublin 4, Ireland. 3Laboratory of Animal Reproduction, Department of Biological

9 Sciences, University of Limerick, Limerick, Ireland. 4Institute for Animal Science,

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Physiology and Hygiene Unit, University of Bonn, Bonn, Germany, 5School of
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11 Veterinary Medicine, University College Dublin, Belfield, Dublin 4, Ireland.
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12 Corresponding author: David A. Kenny ([email protected])
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13 Abstract
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14 The onset of puberty in the bull is regulated by the timing of early GnRH pulsatility

15 release from the hypothalamus, which has been demonstrated to be affected by

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16 plane of nutrition during calf-hood. The aim of this study was to determine the effect

17 of plane of nutrition on growth rate, scrotal development, metabolite concentrations

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18 and exogenous gonadotrophin (GnRH) induced release of luteinizing hormone (LH),

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19 follicle stimulating hormone (FSH) and testosterone (TT) in pre-pubertal bulls of two

20 contrasting dairy breeds. Holstein-Friesian and Jersey bull calves were assigned to

21 either a high or low plane of nutrition from 3 to 49 weeks of age. Intensive blood

22 sampling was conducted at 16, 24 and 32 weeks of age, every 15 min from 30 min

23 prior to intravenous administration of exogenous GnRH to 135 min after. Monthly

24 blood samples were also collected and analyzed for insulin like growth factor 1 (IGF-

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25 1), insulin, leptin, adiponectin and metabolite concentration. Insulin and IGF-1 were

26 higher in bulls on a high plane of nutrition (P<0.001) but were not affected by breed

27 (P>0.05). Leptin was not affected by plane of nutrition or breed (P>0.05).

28 Adiponectin tended to be higher in bulls on a high plane of nutrition (P=0.05), but

29 was not affected by breed (P>0.05). Bulls on a high plane of nutrition had a greater

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30 concentration of LH in response to GnRH (P<0.05) but there was no effect of breed

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31 (P>0.05). FSH concentration was not influenced by breed or plane of nutrition but

32 FSH concentrations did decrease with age (P<0.01), while, LH was not affected by

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33 age (P>0.05). Jersey bulls, particularly those on a high plane of nutrition, had higher

34 TT production in the pre-pubertal period (P<0.001). Using 28 cm as a proxy for age

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at puberty, bulls on a high plane of nutrition were predicted to reach puberty earlier
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36 than bulls on a low plane. In conclusion, the data clearly demonstrate that a high
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37 plane of nutrition positively affects several key nutritional and reproductive hormones

38 which are critical to the endocrinological functionality of the hypothalamic-pituitary-

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39 testicular axis in dairy-bred bull calves.

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40 Keywords: Calf-hood nutrition, puberty, insulin, IGF-1, hypothalamus,

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41 endocrinology.

42 1. Introduction
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43 The advent of genomically-assisted selection has facilitated the early identification of

44 elite sires within weeks of birth leading to an increased demand for their semen at a

45 young age. This necessitates that bulls reach puberty as early as possible and

46 produce an adequate volume of high quality semen to meet this demand. It is well

47 established that a number of factors including breed and nutritional status may

48 influence the timing of puberty in farm animals [1]. Management factors such as

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49 nutrition can be used to hasten the onset of puberty [2] and sexual maturation in the

50 bull [3], leading to an earlier availability of semen from these genetically elite sires

51 which are in high demand. The timing of the early transient rise in anterior pituitary

52 luteinizing hormone (LH) pulsatile activity is a critical factor in determining the age at

53 which sexual maturity is reached [4]. This early rise has been reported to occur

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54 between weeks 10 and 18 of age in Angus and Angus x Charolais bull calves [5] and

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55 is thought to induce responsiveness of testicular Leydig cells to LH, leading to an

56 increase in testosterone (TT) production [6]. This increase in TT is necessary for the

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57 differentiation of Sertoli cells and initiation of spermatogenesis [6]. The timing and

58 magnitude of this early LH rise has been shown to be controlled by the metabolic

59 status of the animal [7, 8].

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60 The effect of early life plane of nutrition on sexual development in beef bulls
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61 has been demonstrated in two related studies [7, 8] in which bulls offered a low

62 plane of nutrition from 10 weeks of age experienced a delay in the onset of puberty
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63 compared to those offered either a medium or high plane of nutrition. This delay was
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64 associated with lower TT concentrations during the peri-pubertal period. These

65 studies demonstrate that testicular size in mature animals is positively influenced by

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66 increased systemic FSH, TT and particularly LH in early life. Other authors

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67 concluded that daily gains of greater than 1.2 kg/day during calf-hood should be
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68 targeted to ensure earlier attainment of puberty in bulls [9]. More recently, a higher

69 plane of nutrition led to a greater scrotal circumference (SC) and thus hastened age

70 at puberty in Holstein-Friesian bulls [10]. It has also been suggested that SC can be

71 used as a predictor of sperm output potential in young dairy bulls [11]. The effect of

72 pre-pubertal growth rate relative to estimated mature weight on the age at onset of

73 puberty has been documented for Simmental, Angus and Hereford heifers [12, 13]. A

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74 high plane of nutrition has also been used to achieve a heavier weight, at a younger

75 age in heifers, leading to an associated decrease of approximately four months in

76 age at puberty [14]. Research on age at puberty onset in beef-bred bulls has also

77 shown that early maturing breeds attain puberty at a younger age than late-maturing

78 breeds [4]. While the effect of breed type (early v late maturing) on age at onset of

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79 puberty has been well defined in heifers; there has been less work carried out,

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80 however, in bulls, possibly due to the greater challenge involved in assessing

81 attainment of puberty.

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82 Metabolic hormones such as leptin, adiponectin, insulin and insulin-like

83 growth factor-1 (IGF-1) signal the nutritional status of the animal to the hypothalamus

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via receptors in the arcuate, neuro-peptide Y and pro-opiomelanocortin nuclei [15],
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85 which regulates the secretion of LH and FSH, mediated through the action of
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86 gonadotrophin releasing hormone (GnRH). These hormones are necessary for

87 testicular and sexual development and in turn dictate the age at which puberty is
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88 attained [16-18]. Systemic concentrations of metabolic hormones such as IGF-1

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89 have been shown to vary according to breed [19] and higher IGF-1 was consistent

90 with greater LH pulsatility in Angus heifers, leading to more precocious puberty,

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91 when compared to other breeds. Additionally, IGF-1 concentrations were higher in

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92 Holstein-Friesian bulls offered a high plane of nutrition up to 31 weeks of age [10].

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93 The influence of other metabolic hormones such as leptin on the functional

94 development of the hypothalamic-pituitary-gonadal axis is not as clear [20, 21]. We

95 hypothesized that increasing the plane of nutrition offered to bull calves of different

96 breeds would positively influence metabolic hormones associated with increasing

97 secretion of reproductive hormones. Due to a lower mature bodyweight we also

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98 hypothesized that Jersey bulls would have an earlier response to plane of nutrition

99 than Holstein-Friesians.

100 The aim of this study was to determine the effect of age, breed and plane of

101 nutrition on systemic growth rate, scrotal development, metabolite concentrations, as

102 well as exogenous GnRH-induced release of LH, FSH and TT in pre-pubertal

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103 Holstein-Friesian and Jersey bulls.

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104 2. Materials and Methods

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105 All animal procedures performed in this study were conducted under experimental

106 licence from the Irish Department of Health and Children (licence number

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B100/4516). Protocols were developed in accordance with the Cruelty to Animals Act
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108 (Ireland 1876, as amended by European Communities regulations 2002 and 2005)

109 and the European Community Directive 86/609/EC.

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110 2.1. Animals and treatments

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111 Holstein-Friesian (F) and Jersey (J) bull calves (n=34) with a mean (±S.D.) age of 21

112 (±10.3) days and bodyweight of 47.4 (±13.0) kg and 33.1 (±5.13) kg, respectively,
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113 were sourced from commercial dairy farms and were blocked on breed, age, sire and

114 bodyweight and assigned to either a high or low plane of nutrition. All calves were
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115 group housed indoors at Teagasc, Grange Beef Research Centre on sawdust-
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116 floored pens (balanced for treatment and breed) with a space allowance of 1.6

117 m2/calf. Calves were individually fed milk replacer and concentrate (Table 1) using

118 an electronic feeding system (Förster-Technik, Vario, Engen, Germany). After five

119 days acclimatization, at 26 (±10.3) days of age, high F (n=9) and high J (n=8)

120 received 1200 g (8 L at 150 g/L) and 800 g (6 L at 133.33 g/L) of milk replacer (23%

121 crude protein, 18% lipid; Blossom Easymix; Volac, Co. Cavan, Ireland) daily,

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122 respectively, together with concentrate ad libitum (Lakeland Dairies, Monaghan,

123 Ireland). Low F (n=11) were allocated 500 g (4 L at 125 g/L) of milk replacer plus a

124 maximum of 1 kg of concentrates daily while low J (n=6) were allocated 350 g (3.5 L

125 at 100 g/L) of milk replacer plus a maximum of 1 kg of concentrates daily. Treatment

126 diets were designed using National Research Council (2001) guidelines [22]. Bulls

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127 were weaned when consuming a minimum of 1 kg of concentrate for 3 consecutive

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128 days, at a mean age (±S.D.) of 83 (±10.5) days. Following weaning, high F and high

129 J were offered ad libitum concentrates, while low F received 1.7 kg and low J

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130 received 1.4 kg of concentrate daily. All bulls had daily access to approximately 0.5

131 kg of straw each from pre-weaning to turn-out. This equates to HF calves on the low

132
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plane of nutrition being offered 1.26 times the metabolisable energy requirements
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133 required for maintenance (MEm) requirements and J calves on the low plane of
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134 nutrition being offered 1.17 x MEm requirements. Bulls were turned out to grass at 16

135 weeks of age where high F and high J received grass and concentrate ad libitum
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136 while low F and low J both received grass ad libitum plus 0.5 kg of concentrates per
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137 day. All animals had constant access to fresh water. Bulls were weighed weekly until

138 weaning and were weighed at 14 day intervals thereafter (Fig 1). Scrotal
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139 circumference measurements were taken at 14 day intervals using a scrotal

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140 measuring tape (Neogen®, Lexington, KY, USA.), beginning at 19 weeks of age (Fig
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141 1). Both SC and weight measurements continued until bulls reached 49 weeks of

142 age. A SC of 28 cm was used a proxy for age at puberty [23].

143 The pre-weaning intakes and feeding behavior of these animals has been previously

144 reported [24]. In addition, the health status of these animals from initiation of dietary

145 treatments until 14 days post-weaning has been characterized in detail [25].

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146 2.2. Blood sampling and analysis

147 Monthly bloods samples were collected via jugular venipuncture, beginning at

148 approximately 4 weeks of age (Fig 1). On each occasion, blood was collected into

149 either a 9 mL evacuated tube containing lithium heparin (Greiner Vacuette; Cruinn

150 Diagnostics, Dublin, Ireland) for adiponectin, IGF-1, metabolite and leptin analysis or

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151 into a 6 mL K3 ethylenediaminetetraacetic acid (K3 EDTA; Vacuette, Cruinn

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152 Diagnostics) for insulin analysis. Blood was centrifuged at 1750 g for 15 min; plasma

153 was harvested and stored at -20 ˚C until analysis was performed.

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154 Insulin-like growth factor-1 concentrations were determined using a

155 radioimmunoassay (RIA) following acid–ethanol extraction using the method

156
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previously described by Beltman et al. [26]. The intra- and inter- assay coefficients of
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157 variation (CVs) were determined by replicating a low, normal and high reference
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158 sample, at the beginning, middle and end of each assay. Intra- and inter-assay CVs

159 for IGF-1 were 12.7, 6.9, 5.1% and 7.5, 1.6, 4.1% for low, medium and high,
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160 respectively. The sensitivity of each assay was defined as the lowest concentration
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161 detectable. The sensitivity of the IGF-1 assay was 4 ng/mL. Insulin concentrations

162 were determined using INS-IRMA (immunoradiometricassay) kits (DIAsource

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163 Immunoassays, Louvain-la-Neuve, Belgium), following the manufacturer’s

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164 instructions. Intra- and inter-assay CVs for insulin were 5.5, 4.7, 4.1% and 13.9,
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165 10.8, 11.2% for low, medium and high, respectively. The sensitivity of insulin assays

166 was 1 ng/mL. Leptin concentrations were determined using enzyme immunoassay,

167 previously described by Sauerwein et al. [27]. The sensitivity of the leptin assay was

168 0.6 ng/mL. Intra- and interassay CVs were 5% and 9%, respectively. Adiponectin

169 concentrations were determined using enzyme immunoassay, previously described

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170 by Mielenz et al. [28]. The sensitivity of the adiponectin assay was 0.03 ug/mL. Intra-

171 and interassay CVs were 5% and 10%, respectively.

172 Concentrations of albumin, urea, total protein, β-hydroxybutyrate (BHB),

173 glucose, non-esterified fatty acid (NEFA), triglycerides, and creatinine were

174 determined as described by Lawrence et al. [29]. All metabolite concentrations were

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175 measured on an automatic analyzer (AU 400; Olympus, Tokyo, Japan). The inter

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176 assay CVs for low, medium and high standards were 1.65, 1.77 and 0.72%,

177 respectively. The sensitivities were as follows: glucose: 0.02 mmol/L, urea 0.9:

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178 mmol/L, BHB: 0.1 mmol/L, NEFA: 0.072 mmol/L, triglycerides: 0.004 mmol/L, total

179 protein: 0.8 g/L, albumin: 0.1 g/L, creatinine: 2.3 µmol/L. Globulin concentration was

180
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calculated as the difference between total protein and albumin concentrations [30].
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181 A GnRH intravenous challenge was carried out using all bulls at 16, 24 and 32
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182 weeks of age (Fig 1). At each challenge, a GnRH agonist (Buserelin Receptal®;

183 Intervet Ireland Limited, Dublin, Ireland) was administered (0.05 mg/kg bodyweight,
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184 i.v.), immediately after the third blood sample was taken. Blood samples were
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185 collected at 15 min intervals for LH, FSH and at 30 min intervals for TT. Blood was

186 allowed to clot at room temperature overnight and was then centrifuged at 780 g for
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187 10 min. Serum was harvested and stored at -20˚C until analysis. Sampling at 16
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188 weeks of age was done based on the documented early gonadotropin rise normally
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189 occurring between 12 and 20 weeks of age [5]. Sampling was carried out at 24

190 weeks of age to profile the end of the first transient LH rise [5]. Bulls were sampled at

191 32 weeks as this time-point is associated with an increase in serum TT and a second

192 rise in LH secretion associated with puberty in beef bulls [31].

193 All three hormones were analyzed in duplicate using RIA. Follicle stimulating

194 hormone concentrations were determined using the method described by Crowe et

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195 al. [32]. The sensitivity of the assay was 0.05 ng/mL. Intra- and inter-assay CVs were

196 12.5, 9.4, 9.3% and 12.8, 6.9, 12.8% for low, medium and high, respectively.

197 Luteinizing hormone concentrations were determined using the method described by

198 Cooke et al. [33]. The sensitivity of the assay was 0.2 ng/ml. Intra- and inter-assay

199 CVs were 8.7, 8.9, 8.7% and 8.4, 6.0, 8.4% for low, medium and high, respectively.

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200 Testosterone concentrations were determined using solid-phase RIA kits (Siemens,

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201 Los Angeles, CA, USA); following the manufacturer’s instructions. The sensitivity of

202 the assay was 0.1 ng/mL. Intra- and inter-assay CVs were 5.0, 3.5, 2.4% and 16.2,

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203 5.3, 4.9% for low, medium and high, respectively.

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204 2.3. Statistical analysis
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205 Area under the curve (AUC) was determined for FSH, LH and TT between 0 and 135

206 min relative to GnRH administration using Sigma Plot, version 11 (Systat Software,
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207 San Jose, CA). Basal serum concentration of each hormone (mean of two samples

208 taken prior to GnRH injection) for each animal was included as a covariate in the
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209 statistical model. All data, including weight, SC, monthly blood samples and GnRH
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210 challenges were analyzed using the following procedures of Statistical Analysis
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211 Software (SAS version 9.3, Cary, NC, USA). Data were tested for normality

212 (UNIVARIATE procedure) and, post-weaning bodyweight, metabolic hormone,

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213 metabolite, with the exception of albumin and glucose and reproductive hormone,
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214 with the exception of LH data were transformed to the power of lambda (TRANSREG

215 procedure). Data were then subjected to repeated measures ANOVA (MIXED

216 procedure). Age was included as the repeated term. Differences in individual least-

217 square means were evaluated using the Tukey-Kramer adjustment. Diet, block,

218 breed, age and their interactions were included in the model. The interaction term, if

219 not statistically significant (P > 0.05), was subsequently excluded from the final

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220 model. The covariance matrix, namely simple, compound symmetry or toeplitz was

221 determined for each variable by examining the Bayesian Information Criteria (BIC)

222 (smaller is better). All results are presented as mean ± s.e.m., unless stated

223 otherwise.

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224 3. Results

225 Bodyweight and scrotal circumference

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226 There were no plane of nutrition by breed by age interactions among the main

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227 effects for either pre- or post-weaning bodyweight measurements (P>0.05). There

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228 was a plane of nutrition by age interaction for pre-weaning bodyweight (P<0.001; Fig
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229 2). An effect of plane of nutrition on bodyweight was not evident until animals

230 reached 4 weeks of age, after which bulls on the high plane of nutrition remained
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231 heavier than those on low plane of nutrition. There was also a breed by age

232 interaction for pre-weaning bodyweight (P<0.05); F bulls were heavier than J at the
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233 start of the study (3 weeks of age); this difference did not reach statistical
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234 significance at either 4 or 5 weeks of age but was detectable again at 6 weeks of age

235 after which F remained heavier than J until weaning. There was a plane of nutrition
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236 by age interaction for post-weaning bodyweight (P<0.001; Fig 3). Consistent with the
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237 pre-weaning period, bulls on a high plane of nutrition were heavier than those on a
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238 low plane; the magnitude of this difference increased after weaning and continued to

239 increase until 49 weeks of age (P<0.01). There was a breed by age interaction for

240 post-weaning bodyweight (P<0.001). There were small differences between breeds

241 from 13 to 21 weeks of age; however, from 23 weeks of age, the bodyweight

242 difference between F and J bulls increased until the end of the study at 49 weeks of

243 age.

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244 There was a plane of nutrition by breed by age interaction for SC (P<0.01; Fig

245 4). Holstein-Friesian bulls on a high plane of nutrition had a larger SC than those on

246 a low plane of nutrition at 33, 37, 41 and 43 weeks of age. Scrotal circumference

247 was also larger for F than J bulls on a low plane of nutrition from 31 to 49 weeks of

248 age. Jersey bulls on a high plane of nutrition had a larger SC than either F or J bulls

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249 on a low plane of nutrition from 25 to 49 weeks of age, with the exception of low F

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250 from 45 to 49 weeks of age. Within plane of nutrition, there was no difference

251 between either high F or J bulls (P>0.05). Low F had a larger SC than low J at 47

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252 and 49 weeks of age (P<0.05). Predicted age at puberty, based on a threshold SC of

253 28 cm [34] and proportion of bulls predicted to be pubertal at 49 weeks of age from

254
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each diet group are presented in Table 2. Although not statistically significant, J bulls
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255 on a high plane of nutrition were predicted to reach a threshold SC of 28 cm 3 weeks
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256 younger than F bulls on a high plane of nutrition (P>0.05) and 9 weeks younger than

257 F bulls on a low plane of nutrition (P<0.01). Holstein-Friesian bulls on a high plane of
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258 nutrition were predicted to reach a threshold SC of 28 cm 6.5 weeks younger than F
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259 bulls on a low plane of nutrition (P<0.05). Based on SC measurements, only one J

260 bull from the low plane of nutrition reached a threshold SC of 28 cm at 49 weeks of
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261 age; therefore, this group could not be directly compared to other groups.
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262 Metabolic hormones and metabolites

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263 There was a breed by age interaction for systemic concentrations of IGF-1 (P<0.01;

264 Fig 5); F bulls had higher IGF-1 than J bulls at the start of the study. However, serum

265 IGF-1 concentrations converged at 8 weeks of age and were similar between the two

266 breeds for the remainder of the study. There was also a plane of nutrition by age

267 interaction for IGF-1 (P<0.001); concentrations were higher in bulls fed the high

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268 plane of nutrition at 8 weeks of age, compared to those on the low plane of nutrition.

269 Insulin-like growth factor-1 concentrations then converged until 24 weeks of age.

270 From 24 to 28 weeks of age, bulls on the high plane of nutrition had higher IGF-1

271 concentrations than those on the low plane of nutrition. There was a plane of

272 nutrition by age interaction for insulin concentration (P<0.001; Fig 5). Bulls on a high

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273 plane of nutrition had higher insulin than those on low, at both 32 and 36 weeks of

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274 age. There was no effect of breed on insulin concentration (P>0.05).

275 There was a breed by age interaction for adiponectin concentration (P<0.001;

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276 Fig 6). Holstein-Friesian bulls had a higher concentration of adiponectin at 12 weeks

277 of age in comparison to J bulls. Bulls on a high plane of nutrition tended to have

278
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higher adiponectin concentrations compared to bulls on a low plane of nutrition
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279 (P=0.05). There was no effect of plane of nutrition or breed on systemic
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280 concentrations of leptin (P>0.05; Fig 6). There was an effect of age (P<0.001) on

281 leptin with concentrations being higher in all bulls at 20 weeks than at 16 weeks of
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282 age (P<0.01); no differences were detected at any other age (P>0.05).
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283 There was no plane of nutrition by breed by age interaction or breed effect for

284 albumin concentration (P>0.05; Fig 7). Bulls on a high plane of nutrition tended to
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285 have higher albumin concentrations compared to those on a low plane of nutrition
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286 (P=0.07). There was a quadratic effect of age on albumin concentration (P<0.001)
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287 which decreased between 4 and 12 weeks of age and then, following a large

288 increase at 16 weeks of age, decreased to levels similar to those observed at the

289 beginning of the experimental period. There was no plane of nutrition by breed by

290 age interaction or plane of nutrition effect on creatinine concentration (P>0.05; Fig 7).

291 There was a breed by age interaction for creatinine (P<0.001); J bulls had higher

292 creatinine concentrations at all blood sampling time-points compared to F bulls.

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293 However, the magnitude of this difference between F and J began to decrease from

294 24 weeks of age with smaller differences particularly evident at 24 and 39 weeks of

295 age (P<0.05).

296 There was no plane of nutrition by breed by age interaction for globulin

297 concentration (P>0.05, Fig 7). There was a plane of nutrition by age interaction

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298 (P<0.05); bulls on the high plane of nutrition tended to have lower globulin

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299 concentrations compared to those on the low plane of nutrition at 8 (P=0.06) and 12

300 weeks (P=0.09) of age. Jersey bulls had a higher globulin concentration than F bulls

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301 (P<0.001). There was no plane of nutrition by breed by age interaction or effect of

302 breed on glucose concentration (P>0.05; Fig 7). There was a plane of nutrition by

303
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age interaction (P<0.01), manifested as bulls on a high plane of nutrition having a
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304 higher glucose concentration compared to bulls on a low plane at 8 weeks of age.
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305 There was no plane of nutrition by breed by age interaction for total protein

306 concentration (P>0.05). There was a plane of nutrition by age interaction for total
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307 protein (P<001; Fig 7). Bulls on a high plane of nutrition had a lower total protein
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308 concentration than those on a low plane of nutrition at 8 weeks of age. Differences

309 between planes of nutrition were not detected at any other age. Total protein
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310 concentrations were affected by breed (P<0.01) with J having a higher concentration
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311 compared to F bulls. There was no plane of nutrition by breed by age interaction for
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312 triglyceride concentration (P>0.05; Fig 7). There was a tendency for a plane of

313 nutrition by breed interaction of triglyceride concentration (P=0.08). Jersey bulls on a

314 low plane of nutrition had lower triglyceride concentrations than F bulls on a low

315 plane of nutrition (P<0.05) and F bulls on a high plane of nutrition (P<0.05). Jersey

316 bulls on a low plane of nutrition also tended to have lower concentrations than J bull

317 on a high plane of nutrition (P=0.09). There was an effect of age on triglyceride

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318 concentration (P<0.001) as a result of a significant decrease in triglycerides between

319 12 and 16 weeks of age and also between 20 and 24 weeks of age.

320 There were no differences between the main effects of either plane of nutrition

321 or breed on NEFA concentration (P>0.05; Fig 8). There was an effect of age on

322 NEFA concentrations (P<0.001) which increased from 8 to 12 weeks, decreased

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323 from 12 to 20 weeks, increased again from 20 to 24 weeks and finally decreased

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324 from 24 to 28 weeks and thereafter remained unchanged to the end of the trial

325 period. There was a strong tendency for a plane of nutrition by breed by age

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326 interaction for BHB concentration (P=0.06; Fig 8). At 12 weeks of age, F bulls on a

327 high plane of nutrition had a lower concentration of BHB than both F and J bulls on a

328
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low plane of nutrition. Concentrations of BHB increased from 4 to 20 weeks of age
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329 (P<0.01), after which BHB concentrations plateaued and were not different. No plane
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330 of nutrition by breed by age interaction was detected for urea concentration (P>0.05;

331 Fig 8). There was a plane of nutrition by age interaction (P<0.001) manifested as
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332 bulls on a high plane of nutrition having a higher urea concentration at 24 weeks of
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333 age compared to those on a low plane of nutrition. There was an effect of breed on

334 urea concentration (P<0.05) as F bulls had higher concentrations than J bulls.
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335 Intravenous GnRH challenge

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336 For statistical analysis, concentrations of each hormone were represented by AUC
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337 data; as such, results below are total AUC following GnRH administration. There

338 were no plane of nutrition by breed by age interactions for concentrations of either

339 FSH or LH (P>0.05; Fig 9, Fig 10). FSH concentrations were not affected by plane of

340 nutrition or breed but were affected by age (P<0.01). Concentrations of FSH were

341 highest at 16 weeks of age and decreased subsequently. Irrespective of breed, bulls

342 on the high plane of nutrition had higher concentrations of LH compared with those

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343 on the low plane of nutrition (P<0.05). There was no effect of age on LH

344 concentrations (P>0.05). There was a three-way interaction of plane of nutrition by

345 breed by age for serum concentrations of TT (P<0.001; Fig 11). All bulls had low

346 concentrations of TT at 16 weeks of age compared with either 24 or 32 weeks of

347 age. Concentrations increased between 9 and 10-fold at 24 and 32 weeks of age,

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348 after a GnRH challenge, with J having consistently higher concentrations of TT than

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349 F (P<0.01), particularly those on a high plane of nutrition at 24 weeks of age.

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350 4. Discussion

351 Increasing the plane of nutrition offered in the early calf-hood period has a positive

352
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effect on the metabolic status of bull calves. This, in turn, leads to an increase in
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353 concentrations of metabolic hormones such as IGF-1, which in turn stimulate GnRH

354 secretion [35] and thus evoke a cascade of trans-tissue endocrinologically-mediated

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355 events, necessary for puberty to occur in the bull. The results of this study clearly
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356 demonstrate that both plane of nutrition and breed affect TT production in early calf-
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357 hood with higher systemic concentrations evident in calves offered a high compared

358 to low plane of nutrition and J calves having higher concentrations than F calves.
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359 Heavier bodyweight and a larger SC were observed in bulls fed a high

360 compared with a low plane of nutrition, consistent with previous reports [7-9].
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361 However, between weeks 41 and 49 the growth of J bulls on a low plane of nutrition
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362 plateaued. Grass was offered to all animals ad libitum and in addition the bulls on a

363 low plane of nutrition were offered 0.5 kg of concentrate. The diet offered should

364 have supported a growth rate in excess of 0.5 kg/day according to the national

365 research council guidelines. Grazing groups were balanced for breed so the same

366 diet was available to both breeds on a low plane of nutrition. Therefore, it is unclear

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367 why the growth rate of Jersey bulls was static while the Holstein-Friesians

368 maintained an upward growth trajectory. The lack of a breed difference for SC was

369 surprising considering the difference in bodyweight between the two breeds.

370 However, when SC was expressed as a percentage of bodyweight, in order to

371 account for differences in mature bodyweight, SC accounted for a greater

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372 percentage of bodyweight in J compared to F bulls (Table 2). It is also interesting

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373 that J bulls had a larger SC on a high plane of nutrition compared to that of F bulls

374 while J bulls on a low plane of nutrition responded in the opposite manner, with a

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375 smaller SC than their F contemporaries. This testicular growth retardation had a

376 detrimental effect on the proportion of bulls which could be categorized as pubertal

377
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at 49 weeks of age, using the definition of achieving a SC of 28 cm [23].
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378 In the present study, systemic concentrations of IGF-1 were not affected by
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379 plane of nutrition until 24 weeks of age, after which bulls on a high plane of nutrition

380 had higher IGF-1 than bulls on low. Similar results have been shown in beef-bred
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381 bull calves fed either a control or restricted (75% of ad libitum control) diet, whereby,
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382 those on a control diet had higher IGF-1 concentrations at 26 weeks of age [8]. In

383 contrast to these findings, nutritional effects on IGF-1 have been reported at 11
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384 weeks of age in F bulls, where bulls on a high plane of nutrition had higher IGF-1
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385 than those on low during 10 hours of intensive blood sampling [10]. The results of
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386 the two aforementioned studies have shown temporal associations between

387 systemic concentrations of IGF-1 and LH. While no temporal relationships were

388 observed between IGF-1 and LH in the current study, the overall effect of a higher

389 plane of nutrition on LH is likely to have been mediated, to some extent, by increases

390 in hepatic IGF-1 production. Indeed, receptors for IGF-1 have been detected in the

391 pre-optic area of the hypothalamus, stimulating GnRH secretion [36] and also in

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392 anterior pituitary cell lines of rats; an increase in LH responsiveness to GnRH has

393 been detected when pituitary cell lines are incubated in IGF-1 [37], highlighting the

394 important association between IGF-1 and gonadotrophin secretion.

395 The effects of insulin on gonadotrophin secretion are inconsistent; in ewes

396 intra-cerebral-ventricular infusion of exogenous insulin has resulted in both an

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397 increase [38] and decrease [39] in LH pulsatility. In heifers, a decrease in insulin,

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398 induced by fasting, has been associated with a reduction in LH pulsatility [40]. In the

399 current study, a high plane of nutrition led to higher insulin concentration, though

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400 concentrations were within the normal range low plane of nutrition did not display

401 extremely suppressed levels of insulin. In this instance, insulin would be available to

402
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regulate blood glucose levels allowing maintenance of glucose homeostasis [41].
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403 Animals in greater energy balance should have higher adiposity and therefore higher
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404 concentrations of leptin [42]. Nonetheless, leptin was unaffected by either plane of

405 nutrition or breed in the current study and this lack of an effect of nutrition has
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406 previously been reported in young bulls that were offered different planes of nutrition
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407 [8, 10] perhaps due to the low levels of subcutaneous fat in these growing animals.

408 Similar to insulin, leptin only plays an important, short-term role, in terms of GnRH
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409 secretion, during periods of fasting. In cows, short-term fasting appears to sensitize
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410 the hypothalamic-pituitary axis to exogenous leptin, which results in heightened

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411 secretion of both LH and insulin following treatment with exogenous leptin [43]. Since

412 leptin receptors are not found on GnRH neurons, it is likely that kisspeptin acts as an

413 intermediary between leptin and GnRH neurons; signaling metabolic status [44]. The

414 effects of nutrition and breed on adiponectin concentrations in bulls have not been

415 well characterized. In the current study, F bulls had higher adiponectin

416 concentrations than J bulls at 12 weeks of age. In addition, bulls on a high plane of

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417 nutrition tended to have higher concentrations than those on low; both findings are in

418 contrast to the previous reports. Similar to leptin, adiponectin is secreted by brown

419 and white adipose tissue, but at higher concentrations than leptin [45] and in contrast

420 to leptin, adiponectin is inversely related to body fat mass, in cattle [46]. Therefore, it

421 would be expected that young dairy bulls with a higher potential for fat deposition

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422 (high vs low plane of nutrition) would have lower adiponectin concentrations.

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423 Increased adiponectin concentrations are associated with disturbances in GnRH

424 pulsatility, in men [47] via down-regulation of KISS1 gene; in in vitro hypothalamus

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425 cell lines [48], indicating that adiponectin has an opposing effect to that of leptin on

426 GnRH pulsatility.

427
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Many of the metabolites analyzed in the current study are known to have an
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428 indirect role in reproduction, primarily as metabolic signals to the hypothalamus.
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429 Circulating leptin concentrations have been shown to be related to body adiposity

430 [42] and triglyceride concentration [49] in cattle. This does not appear to be the case
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431 in the current study. Triglycerides are mobilized in response to negative energy
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432 balance-induced lipolysis [50]. If triglycerides are stored, there is a greater potential

433 for adipose tissue to produce leptin; however, if triglycerides are mobilized, adipose
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434 tissue reserves may be reduced leading to a lower leptin concentration and thus
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435 reduce the stimulatory effect of GnRH secretion. The tendency towards lower
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436 triglycerides in the F bulls on a high plane of nutrition indicates a positive metabolic

437 status, potentially facilitating greater leptin synthesis and secretion. The higher

438 concentration of glucose in bulls on a high plane of nutrition is consistent with their

439 greater feed intake and overall better metabolic status. The effects of glucose on the

440 hypothalamus have been reviewed by Burdakov et al. [51] who concluded that

441 glucose stimulated the pro-opiomelanocortin nuclei and inhibited the appetite-

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442 promoting neuro-peptide Y nuclei, processes which have been shown in sheep to be

443 important for the secretion of GnRH. [52]. The higher urea concentration in bulls on

444 the high plane of nutrition is likely as a result of higher protein intake. It has been

445 shown that protein supplementation which leads to an increase in blood urea

446 concentrations, has no apparent effect on steroid hormone concentrations or

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447 pregnancy rates in cows [53] or heifers [54]. In cows, an increase in blood urea

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448 concentrations has been shown to coincide with an increase in LH secretion [55],

449 while others have reported that LH secretion is unaffected by changes in urea [56,

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450 57]. It is more plausible that the increased LH secretion in calves on a high plane of

451 nutrition, observed here, was more related to the increased systemic IGF-1

452 concentrations recorded.

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453 There was no effect of breed or plane of nutrition on systemic concentrations
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454 of FSH, following GnRH administration, but FSH did decrease with age. At birth,

455 FSH concentrations are typically high and begin to decline at 14 weeks of age and
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456 are at their lowest concentrations by around 25 weeks of age in the bull [35]. This
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457 period coincides with a period of rapid proliferation of immature Sertoli cells [34]

458 suggesting that FSH concentration in the young bull calf plays a role in determining
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459 future semen production potential. This proliferation period may even be limited to
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460 the initial post-natal period; as administration of exogenous FSH to bull calves
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461 between 4 and 8 weeks of age increased the number of Sertoli cells per

462 seminiferous tubule [58], observed following castration at 56 weeks of age. This

463 would suggest that the early effects of nutrition on GnRH pulsatility may influence the

464 proliferation of Sertoli cells in the young bull calf. It has been recently reported [10]

465 that once FSH concentrations have declined they remain low, in agreement with the

466 findings of this study. However, others [31] reported a second rise in FSH, beginning

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467 at 30 weeks of age and continuing to 45 weeks of age for Hereford and Hereford x

468 Charolais bulls offered a common plane of nutrition. There was no evidence of such

469 a rise at the 32-week sampling point of this study or it may have occurred after the

470 final sampling point of this study. As FSH appears to be unaffected by breed or level

471 of nutrition post-natally, it may be that concentrations reflect pre-natal in utero

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472 environment. For example, it has been shown, in cattle, that systemic FSH

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473 concentrations are increased when a low diet is offered, to the dam, pre-natally [59].

474 However, the authors acknowledged that energy supplied, to dams, by the high diet

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475 may have been insufficient to cause an appreciable increase in FSH, in their

476 offspring [59].

477
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Age had no effect on systemic concentrations of LH. This result was
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478 unexpected, as other studies have shown that LH is highest between 12 and 20
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479 weeks, after which concentrations begin to decline [31, 34]. This rise could have

480 occurred either before or after the 16 week sampling point employed in this study.
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481 However, as 16 weeks is in the center of the documented LH rise window it should
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482 have been the optimal time to detect a rise in LH. The higher secretion of LH in bulls

483 offered a high plane of nutrition indicates that they had a greater store of LH within
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484 the anterior pituitary during the period of the brain’s highest plasticity (the infantile
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485 period). This represents a positive effect of offering a higher plane of nutrition when
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486 trying to increase LH secretion, given that early increases in LH can hasten puberty

487 and sexual maturity [8]. The relationship between plane of nutrition and IGF-1 in F

488 bulls [10] and Angus and Angus x Charolais bulls [60] has been documented. Brito et

489 al. (2007) found a moderate positive correlation between IGF-1 and leptin in beef-

490 bred bulls. As reproductive hormones are regulated by the metabolic status of the

491 animal, the higher energy intake of animals, on ad libitum intake is likely to lead to a

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492 stimulatory effect on the hypothalamus, leading to increased GnRH secretion and

493 thus a higher pulsatile release of LH.

494 Serum TT concentrations were highest in bulls on the high plane of nutrition.

495 Jersey bulls on this plane of nutrition produced more TT than F at 24 and 32 weeks

496 of age and TT concentrations increased significantly with age for both breeds and

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497 nutritional groups. The level of TT detected at 24 weeks in the present study for J

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498 bulls is in agreement with other studies using dairy bulls [61]. Other studies also

499 reported highest TT concentrations at 24 weeks when Angus or Angus x Charolais

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500 bull calves were either offered either a high or a moderate plane of nutrition [62] and

501 also when feeding maintenance diets to F bulls [6]. The overall increase in TT

502
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concentrations observed in these bulls coincides with a decrease in LH and FSH
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503 secretion at the same time-points. This observation is supported by the findings of
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504 others [5], where androgens were shown to have a negative feedback on

505 gonadotrophin release in bulls. The treatment response of higher TT secretion at 24

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506 and 32 weeks in these animals supports the argument for feeding bulls a higher
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507 plane of nutrition in the pre-pubertal period as TT plays a critical role in

508 spermatogenesis [63]. While the importance of high concentrations of TT to

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509 spermatogenesis is not fully understood, low concentrations of TT are associated

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510 with a dramatic reduction in sperm production post puberty [64]. It is possible that the
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511 greater TT in J bulls is associated with SC; however; there was no effect of breed on

512 SC. Furthermore, there is no evidence in the literature to suggest that J bulls have a

513 greater population of Leydig cells than F bulls. There is evidence, however, that J

514 bulls will reach puberty at a younger age than F bulls when both breeds are fed ad

515 libitum diets [65]; therefore this increase in TT may be associated with earlier sexual

516 development.

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517 While measurement of age at onset of puberty, using traditional approaches

518 [66] was beyond the scope of the current study the higher production of LH and TT

519 observed, along with larger scrotal circumference suggests that bulls on a higher

520 plane of nutrition would reach puberty and thus sexual maturation at a younger age,

521 compared to their contemporaries offered a lower plane of nutrition based on the

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522 findings of [4, 10, 62]. For example, based on reaching a threshold SC of 28 cm, we

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523 estimate that bulls on a high plane of nutrition would be 7.5 weeks younger at

524 puberty than those on the low plane of nutrition. This difference in age at puberty

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525 using a SC of 28 cm as a proxy has been reported in F bulls when offered a high or

526 low plane of nutrition for 31 weeks followed by a common, moderate plane of

527
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nutrition [10]. Ours is the first published study comparing F and J bulls of different
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528 planes of nutrition. Differences between breeds in estimated age at puberty were not
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529 found in the current study, mostly likely due to small sample size when the low J

530 bulls were excluded. There was a numerical difference for age at puberty between F
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531 and J bulls (6 weeks) and this trend of J bulls being younger at puberty, than F has
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532 been reported previously and has been associated with the lower proportion of

533 mature bodyweight that J bulls have to reach [65]. Notwithstanding this, within the
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534 confines of the current study, when maintained on a lower plane of nutrition F were
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535 estimated to be pubertal before J bulls.

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536 In conclusion, this study has demonstrated that young dairy bulls on a high

537 plane of nutrition have a better metabolic status as evidenced by higher systemic

538 concentrations of IGF-1 and insulin and as a result have a greater potential for

539 synthesis and secretion of LH than their contemporaries maintained on a lower plane

540 of nutrition. This was consistent with higher TT production in the pre-pubertal period

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541 and coincided with increased testicular development and most likely earlier onset of

542 puberty.

543 Acknowledgements

544 This research was made possible by funding from the Department of Agriculture,

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545 Food and Marine Research Stimulus Fund (Project 11/S/116). The authors are

546 grateful for the skilled technical assistance of Mr. E. Mulligan and Dr. E. Matthews.

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547 References

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705 [55] Jordan ER, Swanson LV. Effect of Crude Protein on Reproductive Efficiency,
706 Serum Total Protein, and Albumin in the High-Producing Dairy Cow. J Dairy Sci.
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707 1979;62:58-63.
708 [56] Blauwiekel R, Kincaid RL, Reeves JJ. Effect of High Crude Protein on Pituitary
and Ovarian Function in Holstein Cows1. J Dairy Sci. 1986;69:439-46.
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709
710 [57] Elrod CC, Butler WR. Reduction of fertility and alteration of uterine pH in heifers
711 fed excess ruminally degradable protein. J Anim Sci. 1993;71:694-701.
712 [58] Mann DR, Akinbami MA, Wallen K, Gould KG, Groome NP, Swanston IA, et al.
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713 Inhibin-B in the male Rhesus monkey: impact of neonatal gonadotropin-releasing

714 hormone antagonist treatment and sexual development. J Clin Endocrinol Metab.
715 1997;82:1928-33.
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716 [59] Sullivan TM, Micke GC, Greer RM, Perry VE. Dietary manipulation of Bos
717 indicusxheifers during gestation affects the prepubertal reproductive development of
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718 their bull calves. Anim Reprod Sci. 2010;118:131-9.

719 [60] Brito LFC, Barth AD, Rawlings NC, Wilde RE, Crews DJ, Mir PS, et al.
720 Circulating metabolic hormones during the peripubertal period and their association
721 with testicular development in bulls. Reprod Domest Anim. 2007;42:502-8.
722 [61] Curtis SK, Amann RP, Walker OA. Testicular development and establishment of
723 spermatogenesis. J Anim Sci. 1981;53:1645- 54.
724 [62] Brito LF, Barth AD, Rawlings NC, Wilde RE, Crews DH, Jr., Mir PS, et al. Effect
725 of improved nutrition during calfhood on serum metabolic hormones, gonadotropins,
726 and testosterone concentrations, and on testicular development in bulls. Domest
727 Anim Endocrinol. 2007;33:460-9.
728 [63] Smith LB, Walker WH. The regulation of spermatogenesis by androgens. Semin
729 Cell Dev Biol 2014;30:2-13.

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730 [64] Zirkin BR, Santulli R, Awoniyl CA, Ewing LL. Maintenance of advanced
731 spermatogenic cells in the adult rat testis: quantitative relationship to testosterone
732 concentration in the testis. Endocrinology. 1989;124:3043-9.
733 [65] Stewart TS, Long CR, Cartwright TC. Characterization of cattle of a five-breed
734 diallel. III. Puberty in bulls and heifers. J Anim Sci. 1980;50:808-20.
735 [66] Wolf FR, Almquist JO, EB. H. Prepuberal behavior and puberal characteristics of
736 beef bulls on high nutrient allowance. J Anim Sci. 1965 24:761-5.
737

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753 Figure titles

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754 Fig 1. Schedule of procedures carried out for the experiment. IGF-1: insulin-like

755 growth factor 1; GnRH: gonadotrophin releasing hormone.

756 Fig 2. Effect of plane of nutrition and breed on pre-weaning bodyweight of pre-

757 pubertal dairy bulls measured weekly; from the start of the trial at 3 weeks to

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758 weaning at 11 weeks of age. T= effect of plane of nutrition; B= effect of breed; A=

759 effect of age; T*B*A= interaction effects; Error bars = ±S.E.M. NS = non-significant

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760 Fig 3. Effect of plane of nutrition and breed on post-weaning bodyweight of young

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761 dairy bulls measured bi-weekly from post-weaning to 49 weeks of age. T= effect of

762 plane of nutrition; B= effect of breed; A= effect of age; T*B*A= interaction effects;

763 Error bars = ±S.E.M. NS = non-significant

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764 Fig 4. Effect of plane of nutrition and breed on scrotal circumference of young dairy
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765 bulls measured bi-weekly from 19 to 49 weeks of age. T= effect of plane of nutrition;

766 B= effect of breed; A: effect of age; T*B*A= interaction effects. Error bars = ±S.E.M.
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767 Fig 5. Mean insulin-like growth factor-1 (IGF-1; upper pane) and insulin (lower pane)
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768 concentrations in Holstein-Friesian and Jersey bulls; offered a high or low plane of
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769 nutrition. T= effect of plane of nutrition; B= effect of breed; A: effect of age; T*B*A=

770 interaction effects. Error bars = ±S.E.M. NS = non-significant.

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771 Fig 6. Mean leptin (upper pane) and adiponectin (lower pane) concentrations in
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772 Holstein-Friesian and Jersey bulls; offered a high or low plane of nutrition. T= effect

773 of plane of nutrition; B= effect of breed; A: effect of age; T*B*A= interaction effects.

774 Error bars = ±S.E.M. NS = non-significant

775 Fig 7. Mean plasma metabolite concentrations in Holstein-Friesian and Jersey bulls;

776 offered a high or low plane of nutrition. T= effect of plane of nutrition, B= effect of

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777 breed, A= effect of age, T*B*A= interaction effects. Error bars = ±S.E.M. NS = non-

778 significant

779 Fig 8. Mean plasma non-esterified fatty acids (upper panel), beta-hydroxybutyrate

780 (middle panel) and urea concentrations (lower panel) in Holstein-Friesian and Jersey

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781 bulls; offered a high or low plane of nutrition. T= effect of plane of nutrition, B= effect

782 of breed, A= effect of age, T*B*A= interaction effects. Error bars = ±S.E.M. NS =

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783 non-significant

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784 Fig 9. Mean serum concentration of follicle stimulating hormone (FSH), before and

785 after a GnRH challenge (marked by the arrow), in Holstein-Friesian and Jersey bull

786

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787 offered a high or low plane of nutrition. T= effect of plane of nutrition, B= effect of

788 breed, A= effect of age, T*B*A= interaction effects. Error bars = ±S.E.M. NS = non-
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789 significant
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790 Fig 10. Mean serum concentration of luteinizing hormone (LH) before and after a
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791 GnRH challenge (marked by an arrow), in Holstein-Friesian and Jersey bulls, at 16

792 (upper panel), 24 (middle panel), and 32 (lower panel) weeks of age; offered a high
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793 or low plane of nutrition. T= effect of plane of nutrition; B= effect of breed; A= effect
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794 of age; T*B*A= interaction effects. Error bars = ±S.E.M. NS = non-significant

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795 Fig 11. Mean serum concentrations of testosterone (TT) before and after a GnRH

796 challenge (marked by an arrow), in Holstein-Friesian and Jersey bull calves, at 16

797 (top), 24 (middle), and 32 (bottom) weeks of age; offered a high or low plane of

798 nutrition. T= effect of plane of nutrition, B: effect of breed, A= effect of age, T*B*A=

799 interaction effects. Error bars = ±S.E.M. NS = non-significant

800

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801

802

Table 1. Ingredient and chemical composition of concentrate offered to calves.

Ingredient (%) Concentrate
Barley 26.5
Soya Bean (dehulled) 25
Maize 15
Beet pulp 12.5

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Soya hulls 12.5
Molasses 5
Minerals and Vitamins 2.5

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Vegetable oil 1
DM 91.2
Energy (MJ ME/kg DM)1 11.06
Crude Protein (g/kg DM)2 188

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Crude Fiber (g/kg DM) 80
Ash (g/kg DM) 71
Crude oil (g/kg DM) 33
1

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megajoules of metabolisable energy per kilogram of dry matter
2
grams per kilogram of dry matter
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815

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816

Table 2. Effect of plane of nutrition and breed on estimated age at puberty in dairy bulls
High F Low J Low F Diet Breed
High J
Scrotal
circumference
13 ± 0.15ac 10 ± 0.10b 14 ± 0.20a 12 ± 0.10c *** ***
(% of
bodyweight)

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8/8 9/9 1/6 8/11
Pubertal at 49 - -
(100%) (72%)

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weeks of age (100%) (17%)

Age at
puberty 34 ± 1.5a 37 ± 2.5a - 43 ± 1.1b * ns

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(weeks)1
a,b,c
= Different superscripts differ significantly within row. *=P<0.05; ***=P<0.001.

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1
Based on attainment of scrotal circumference of 28 cm, measured bi-weekly from 19 weeks
of age (Lunstra et al., 1978).
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Mean ± standard error of mean.

J= Jersey. F=Holstein-Friesian.
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820
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Highlights

• An increased plane of nutrition led to increased growth rate in both Holstein-Friesian

and Jersey and thus a larger scrotal circumference.
• Metabolic hormones; insulin-like growth factor-1 and insulin were increased on a higher
plane of nutrition.
• A gonadotrophin releasing hormone challenge highlighted that bulls on a high plane of
nutrition had greater potential to store and secrete luteinizing hormone compared to
bulls on a low plane of nutrition.

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• There was higher secretion of testosterone in bulls on a high plane of nutrition; this was
particularly evident in Jersey bulls.

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