Association between pre-breeding metabolic profiles and reproductive performance in heifers and lactating dairy cows

Accepted Manuscript Association between pre-breeding metabolic profiles and reproductive performance in heifers and lactating dairy cows Yasmin Schuermann, Gerald Eastman Welsford, Evan Nitschmann, Linda Wykes, Raj Duggavathi PII:

S0093-691X(18)30915-4

DOI:

https://doi.org/10.1016/j.theriogenology.2019.03.018

Reference:

THE 14928

To appear in:

Theriogenology

Received Date: 11 October 2018 Revised Date:

9 March 2019

Accepted Date: 20 March 2019

Please cite this article as: Schuermann Y, Welsford GE, Nitschmann E, Wykes L, Duggavathi R, Association between pre-breeding metabolic profiles and reproductive performance in heifers and lactating dairy cows, Theriogenology (2019), doi: https://doi.org/10.1016/j.theriogenology.2019.03.018. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT

1

Association between pre-breeding metabolic profiles and reproductive performance in heifers and

2

lactating dairy cows

3

RI PT

4

Yasmin Schuermanna, Gerald Eastman Welsforda, Evan Nitschmannb, Linda Wykesb, and Raj

6

Duggavathia,*

SC

5

7 8

1

9

2School

M AN U

Department of Animal Science, McGill University, Sainte-Anne-de-Bellevue, QC H9X 3V9, Canada

of Human Nutrition, McGill University, Sainte-Anne-de-Bellevue, QC H9X 3V9, Canada

10 11

*

12

Raj Duggavathi,

13

[email protected],

14

McGill University, Animal Science, 21111, Lakeshore road, Ste-Anne-de-Bellevue, QC, CAN H9X3V9

15

Tel: 514-398-7803

16 17

AC C

EP

TE D

Corresponding author:

18

Key words: heifers; primiparous cows; multiparous cows; plasma; metabolic parameters; oxidative

19

stress;

20 21 22 23

ACCEPTED MANUSCRIPT Schuermann et al 24

ABSTRACT Lactating cows and nulliparous heifers are in distinctive and unique physiological conditions

26

when they are approaching the planned time of breeding, at approximately 60 days in milk and 13-15

27

months of age, respectively. This study aimed to profile the metabolic milieu in heifers (N=14) and

28

lactating cows (N=15) in the weeks leading up to planned time of breeding. All cows were followed for a

29

period of 15 weeks, from 3 weeks pre-calving to 12 weeks post-calving, while heifers were monitored for

30

a period of 4 weeks leading up to the tentative week of breeding (pre-breeding period). For data analysis,

31

we further divided cows into primiparous (N=8) and multiparous (N=7) cows owing to the significant

32

difference in their milk yield. Assessment of reproductive performance showed that primiparous and

33

multiparous cows tended to have lower pregnancy rates compared to heifers (P<0.1). Plasma

34

concentrations of β-hydroxybutyric acid were about 2-fold higher in multiparous cows than those of

35

heifers in the week leading up to planned time of breeding (P<0.05). Total bile acid levels during the pre-

36

breeding period were higher in all lactating cows compared to heifers (P<0.05) and glucose levels were

37

lower in lactating cows (P<0.05). Triglyceride concentrations were lowest in multiparous cows compared

38

to both primiparous cows and nulliparous heifers (P<0.05). In addition, lactating cows had higher

39

concentrations of total-cholesterol and the high-density lipoprotein and low-density lipoprotein compared

40

to heifers (P<0.05). Conversely, concentrations of very low-density lipoprotein were lower in multiparous

41

cows than primiparous cows and nulliparous heifers (P<0.05). There were no differences in plasma

42

glutathione levels, as measured by liquid chromatography-tandem mass spectrometry, between the

43

groups, but the ferric reducing ability of plasma was higher in lactating cows compared to heifers

44

(P<0.05). These data establish the differences in the profile of metabolic and oxidative markers during the

45

period approaching planned time of breeding in lactating cows compared to nulliparous heifers. As

46

certain metabolites in the plasma have been shown to be represented in the ovarian follicular

47

microenvironment, the unique profiles may influence reproductive performance in dairy cattle in different

48

physiological stages.

AC C

EP

TE D

M AN U

SC

RI PT

25

49 2

ACCEPTED MANUSCRIPT Schuermann et al 50

1. Introduction Achieving economical sustainability on dairy herds is largely dependent on cow longevity and

52

reproductive success [1]. On North American dairy herds, the average age of milking cows has been

53

reported as 4 years and 3 months, which translates to slightly over 2 lactations [2]. Yet, these cows have

54

the potential to live for 9.1 years, which translates to slightly less than 6 lactations [3]. Most importantly,

55

these cows only begin contributing to the financial success of the herd by the third lactation [4]. Reduced

56

longevity is a consequence of involuntary culling most typically related to poor reproductive

57

performance, which represents 15.99% of all involuntary culling [5].

SC

RI PT

51

The transition period, defined as three weeks pre-calving to three weeks post-calving, comprises a

59

time of enhanced susceptibility to diseases including milk fever, metritis, displaced abomasum, and

60

ketosis [6, 7]. Moreover, 75% of metabolic related diseases occur within the first month after calving [8].

61

Cumulative effects of disorders in the transition period can extend for more than 4 months, which can

62

severely compromise reproductive performance [9]. Excessive lipid mobilization to meet the energy

63

demands of lactation leads to metabolic stress characterized by well-defined increased circulating levels

64

of non-esterified fatty acids (NEFAs) and β-hydroxybutyric acid (BHBA) and decreased levels of

65

circulating glucose [6, 10]. Oxidative stress evaluated through the presence of reactive oxidative species

66

(ROS) and antioxidant systems provides an insight to a cow’s health status, where high levels of ROS and

67

low levels of antioxidants have been reported in cows experiencing negative energy balance [11]. Both

68

elevated BHBA and NEFA during the transition period have shown to decrease reproductive performance

69

[12, 13], as described by anovulation [14], decreased conception [15] increased number of days open,

70

and increased number of services per pregnancy [16].

TE D

EP

AC C

71

M AN U

58

Fertility is among the most important factors evaluated on a dairy operation. Moreover, fertility of

72

dairy heifers is higher than that of lactating dairy cows, which is attributed to the fact that nulliparous

73

heifers do not experience the stress of the transition period [17, 18]. One study in particular, showed that

74

the first and second insemination pregnancy rates were 84.3% and 19.5% for heifers and 3rd/4th parity

75

cows, respectively [19]. Following calving, it is common practice amongst North American dairy herds to

3

ACCEPTED MANUSCRIPT Schuermann et al allow for a voluntary waiting period (VWP) of approximately 60 days (8.5 weeks) from calving to

77

breeding [20, 21]. During this time, the animal is expected to recover from calving, with respect to uterine

78

involution, resumption of ovarian activity, and estrous behavior [20]. Nonetheless, the process of

79

activation of an oocyte from the ovarian reserve until it becomes of ovulatory capacity requires

80

approximately 80 to 100 days. This time course suggests that the ovulatory follicle ready at breeding (60

81

DIM) developed during the transition period. The metabolic changes during that time are expected to

82

affect the follicular microenvironment as put forth by the Britt Hypothesis [22-24].

SC

RI PT

76

Although successful pregnancy is multifactorial, the female must produce a developmentally

84

competent oocyte. Both granulosa cells and the follicular fluid in the ovary are part of the

85

microenvironment responsible for the production of a fertilizable oocyte [22, 25]. In cattle, it has been

86

previously shown that the presence of metabolites in circulating blood is reflected in the follicular fluid

87

[26], where for example cholesterol levels are approximately 55% lower in follicular fluid compared to

88

blood, while the level of BHBA is nearly identical between blood and the follicular fluid [27]. On another

89

note, elevated levels of circulating BHBA have shown to be associated with decreased fertility through

90

changes in the steroidogenic profile [28] and decreased success of conception [15]. Overall, blood

91

analyses provide a mirror into the follicular microenvironment, which in turn shed light on the changes in

92

metabolic indicators and oxidative stress markers that have the potential to impact follicle development.

EP

TE D

M AN U

83

Characterizing plasma metabolic parameters and oxidative stress markers of dairy cattle in

94

different physiological stages (nulliparous heifers, primiparous cows, and multiparous cows) in the month

95

leading up to planned time of breeding can open a window into the follicular microenvironment in which

96

the oocyte is developing. Therefore, the aim of this study was to follow lactating dairy cows through the

97

transition period to the subsequent breeding period and breeding aged heifers for a one month sampling

98

period and to superimpose the pre-breeding period from all groups of cattle.

AC C

93

99 100 101

2. Materials and methods

4

ACCEPTED MANUSCRIPT Schuermann et al 102

2.1 Ethics statement All animal procedures were approved by the Animal Care Committee of the Faculty of

104

Agricultural and Environmental Sciences of McGill University. This study was conducted in 2014 on two

105

Canadian dairy farms with Holstein cattle applying similar herd management practices: 1) the Macdonald

106

Campus Farm from McGill University (Farm 1) and 2) a commercial dairy farm on the outskirts of

107

Montreal (Farm 2).

108

2.2 Animals and husbandry

SC

RI PT

103

Primiparous (P) cows (N=8), multiparous (M) cows (N=7) and nulliparous heifers (N) (N=14)

110

were used in this study. Cows entering their first lactation (P) calved at 24.65 ± 1.01 months of age, while

111

multiparous cows (M) were of 50.54 ± 7.45 months of age (entering their 3.14 ± 0.55 lactation), and

112

heifers were 13.15 ± 0.17 months of age. We did not monitor the body condition score of these animals

113

and we acknowledge that our sample size is small, both of which represent limitations in the overall

114

study. However, the small sample size was mainly due to the complex nature of sample processing,

115

analysis and cost of liquid chromatography-tandem mass spectrometry. Animals from both farms, were

116

housed under similar conditions, where all lactating cows were housed in a tie-stall setting and all heifers

117

were kept in a free-stall setting. At the time of this study,, Farm 1 was milking 68 cows producing on

118

average 9580 kg/cow/lactation, with the first breeding at 71 days in milk (DIM), and a calving interval of

119

408 days, while Farm 2 was milking 66 cows producing on average 9528kg/cow/lactation, with the first

120

breeding at 72 DIM, and a calving interval of 430 days. Cows on both farms were fed a base diet of

121

alfalfa silage, corn silage, and soybean meal at the onset of lactation, while alfalfa silage and dry hay were

122

predominantly fed during the dry period. Heifer rations on both farms were similar, which was lower in

123

energy and higher in fiber when compared to lactating cows. At the onset of lactation, cows from both

124

farms were milked twice daily at approximately 0600h and 1730h, where individual milk yields were

125

recorded at each milking. Reproductive parameters were monitored for subsequent breeding: percent

126

pregnancy rates, calculated based on the number of services, and the calving interval in days (restricted

127

measurement for only the two groups of lactating cows).

AC C

EP

TE D

M AN U

109

5

ACCEPTED MANUSCRIPT Schuermann et al 128

2.3 Blood collection The experimental design is shown in Figure 1. Weekly blood samples were collected from heifers

130

(N=14) for approximately 1 month starting at 4 weeks prior to the week of planned time of breeding, and

131

primiparous (N=8) and multiparous (N=7) cows for fifteen weeks starting from 3 weeks pre-partum to 12

132

weeks post-partum. In our model, we addressed a typical VWP, where the onset of breeding for lactating

133

dairy cows commences at approximately 8 weeks in milk. We superimposed the weekly blood collections

134

from nulliparous heifers onto the 4 weeks prior to planned time of breeding for lactating cows (i.e. 4

135

weeks to 8 weeks post-calving) as identified by the “pre-breeding period”.

SC

RI PT

129

Blood samples were collected from all animals from the coccygeal vein between 1300h and

137

1500h on the day of sampling. This time corresponded to the period between morning (6h30) and evening

138

(18h30) feedings in order to avoid spikes in metabolic parameters due to feed intake. Blood samples were

139

collected in EDTA treated vacutainers and all samples were cooled down on ice, centrifuged for 10 min at

140

1500 rpm, and immediately frozen in liquid nitrogen until arrival at the laboratory, where they were

141

stored in a -80°C freezer. Blood samples for glutathione analysis were processed on farm as described

142

below.

143

2.4 Metabolic parameters

TE D

M AN U

136

We measured the plasma concentration of the following metabolites: β-hydroxybutyric acid

145

(BHBA), glucose, triglycerides, total bile acids, total cholesterol, high density lipoprotein-cholesterol

146

(HDL-C), low density lipoprotein-cholesterol (LDL-C), and very low-density lipoprotein-cholesterol

147

(VLDL-C). All analytes were measured in duplicate.

AC C

148

EP

144

Due to the fact that BHBA profiles in transition cows are well-established, they were measured

149

by clinical analyzer at the Prairie Diagnostic Services Inc. (Saskatoon, Canada) in samples of select time-

150

points. The time-points for the cows included 3 weeks pre-calving, the week of calving, and weeks 3, 7,

151

and 12 weeks post-calving. In heifers, BHBA was measured in one sample collected at 1 week prior to

6

ACCEPTED MANUSCRIPT Schuermann et al 152

planned time of breeding, which in our model would be the equivalent to 7 weeks post-partum in lactating

153

cattle. The other target metabolites were measured in all weekly samples of cows and heifers using

155

colorimetric/enzymatic assays following the manufacturer’s protocol unless otherwise specified: 1)

156

Glucose - Glucose kit from Sekisui Diagnostics (220-32, BioPacific Diagnostics Inc, North Vancouver,

157

BC, Canada). The intra- and inter-assay coefficients of variation were 2.59% and 3.10%, respectively. 2)

158

Triglycerides - Triglycerides-SL kit from Sekisui Diagnostics (236-60, BioPacific Diagnostics Inc., North

159

Vancouver, BC, Canada). The intra- and inter-assay coefficients of variation were 1.16% and 3.71%,

160

respectively. 3) Total bile acids - Total Bile Acid kit from Diazyme Laboratories (DZ042A-K, BioPacific

161

Diagnostics Inc., North Vancouver, BC, Canada). The intra- and inter-assay coefficients of variation were

162

16.77% and 19.76%, respectively. 4) Total Cholesterol -Cholesterol-SL kit from Sekisui Diagnostics

163

(234-60, BioPacific Diagnostics Inc., North Vancouver, BC, Canada). The intra- and inter-assay

164

coefficients of variation were 7.15% and 16.24%, respectively. 5) HDL-C - HDL-C kit from Wako

165

Diagnostics (997-72591 and 993-72691, Wako Life Sciences, Inc., Mountain View, CA, USA), where the

166

second incubation period was increased by 1.5h to assess a stable reaction. The absorbance in individual

167

plasma samples was measured using the FilterMaxTM F5 Multi-Mode. The intra- and inter-assay

168

coefficients of variation were 2.47% and 5.43%, respectively.

SC

M AN U

TE D

EP

170

Concentrations of VLDL-cholesterol (VLDL-C) and LDL-cholesterol (LDL-C) were calculated using the following validated equations:

AC C

169

RI PT

154

171

A) VLDL-C = Triglycerides/5 [29]

172

B) LDL-C = Total cholesterol – (HDL-C + VLDL-C) [29, 30]

173 174 175

2.5 Assessment of oxidative stress markers Circulating levels of glutathione (GSH) values and the Ferric Reducing Ability of Plasma (FRAP) assay were used to assess the antioxidant systems in cattle.

176

GSH being very reactive and unstable required immediate derivatization from blood samples

177

upon collection. First, 500µl of whole blood (from EDTA treated vacutainers) was placed in a 2ml tube

7

ACCEPTED MANUSCRIPT Schuermann et al with 10µl of 10mM γ-glutamyl-leucine (γGL), which was used as an internal standard, and 200µl of

179

100mM N-ethylmaleimide (NEM). This mixture was quickly vortexed, then placed on ice for 5-min.

180

Following incubation, 50µl of zinc sulphate (ZnSO4 .7H2O) was used to lyse the NEM-treated blood and

181

1ml of ice cold methanol was added to the mixture for protein-precipitation, where the mixture was

182

centrifuged at 800 rpm for 1-2 min. The supernatant was stored in liquid nitrogen, until arrival at the

183

laboratory, where it was transferred to a -80°C freezer. Samples were analyzed by liquid chromatography-

184

tandem mass spectrometry (LC/MS) as previously described [31]. In brief, chromatography was

185

performed using the Agilent 1290 UHPLC system (Agilent Technologies) and mass spectrometry was

186

accomplished using the Agilent 6460 Triple Quadrupole (QQQ) system (Agilent Technologies) in

187

positive electrospray mode. MassHunter Qualitative and Quantitative Analysis software (Agilent

188

Technologies) was used to the measure GSH by means of integrating chromatographic peaks, followed by

189

the creation of a standard curve, and the response ratios calculated against γGL.

M AN U

SC

RI PT

178

To measure the total antioxidant activity, we performed the FRAP assay as described previously

191

[32, 33], with some modifications for bovine plasma samples. Briefly, we prepared a series of standards

192

of ferrous sulfate (FeSO4. 7H2O) from 0.1 mM to 1mM. Next, we prepared the FRAP reacting reagent

193

from: 200ml of 300mM Acetate Buffer (at pH 3.6), 20 ml of 10mM TPTZ (2, 4, 6-tripyridyl-s- triazine),

194

20ml of 20mM Iron (III) chloride hexahydrate (FeCl3. 6H2O), and 24 ml of distilled water. The straw

195

coloured FRAP reagent was subsequently incubated in a water bath at 37°C for 10 minutes. The assay

196

uses antioxidants as reductants in a redox-linked colorimetric method, whereby ferric tripyridyl triazine

197

(Fe III TPTZ) is reduced to its ferrous form. This reaction causes the straw coloured substance to become

198

blue, and thus, absorbance is measured at 593 nm. Thus, by means of a microtiter plate, we added 30 µl

199

H2O and 10µl standards or samples, followed by 200 µl FRAP-solution, mixed for 10 seconds and the

200

absorbance was measured after approximately 8 minutes. The intra- and inter-assay coefficients of

201

variation were 4.84% and 3.10%, respectively.

202

2.6 Statistical analysis

AC C

EP

TE D

190

8

ACCEPTED MANUSCRIPT Schuermann et al The data are presented as LS means ± standard errors of the mean (SEM). All data (milk

204

production, reproductive performance, metabolites, and oxidative stress markers) with repeated measures

205

were analyzed using the MIXED model procedure with SAS (version 9.4, SAS Institute Inc., Cary, NC,

206

USA). For BHBA analysis, where the various time points were not equally spaced, an exponential

207

correlation covariance structure SP (POW) was used for repeated measures. To compare the week prior to

208

the onset of breeding between all three groups were compared by analysis of variance. Metabolite and

209

oxidative stress data were analyzed to compare primiparous cows to multiparous cows from 3 weeks pre-

210

calving to 12 weeks post-calving, while analysis of the pre-breeding period was used to compare

211

primiparous cows, multiparous cows and nulliparous heifers. All data was tested for normality using the

212

Shapiro-Wilk test (PROC UNIVARIATE) and if needed were log-transformed. Post-analysis, all data

213

were back transformed for graphical representation. The following model was used:

214

Yijkm = µ + Gi + Tj + Fk + GTij + Cm(ik) + eijkm

215

Where Yijkm represents the parameter being measured, µ =represents the overall mean Gi is the effect of

216

the ith group (three stages of production: primiparous (P), multiparous (M), and heifers (H)), Tj is the

217

effect of the jth time (in weeks), Fk is the effect of the kth farm (two farms), GTij is the effect of the

218

interaction between the ith group and the jth time point, Cm(ik) is the effect of the mth cow within the ith

219

group and the kth farm and, eijkm represents the residual error. The random effect was cow nested within

220

stage of production (primiparous (P), multiparous (M), and heifers (H)) and farm, while the fixed effect

221

included the farm, the weekly blood samples, and the interaction between the weekly blood samples and

222

the stage of production. Differences between groups were detected by Scheffés method. The P-values of

223

<0.05 were declared significant, while P-values of <0.1 tended to be different.

225 226

SC

M AN U

TE D

EP

AC C

224

RI PT

203

3. Results 3.1 Production and reproduction

9

ACCEPTED MANUSCRIPT Schuermann et al We first compared the milk yield from week 1 post-partum to week 12 post-partum for cows

228

entering their first lactation with those entering the second or greater lactation. As expected, the milk

229

yield during the sampling period of multiparous cows was higher than that of primiparous cows (Figure 2;

230

P<0.05). We then compared the pregnancy rates and calving interval, two principal reproductive

231

management parameters, between heifers and cow groups. While primiparous and multiparous cows had

232

similar pregnancy rates, nulliparous heifers had a tendency for higher pregnancy rates than lactating cows

233

(Table 1; P<0.1). Lactating cows required approximately one additional service per pregnancy compared

234

to nulliparous heifers (about 2.4 services for lactating cows and 1.5 services for nulliparous heifers).

235

There was no difference in calving interval between lactating cows (Table 1; P>0.05). Even though the

236

calving interval was on average 18 days longer for multiparous than primiparous cows.

237

3.2 Blood metabolites

M AN U

SC

RI PT

227

Multiparous cows had higher plasma concentrations of BHBA during the experimental period

239

than primiparous cows (Figure 3A, P<0.05). Because heifers were not monitored for fifteen weeks, we

240

compared BHBA levels during the week prior to the anticipated week of breeding. Multiparous cows had

241

2-fold higher levels of BHBA than heifers (Figure 3B, P<0.05) with primiparous cows having

242

intermediate levels that were not different from either group of animals (Figure 3B, P>0.05). The plasma

243

glucose levels were lower in lactating cows than heifers during the pre-breeding period (Figure 4A,

244

P<0.05). On average, primiparous and multiparous cows had 29.89% and 40.99% less circulating

245

glucose, respectively, compared to heifers.

EP

AC C

246

TE D

238

There were fluctuations in the total bile acids in the weekly plasma samples throughout the

247

sampling period. Nonetheless, the overall concentrations of total bile acids during the pre-breeding

248

period, were higher in lactating cows compared to heifers (Figure 4B, P<0.05). The plasma

249

concentrations of triglycerides reached a nadir at one week post-calving in both groups of lactating cows.

250

Overall, the triglyceride levels in lactating cows remained lower post-calving compared to pre-calving.

251

Multiparous cows had the lowest concentrations of plasma triglycerides in the pre-breeding period

252

compared to both primiparous cows and nulliparous heifers (Figure 4C, P<0.05).

10

ACCEPTED MANUSCRIPT Schuermann et al The total cholesterol in circulation was at its lowest concentration in the weeks around calving for

254

both primiparous and multiparous cows. With the onset of lactation, there was a gradual increase in total

255

cholesterol in both groups of cows reaching a plateau between 6 through 12 weeks in milk. We also

256

observed a significant group and time (weeks) interaction for the lactating cows (Figure 5A, P<0.05).

257

During the pre-breeding period, there were significantly higher concentrations of total cholesterol in both

258

primiparous and multiparous cows compared to heifers. On average, there was 36.36% and 50.65% more

259

total cholesterol in primiparous and multiparous cows than heifers, respectively (Figure 5A, P<0.05). We

260

then analyzed the profiles of cholesterol fractions, HDL-C, LDL-C and VLDL-C. Levels of HDL-C were

261

higher in both primiparous and multiparous cows when compared to heifers during the pre-breeding

262

period (Figure 5B, P<0.05). Levels of plasma HDL-C were 31.82% and 39.05% higher for primiparous

263

and multiparous cows, respectively, compared to heifers. The LDL-C levels were higher in both

264

primiparous and multiparous cows than heifers, respectively, during the pre-breeding period (Figure 5C,

265

P<0.05). Levels of VLDL-C in all groups were very low with a nadir at 1 week post-calving in both

266

primiparous and multiparous cattle. Unlike HDL-C and LDL-C levels, the circulating levels of VLDL-C

267

during the pre-breeding period were lowest in multiparous cows compared to both primiparous cows and

268

nulliparous heifers (Figure 5 D, P<0.05).

269

3.3 Biomarkers of oxidative stress markers

EP

TE D

M AN U

SC

RI PT

253

Assays for GSH and FRAP were used to evaluate the ability of the cow to counteract reactive

271

oxidative species. The levels of GSH remained stable in lactating cows during the transition period

272

through the VWP. Furthermore, there was no difference in the concentration of circulating GSH during

273

the pre-breeding period of both lactating cows and nulliparous heifers (Figure 6 A, P>0.05). However,

274

both primiparous and multiparous cows had higher levels of circulating FRAP in the 12 weeks post-

275

calving compared to the 3 weeks pre-calving. Moreover, during the pre-breeding period, FRAP levels

276

were higher for both groups of lactating cows compared to nulliparous heifers (Figure 6 B, P<0.05).

277

AC C

270

4. Discussion

11

ACCEPTED MANUSCRIPT Schuermann et al In this study, we characterized plasma metabolic and oxidative stress indicators of lactating cows

279

from the transition period until 12 weeks post-calving to encompass the pre-breeding period.

280

Furthermore, we collected blood samples from nulliparous heifers during their respective pre-breeding

281

period, which we subsequently superimposed onto the pre-breeding period of lactating cows, separated

282

into primiparous and multiparous cows. The results from this study demonstrated the dramatic changes in

283

profiles of metabolic indicators in plasma of lactating cows and how these profiles are different in heifers

284

during the pre-breeding period. As we [26] and others [34] have shown previously, metabolites in plasma

285

are also found in the ovarian follicular fluid. Results from this study provide a reflection of the metabolic

286

profile of follicles growing during the transition period through the VWP of approximately 8 weeks in

287

milk.

M AN U

SC

RI PT

278

It has been documented that fertility among dairy heifers is higher than that of their lactating

289

counterparts [17]. Evidence exists that first and second insemination pregnancy rates decrease with

290

age/parity, where heifers, 1st parity, 2nd parity, and 3rd/4th parity cows had pregnancy rates of 84.3%,

291

51.5%, 31.4%, and 19.5%, respectively [19]. Similarly, we show a tendency for reduced reproductive

292

performance in lactating cows, where their percent pregnancy rates averaged around 46%, while that of

293

nulliparous heifers averaged around 76%. For the average cow, additional services per pregnancy causing

294

lower pregnancy rates adds to a herd’s cost of production by increased number of days open, which in

295

turn increased the calving interval and thus, increased the days between the peak in milk production [35].

296

More precisely, prolonging the calving interval for an average cow from the anticipated 365 days to 425

297

days would decrease milk production and efficiency since end of lactation cows require more feed per

298

liter of milk produced [35]. Although we did not observe a significant difference between the calving

299

interval of primiparous and multiparous cows with 414.45 ± 26.76 days and 433.39 ± 31.74 days,

300

respectively, they are in both cases longer than economically advantageous. Nonetheless, our data is in

301

line with the average for Quebec Holstein cows (3 521 herds) where lactating cows have a calving

302

interval of 416 days and require approximately 2.4 services to achieve conception [36].

AC C

EP

TE D

288

12

ACCEPTED MANUSCRIPT Schuermann et al Overall, the reduced fertility in lactating cows is in part related to metabolic shifts[13]. While

304

multiple studies have addressed the direct effects of enhanced NEFA and BHBA on oocyte and granulosa

305

cells [28, 37], the potential effects of other metabolic changes cannot be ruled out. Thus, we set out to

306

establish additional blood metabolic indicator and oxidative stress marker differences among heifers and

307

cows of different parities in this study. As expected, here we report higher BHBA levels in multiparous

308

cows compared to primiparous cows throughout the study period. A recent study showed significantly

309

higher levels of BHBA in multiparous cows compared to primiparous cows, with the highest differences

310

at weeks 1 and 4 post-partum [38]. Furthermore, we show that multiparous cows had twice the

311

concentration of BHBA compared to heifers in the week leading up to the planned time of breeding. The

312

concentrations of BHBA are expected to be nearly identical between blood circulation and the follicular

313

fluid [27], suggesting that the ovulating follicle in multiparous cows contains twice the amount of BHBA

314

found in the ovulating follicle of heifers. Higher levels of BHBA have been associated with a longer

315

VWP [15] and pregnancy at first artificial insemination [39]. Increased BHBA was associated with

316

altered expression of a number of genes involved in proliferation and fatty acid metabolism in follicular

317

granulosa cells [40]. Elsewhere, BHBA treatment reduced estradiol and progesterone synthesis in cultured

318

granulosa cells [28]. Our results further contribute to the knowledge that increased age and number of

319

parities enhance the volume of milk produced, this higher demand is likely met through lipid mobilization

320

observed through higher levels of BHBA. It has been suggested that metabolic indicators and oxidative

321

stress markers are closely intertwined, where higher BHBA levels have been linked with lower levels of

322

antioxidants and higher levels of reactive oxidative species addressed later on in the discussion [11]. The

323

negative correlation between BHBA levels and reproductive success is increased in multiparous cows. A

324

number of studies have reported nutritional management techniques to assist in alleviating metabolic

325

stress by decreasing BHB levels, including, rumen-protected methionine and rumen-protected

326

choline[41]. Both of which would be of great potential benefit to the general population of dairy cows in

327

the third or greater lactation.

AC C

EP

TE D

M AN U

SC

RI PT

303

13

ACCEPTED MANUSCRIPT Schuermann et al Although we did not observe an effect of parity, there were lower levels of glucose in lactating

329

cows compared to heifers in the 4 weeks leading up to the breeding period, explained by an elevated

330

demand of glucose for milk production. This is in contrast to a recent study that reported lower levels of

331

glucose in multiparous cows compared to primiparous cows [38]. Levels of glucose are slightly higher in

332

the follicular fluid compared to blood [26, 27]. Cows with ovulatory dysfunction have lower glucose and

333

insulin like-growth 1 (IGF1) levels than reproductively sound cows [42]. Furthermore, pregnancy at first

334

artificial insemination was lower in cows with lower levels of glucose [43, 44].

SC

RI PT

328

Bile acid synthesis has been shown to be increased at the onset of lactation in dairy cattle through

336

presence of increased cholesterol as a precursor for bile acid synthesis and upregulation of the expression

337

of the hepatic cholesterol 7 alpha-hydroxylase (CYP7A1) gene [45]. Although we observed a great deal

338

of fluctuations in plasma bile acids, overall values were higher in lactating cows compared to heifers in

339

the pre-breeding period. Bile acids have been shown in follicular fluid of both human and bovine ovaries

340

[26, 46, 47]. Profiling of bile acid composition found that bovine follicular fluid predominantly contains

341

cholic acid, cheodeoxycholic acid, deoxycholic acid and their glycine and taurine conjugates [47]. We

342

previously showed threefold higher concentrations of total bile acids in the follicular fluid of the

343

dominant follicle in early-lactating cows compared to nulliparous heifers [26]. Furthermore, it has been

344

suggested that cultured bovine granulosa cells treated with 5 nM of cholic acid, a concentration expected

345

in the follicular fluid, inhibits estrogen synthesis [48]. Given that bile acids have recently been shown to

346

act as signals to regulate metabolic functions in humans [49], these initial observations in cows certainly

347

trigger interest in the effects of bile acids on cow fertility.

TE D

EP

AC C

348

M AN U

335

Triglyceride levels reached a nadir at one week after parturition for both groups of lactating cows

349

and remained lower in lactating cows compared to heifers, the difference being more obvious when

350

compared to multiparous cows. Triglyceride levels are low in bovine follicular fluid [27] and remain

351

stable irrespective of concentrations found in circulation [50]. However, cows with higher levels of

352

triglyceride accumulation on the liver had a 35% decreased probability of estrous and a 30% decreased

14

ACCEPTED MANUSCRIPT Schuermann et al 353

chance of pregnancy [51]. In this study, the lower levels of plasma triglycerides persisted through to the

354

pre-breeding period. This study showed a gradual increase in the levels of total cholesterol, HDL-C, and LDL-C in

356

cows from the onset of lactation and reaching a plateau at time of anticipated breeding. Levels of the

357

aforementioned cholesterol fractions and cholesterol itself were significantly higher in the lactating cows

358

compared to heifers. There were no differences between primiparous and multiparous cows in total

359

cholesterol and HDL-C, which is in agreement with a previous report [52]. However, we found a

360

significant effect of parity on LDL-C, which were higher in multiparous than primiparous cows. There is

361

strong correlation between levels of cholesterol in blood and follicular fluid [26, 27] with higher

362

cholesterol entry into the large follicles compared to small follicles [53]. HDL has long been detected in

363

follicular fluid, while LDL was previously believed to not have been localized in the follicular fluid due

364

to its larger size, making it less likely to cross the blood-follicle barrier, but has recently been located in

365

follicular fluid of cattle [54]. Interestingly, higher levels of total cholesterol, HDL, and LDL were found

366

in the follicular fluid of healthy versus atretic follicles [54]. Some in vitro studies showed that addition of

367

LDL and HDL increased progesterone synthesis in bovine granulosa cells [55, 56]. Higher HDL levels in

368

the dry period have been suggested to benefit cows in their transition period as it would indicate improved

369

liver function [57]. However, we did not observe any differences in HDL levels between in the pre-

370

partum period of multiparous and primiparous cows, it has been suggested that cows with higher

371

concentrations of HDL experienced improved calving to first service and calving to conception intervals.

372

The previous study also suggested that those cows had lower risk of retained placenta and/or metritis

373

(although, not significant due to the low number of animals per group) [57]. Moreover, higher levels of

374

HDL have been reported in cows fed with high energy diet during the dry period[58].

AC C

EP

TE D

M AN U

SC

RI PT

355

375

Lactating multiparous cows were shown to have higher BHBA and cholesterol, and lower glucose

376

in circulation than heifers [59]. Altered metabolic profiles were associated with reduced estradiol and

377

progesterone concentrations in the dominant follicles. This study also showed that expression of the gene

378

encoding for steroidogenic acute regulatory protein (STAR), a rate limiting enzyme in cholesterol

15

ACCEPTED MANUSCRIPT Schuermann et al transport, was lower in cows [59]. These observations suggest that while cholesterol alone may have

380

positive effects on ovarian steroidogenesis, their effects could be opposite in association with high BHBA

381

and low glucose. However, the low concentrations of circulating C-VLDL in lactating cows, specifically

382

in multiparous cows, during the pre-breeding period would suggest that the process of removing lipids

383

from the liver in the form of C-VLDL is less efficient in lactating cows and may be in relation to a liver

384

which is not functioning at the same capacity as that of nulliparous heifers.

RI PT

379

GSH and FRAP are key markers used to evaluate the antioxidant system in plasma and they

386

complement each other as the FRAP assay does not take into consideration the level of reduced

387

glutathione [60]. The transition period has been described by a depletion of antioxidant status

388

characterized by an imbalance between the reactive oxygen metabolites and elimination by the

389

antioxidant system leading to oxidative stress [61]. It is important to maintain levels of glutathione as it is

390

a natural antioxidant produced in large amounts by the liver. Glutathione is central for preventing fatty

391

liver and mitochondrial injury as shown in mice lacking the Gclc gene required for GSH synthesis[62].

392

Many previous studies have measured glutathione peroxidase activity (GSH-Px) as opposed to measuring

393

reduced glutathione (GSH), where the latter is considered a more accurate indicator of the antioxidant

394

capacity of a cell [11]. It was shown that GSH-Px activity was higher in post-calving than pre-calving

395

cows suggesting oxidative stress [11]. However, we found no difference in GSH levels at any time of the

396

sampling period, nor between the sampling groups during the pre-breeding period. Our observation of

397

higher FRAP in cows than heifers does suggest that there may be hepatic adjustments in transition cows

398

to alleviate any oxidative stress due to parturition or lactation stress. In line with this, a previous study

399

demonstrated that primiparous cows experienced an increase in FRAP from the pre-calving to post-

400

calving period [63].

AC C

EP

TE D

M AN U

SC

385

401

Taken together, primiparous and multiparous cows, compared to nulliparous heifers, experience

402

greater metabolic imbalance from the transition period through the VWP when the processes of oocyte

403

development, follicle growth, and ovulation (80-100 days) are taking place. Our observations of

404

differences between primiparous and multiparous cows contributes to the knowledge that metabolic

16

ACCEPTED MANUSCRIPT Schuermann et al changes may contribute to the decline in reproductive success with every passing parity. We propose that

406

higher levels of BHBA, cholesterol, bile acids and the lower concentrations of glucose in multiparous

407

cows may hinder the follicular microenvironment and thus influence their reproductive performance.

408

Experiments to test this hypothesis are needed. We emphasize the importance of individualized nutritional

409

management for dairy cows of different parities. Furthermore, the length of the metabolic imbalance from

410

our results highlights the diversion of energy away from reproduction. Cows require energy for body

411

maintenance, production, and health prior to being directed towards reproduction. We would recommend

412

adopting a longer VWP that commensurate with milk production levels and parity such as has been

413

recently suggested [20, 64], but of course the financial implications of increasing the calving interval need

414

to be considered. The latest data from Valacta (A DHI platform for Eastern Canada) showed that farms

415

(n=704) with high-producing cows (average of 11,500 kg/cow/year with 4.00% Fat and 3.29% Protein)

416

had average days in milk at first breeding (VWP) of 76 days and required 2.35 breeding/cow/year [65].

417

Recent research has suggested that cows with similar production performance would benefit from a VWP

418

up to 180 days in milk as it significantly improves first artificial insemination (AI)/conception rates and

419

leads to lower number of AI/conception[64]. These studies along with our data highlight the value of a

420

longer VWP for our high-producing dairies. Nonetheless, further studies are needed to understand how

421

the altered metabolic profiles directly affect reproductive processes during early lactation when the cows

422

are bred.

AC C

423

EP

TE D

M AN U

SC

RI PT

405

424

Conflict of Interest

425

None of the authors of the above manuscript has declared any conflict of interest.

426

Acknowledgments

427

We would like to thank the Macdonald Campus Farm and Ferme Norline for access to their dairy

428

cattle. In addition, we would like to thank Dr. Luis Agellon for use of his facility, as well as Dr. Roger

429

Cue for assistance with statistical analysis. YS was supported by the Réseau Quebecois en Reproduction

430

(RQR)-CREAT scholarship, Department of Animal Science Graduate Excellence Fellowship, and the

17

ACCEPTED MANUSCRIPT Schuermann et al Fonds de recherche du Québec – Nature et Technologie (FRQNT) (B2) and GW was supported by the

432

Natural Science and Engineering Research Council- Undergraduate Student Research Awards (NSERC-

433

USRA). Lastly, this study was supported by both FRQNT and NSERC grants (RD).

434

References:

435

RI PT

431

[1] Delgado HAC, R.I.; Haine, D.; Sewalem, A.; Lacroix, R.; Lefebvre, D.; Dubuc, J.; Bouchard,

437

E.; and Wade, K.M.;. Profitability measures as decision-making tools for Québec dairy herds.

438

Canadian Journal of Animal Science. 2018;98.

439

[2] Murray B. Finding the Tools to Achieve Longevity in Canadian Dairy Cows. WCDS

440

Advances in Dairy Technology. 2013;25:15-28.

441

[3] Van Doormal B. Un regard plus approfondi sur la longévité.: Réseau laitier canadien; 2009.

442

[4] Pellerin D, Adams S, Bécotte F, Cue R, Moore R, Roy R. Pour une vache, l'âge d'or c'est la

443

4e lactation! CRAAQ- 38e Symposium sur les bovins laitiers. Drummondville, Qc2014.

444

[5] Valacta CDEePLQ-A. Évolution de la production laitière québécoise 2016. Le producteur de

445

lait québécois. 2017.

446

[6] Drackley JK. ADSA Foundation Scholar Award. Biology of dairy cows during the transition

447

period: the final frontier? J Dairy Sci. 1999;82:2259-73.

448

[7] Grummer RR. Impact of changes in organic nutrient metabolism on feeding the transition

449

dairy cow. J Anim Sci. 1995;73:2820-33.

450

[8] LeBlanc SJ, Lissemore KD, Kelton DF, Duffield TF, Leslie KE. Major advances in disease

451

prevention in dairy cattle. J Dairy Sci. 2006;89:1267-79.

452

[9] Ribeiro ES, Gomes G, Greco LF, Cerri RLA, Vieira-Neto A, Monteiro PLJ, Jr., et al.

453

Carryover effect of postpartum inflammatory diseases on developmental biology and fertility in

454

lactating dairy cows. J Dairy Sci. 2016;99:2201-20.

455

[10] Leroy J, Vanholder T, Van Knegsel A, Garcia‐Ispierto I, Bols P. Nutrient prioritization in

456

dairy cows early postpartum: mismatch between metabolism and fertility? Reproduction in

457

Domestic Animals. 2008;43:96-103.

458

[11] Bernabucci U, Ronchi B, Lacetera N, Nardone A. Influence of body condition score on

459

relationships between metabolic status and oxidative stress in periparturient dairy cows. Journal

460

of Dairy Science. 2005;88:2017-26.

AC C

EP

TE D

M AN U

SC

436

18

ACCEPTED MANUSCRIPT Schuermann et al [12] Huzzey JM, Mann S, Nydam DV, Grant RJ, Overton TR. Associations of peripartum

462

markers of stress and inflammation with milk yield and reproductive performance in Holstein

463

dairy cows. Preventive veterinary medicine. 2015;120:291-7.

464

[13] Shin EK, Jeong JK, Choi IS, Kang HG, Hur TY, Jung YH, et al. Relationships among

465

ketosis, serum metabolites, body condition, and reproductive outcomes in dairy cows.

466

Theriogenology. 2015;84:252-60.

467

[14] Walsh RB, Kelton DF, Duffield TF, Leslie KE, Walton JS, LeBlanc SJ. Prevalence and risk

468

factors for postpartum anovulatory condition in dairy cows. J Dairy Sci. 2007;90:315-24.

469

[15] Ospina PA, Nydam DV, Stokol T, Overton TR. Associations of elevated nonesterified fatty

470

acids and beta-hydroxybutyrate concentrations with early lactation reproductive performance and

471

milk production in transition dairy cattle in the northeastern United States. J Dairy Sci.

472

2010;93:1596-603.

473

[16] Bertoni G, Trevisi E, Han X, Bionaz M. Effects of inflammatory conditions on liver activity

474

in puerperium period and consequences for performance in dairy cows. J Dairy Sci.

475

2008;91:3300-10.

476

[17] Kuhn MT, Hutchison JL, Wiggans GR. Characterization of Holstein heifer fertility in the

477

United States. J Dairy Sci. 2006;89:4907-20.

478

[18] Wathes DC, Pollott GE, Johnson KF, Richardson H, Cooke JS. Heifer fertility and carry

479

over consequences for life time production in dairy and beef cattle. Animal. 2014;8 Suppl 1:91-

480

104.

481

[19] Balendran AG, M; Pretheeban, T; Singh, R; Perera, R; Rajamahedran, R;. Decreased

482

fertility with increasing parity in lactating dairy cows. Canadian Journal of Animal Science.

483

2008;88:425-8.

484

[20] Stangaferro ML, Wijma R, Masello M, Thomas MJ, Giordano JO. Extending the duration of

485

the voluntary waiting period from 60 to 88 days in cows that received timed artificial

486

insemination after the Double-Ovsynch protocol affected the reproductive performance, herd exit

487

dynamics, and lactation performance of dairy cows. J Dairy Sci. 2018;101:717-35.

488

[21] Miller RH, Norman HD, Kuhn MT, Clay JS, Hutchison JL. Voluntary waiting period and

489

adoption of synchronized breeding in dairy herd improvement herds. J Dairy Sci. 2007;90:1594-

490

606.

AC C

EP

TE D

M AN U

SC

RI PT

461

19

ACCEPTED MANUSCRIPT Schuermann et al [22] Britt JH. Oocyte development in cattle: physiological and genetic aspects. R Bras Zootec.

492

2008;37:110-5.

493

[23] Lucy MC, Butler ST, Garverick HA. Endocrine and metabolic mechanisms linking

494

postpartum glucose with early embryonic and foetal development in dairy cows. Animal. 2014;8

495

Suppl 1:82-90.

496

[24] Britt JH. Impacts of early postpartum metabolism on follicular development and fertility.

497

The Bovine Proceedings1992.

498

[25] Thatcher WW. A 100-Year Review: Historical development of female reproductive

499

physiology in dairy cattle. J Dairy Sci. 2017;100:10272-91.

500

[26] Sanchez R, Schuermann Y, Gagnon-Duval L, Baldassarre H, Murphy BD, Gevry N, et al.

501

Differential abundance of IGF1, bile acids, and the genes involved in their signaling in the

502

dominant follicle microenvironment of lactating cows and nulliparous heifers. Theriogenology.

503

2014;81:771-9.

504

[27] Leroy JL, Vanholder T, Delanghe JR, Opsomer G, Van Soom A, Bols PE, et al. Metabolic

505

changes in follicular fluid of the dominant follicle in high-yielding dairy cows early post partum.

506

Theriogenology. 2004;62:1131-43.

507

[28] Vanholder T, Leroy JL, Van Soom A, Coryn M, de Kruif A, Opsomer G. Effects of beta-

508

OH butyrate on bovine granulosa and theca cell function in vitro. Reproduction in domestic

509

animals = Zuchthygiene. 2006;41:39-40.

510

[29] Friedewald WT, Levy RI, Fredrickson DS. Estimation of the concentration of low-density

511

lipoprotein cholesterol in plasma, without use of the preparative ultracentrifuge. Clin Chem.

512

1972;18:499-502.

513

[30] Kessler EC, Gross JJ, Bruckmaier RM, Albrecht C. Cholesterol metabolism, transport, and

514

hepatic regulation in dairy cows during transition and early lactation. J Dairy Sci. 2014.

515

[31] Vassilyadi P, Harding SV, Nitschmann E, Wykes LJ. Experimental colitis and malnutrition

516

differentially affect the metabolism of glutathione and related sulfhydryl metabolites in different

517

tissues. Eur J Nutr. 2016;55:1769-76.

518

[32] Schlesier K, Harwat M, Bohm V, Bitsch R. Assessment of antioxidant activity by using

519

different in vitro methods. Free Radic Res. 2002;36:177-87.

520

[33] Benzie IF, Strain JJ. The ferric reducing ability of plasma (FRAP) as a measure of

521

"antioxidant power": the FRAP assay. Anal Biochem. 1996;239:70-6.

AC C

EP

TE D

M AN U

SC

RI PT

491

20

ACCEPTED MANUSCRIPT Schuermann et al [34] Sartori R, Haughian JM, Shaver RD, Rosa GJ, Wiltbank MC. Comparison of ovarian

523

function and circulating steroids in estrous cycles of Holstein heifers and lactating cows. J Dairy

524

Sci. 2004;87:905-20.

525

[35] Laven R. Fertility in Dairy Herds - Advanced Part 1: What does poor fertility cost. National

526

Animal Disease Information Service; 2018.

527

[36] Valacta CDEePLQ-A. Évolution de la production laitière québécoise 2017. Le Producteur

528

de lait quebecois. 2018.

529

[37] Vanholder T, Leroy JL, Soom AV, Opsomer G, Maes D, Coryn M, et al. Effect of non-

530

esterified fatty acids on bovine granulosa cell steroidogenesis and proliferation in vitro. Anim

531

Reprod Sci. 2005;87:33-44.

532

[38] Morales Pineyrua JT, Farina SR, Mendoza A. Effects of parity on productive, reproductive,

533

metabolic and hormonal responses of Holstein cows. Anim Reprod Sci. 2018;191:9-21.

534

[39] Chapinal N, Leblanc SJ, Carson ME, Leslie KE, Godden S, Capel M, et al. Herd-level

535

association of serum metabolites in the transition period with disease, milk production, and early

536

lactation reproductive performance. J Dairy Sci. 2012;95:5676-82.

537

[40] Girard A, Dufort I, Sirard MA. The effect of energy balance on the transcriptome of bovine

538

granulosa cells at 60 days postpartum. Theriogenology. 2015;84:1350-61 e6.

539

[41] Sun F, Cao Y, Cai C, Li S, Yu C, Yao J. Regulation of Nutritional Metabolism in Transition

540

Dairy Cows: Energy Homeostasis and Health in Response to Post-Ruminal Choline and

541

Methionine. PLoS One. 2016;11:e0160659.

542

[42] Kawashima C, Sakaguchi M, Suzuki T, Sasamoto Y, Takahashi Y, Matsui M, et al.

543

Metabolic profiles in ovulatory and anovulatory primiparous dairy cows during the first follicular

544

wave postpartum. J Reprod Dev. 2007;53:113-20.

545

[43] Garverick HA, Harris MN, Vogel-Bluel R, Sampson JD, Bader J, Lamberson WR, et al.

546

Concentrations of nonesterified fatty acids and glucose in blood of periparturient dairy cows are

547

indicative of pregnancy success at first insemination. J Dairy Sci. 2013;96:181-8.

548

[44] Green JC, Meyer JP, Williams AM, Newsom EM, Keisler DH, Lucy MC. Pregnancy

549

development from day 28 to 42 of gestation in postpartum Holstein cows that were either milked

550

(lactating) or not milked (not lactating) after calving. Reproduction (Cambridge, England).

551

2012;143:699-711.

AC C

EP

TE D

M AN U

SC

RI PT

522

21

ACCEPTED MANUSCRIPT Schuermann et al [45] Schlegel G, Ringseis R, Keller J, Schwarz F, Eder K. Changes in the expression of hepatic

553

genes involved in cholesterol homeostasis in dairy cows in the transition period and at different

554

stages of lactation. Journal of dairy science. 2012;95:3826-36.

555

[46] Nagy RA, van Montfoort AP, Dikkers A, van Echten-Arends J, Homminga I, Land JA, et al.

556

Presence of bile acids in human follicular fluid and their relation with embryo development in

557

modified natural cycle IVF. Human reproduction (Oxford, England). 2015;30:1102-9.

558

[47] Sanchez-Guijo A, Blaschka C, Hartmann MF, Wrenzycki C, Wudy SA. Profiling of bile

559

acids in bovine follicular fluid by fused-core-LC-MS/MS. The Journal of steroid biochemistry

560

and molecular biology. 2016.

561

[48] Gagnon-Duval L, Salas N, Guerrero Netro H, Duggavathi R, Gévry N, Murphy B. Cholic

562

acid interferes with estrogen synthesis in bovine granulosa cells in vitro. Society for the Sudy of

563

Reproduction 2015 Annual Meeting. San Juan, Puerto Rico, USA2015.

564

[49] Shapiro H, Kolodziejczyk AA, Halstuch D, Elinav E. Bile acids in glucose metabolism in

565

health and disease. J Exp Med. 2018;215:383-96.

566

[50] Wehrman ME, Welsh TH, Jr., Williams GL. Diet-induced hyperlipidemia in cattle modifies

567

the intrafollicular cholesterol environment, modulates ovarian follicular dynamics, and hastens

568

the onset of postpartum luteal activity. Biology of reproduction. 1991;45:514-22.

569

[51] Jorritsma R, Jorritsma H, Schukken YH, Wentink GH. Relationships between fatty liver and

570

fertility and some periparturient diseases in commercial Dutch dairy herds. Theriogenology.

571

2000;54:1065-74.

572

[52] Folnožić I, R. Turk, D. Đuri čić, S. Vince, Z. Flegar-Meštri ć,P. Sobiech, M. Lojkić, H.

573

Valp oti ć, M. Samard žija. The effect of parity on metabolic profile and resumption of ovarian

574

cyclicity in dairy cows. Veterinarski Arhiv. 2016;86:641-53.

575

[53] Leroy JL, Vanholder T, Delanghe JR, Opsomer G, Van Soom A, Bols PE, et al. Metabolite

576

and ionic composition of follicular fluid from different-sized follicles and their relationship to

577

serum concentrations in dairy cows. Anim Reprod Sci. 2004;80:201-11.

578

[54] Schneider A, Absalon-Medina VA, Esposito G, Correa MN, Butler WR. Paraoxonase

579

(PON) 1, 2 and 3 expression in granulosa cells and PON1 activity in follicular fluid of dairy

580

cows. Reproduction in domestic animals = Zuchthygiene. 2013;48:989-94.

AC C

EP

TE D

M AN U

SC

RI PT

552

22

ACCEPTED MANUSCRIPT Schuermann et al [55] O'Shaughnessy PJ, Pearce S, Mannan MA. Effect of high-density lipoprotein on bovine

582

granulosa cells: progesterone production in newly isolated cells and during cell culture. The

583

Journal of endocrinology. 1990;124:255-60.

584

[56] O'Shaughnessy PJ, and D. C. Wathes. Role of lipoproteins and de-novo cholesterol

585

synthesis in progesterone production by cultured bovine luteal cells. Journal of reproduction and

586

fertility. 1985;74.

587

[57] Crociati M, Sylla L, Floridi C, Comin A, Fruganti G, Monaci M, et al. Influence of

588

lipoproteins at dry-off on metabolism of dairy cows during transition period and on postpartum

589

reproductive outcomes. Theriogenology. 2017;94:31-6.

590

[58] Newman A, Mann S, Nydam DV, Overton TR, Behling-Kelly E. Impact of dietary plane of

591

energy during the dry period on lipoprotein parameters in the transition period in dairy cattle. J

592

Anim Physiol Anim Nutr (Berl). 2016;100:118-26.

593

[59] Walsh SW, Mehta JP, McGettigan PA, Browne JA, Forde N, Alibrahim RM, et al. Effect of

594

the metabolic environment at key stages of follicle development in cattle: focus on steroid

595

biosynthesis. Physiological genomics. 2012;44:504-17.

596

[60] Hennipman AJ. The development of a Comprehensive Index (Oxiscore) to assess the

597

Oxidative Status in Periparturient Dairy Cows: Utrecht University; 2014.

598

[61] Abuelo A, Hernandez J, Benedito JL, Castillo C. The importance of the oxidative status of

599

dairy cattle in the periparturient period: revisiting antioxidant supplementation. J Anim Physiol

600

Anim Nutr (Berl). 2015;99:1003-16.

601

[62] Chen Y, Yang Y, Miller ML, Shen D, Shertzer HG, Stringer KF, et al. Hepatocyte-specific

602

Gclc deletion leads to rapid onset of steatosis with mitochondrial injury and liver failure.

603

Hepatology. 2007;45:1118-28.

604

[63] Wullepit N, Raes, K., Beerda, B., Veerkamp, R.F., Fremaut, D., De Smet, S. Influence of

605

management and genetic merit for milk yield on the oxidative status of plasma in heifers.

606

Livestock Science. 2009;123:276-82.

607

[64] Niozas G, Tsousis G, Steinhofel I, Brozos C, Romer A, Wiedemann S, et al. Extended

608

lactation in high-yielding dairy cows. I. Effects on reproductive measurements. J Dairy Sci.

609

2019;102:799-810.

610

[65] Valacta CDEePLQ-A. the evolution of valacta atlantic dairy production 2017 Stats & Tips.

611

2018.

AC C

EP

TE D

M AN U

SC

RI PT

581

23

ACCEPTED MANUSCRIPT Schuermann et al 612 Figure Legends

614

Figure 1. Experimental design. Holstein dairy cows were subject to weekly blood sampling from three

615

weeks pre-calving until twelve weeks post-calving. Dairy heifers were subject to weekly blood sampling

616

for a total of about one month. We designated an analysis overlap between heifers and cows, where the

617

blood samples from heifers were matched to the period from four to eight weeks post-calving. In both

618

scenarios, animals were nearing their respective planned time of breeding and thus, designated as the pre-

619

breeding period. Animals were divided into three groups: Primiparous cows (N=8), Multiparous cows

620

(N=7), and Nulliparous Heifers (N=14).

621

Figure 2. Milk yield of primiparous and multiparous dairy cows. Milk yields collected from week one to

622

week twelve post-calving from primiparous and multiparous dairy cows. Data are expressed as a LS mean

623

± S.E.M. An overall contrast between Groups (Primiparous (P) and Multiparous (M)) is reported as

624

significantly different with P<0.001.

625

Figure 3. Plasma levels of β-hydroxybutyric acid (BHBA) in heifers and dairy cattle. BHBA levels were

626

measured pre-calving, at calving and at three time-points post-calving for lactating dairy cows (A).

627

BHBA levels were compared between heifers, primiparous cows, and multiparous cows at an one week

628

prior to planned time of breeding (B). Data are expressed as a LS mean ± S.E.M. An overall contrast

629

between Groups (Primiparous (P) and Multiparous (M)) is shown (A). Gradual and significant increase in

630

BHBA levels observed in nulliparous heifers compared to multiparous cows as designated by letter

631

superscripts (B), P<0.05.

632

Figure 4. Profile of plasma metabolites from heifers and dairy cows. Glucose (A), total bile acids (B), and

633

triglycerides (C) were measured weekly using commercially available assays from plasma of dairy cows

634

sampled from three weeks pre-partum to twelve weeks post-partum and for a total of about one month in

635

breeding aged heifers. Data are expressed as a LS mean ± S.E.M. An overall contrast between Groups

636

(Primiparous (P), Multiparous (M), and Heifers (N)) in the pre-breeding period is shown by a significant

637

difference, where P<0.05.

AC C

EP

TE D

M AN U

SC

RI PT

613

24

ACCEPTED MANUSCRIPT Schuermann et al Figure 5. Representation of the plasma cholesterol profile from dairy cattle of different ages and

639

physiological stages. Total-cholesterol, HDL-Cholesterol, LDL-Cholesterol, and VLDL Cholesterol were

640

assessed weekly using commercially available assays or previously described calculations from plasma of

641

dairy cows sampled from three weeks pre-partum to twelve weeks post-partum and for a total of five

642

weeks in breeding aged heifers. Data are expressed as a LS mean ± S.E.M. An overall contrast between

643

Groups (Primiparous (P), Multiparous (M), and Heifers (N)) in the pre-breeding period is shown by a

644

significant difference, where P<0.05.

645

Figure 6. Oxidative stress markers measured in plasma of dairy heifers and cows. Blood samples were

646

specifically processed as described in the material and methods to measure the concentration of

647

glutathione in circulation of dairy heifers and cows using liquid chromatography-tandem mass

648

spectrometry (LC/MS). Plasma samples from cattle were measured for FRAP using an adapted protocol

649

as described in the materials and methods. Data are expressed as a LS mean ± S.E.M. An overall contrast

650

between Groups (Primiparous (P), Multiparous (M), and Heifers (N)) in the pre-breeding period is shown

651

by a significant difference, where P<0.05.

655 656

SC

M AN U

TE D

654

Table 1: Effect of age and parity on reproductive performance.

EP

653

AC C

652

RI PT

638

25

ACCEPTED MANUSCRIPT

Figure 1

Schuermann et al

Post-Calving

Pre-Calving Calving (0)

1 2 3 4

5

6

7

8 9 10 11 12

M AN U

SC

-3 -2 -1

RI PT

Weeks

-4 -3 -2 -1 0

TE D

Nulliparous (N) Heifers

AC C

EP

Primiparous (P) & Multiparous (M) Cows

Pre-Breeding Weeks Breeding Period

ACCEPTED MANUSCRIPT

Figure 2

Schuermann et al

Primiparous (P)

M AN U

SC

Group: P<0.001 Week: P<0.001

25 2

3

4

EP

1

TE D

37.5

AC C

Milk Yield (Kg/d)

50

RI PT

Multiparous (M)

5

6 7 8 Weeks Post-Calving

Breeding Period 9

10

11

12

ACCEPTED MANUSCRIPT

Figure 3

Schuermann et al Primiparous (P)

A

Multiparous (M)

RI PT

2.00

SC

0.00

0.50

3 Weeks Relative to Calving

Breeding Period 7

1 week prior to planned time of breeding

EP

1.00

AC C

B

0

TE D

-3

M AN U

1.00

BHBA (mmol/L)

BHBA (mmol/L)

Group: P<0.05 Week: P<0.001

12

b

a,b

a

0.00

Nulliparous (N)

Primiparous (P)

Multiparous (M)

ACCEPTED MANUSCRIPT

Figure 4

Cows (-3 to 12 weeks post-calving) Week: P<0.001

3.00

RI PT

Glucose (mmol/L)

A 4.50

Schuermann et al

P vs. N: P<0.05 M vs. N: P<0.05

40

M AN U

Cows (-3 to 12 weeks post-calving) Week: P<0.01

TE D

C

80

SC

1.50

Total Bile Acids (µmol/L)

B

0

EP

30

P vs. N: P<0.05 M vs. N: P<0.05

15

AC C

TG (mg/dl)

Cows (-3 to 12 weeks post-calving) Week: P<0.01

Group*Week: P<0.05 M vs. N: P<0.05 M vs. P: P<0.05

0 -3

-2

-1

0

1

2

3

4

5

6

7

8

9

10

11

12

Weeks Relative to Calving Pre-Breeding Weeks Nulliparous (N)

Primiparous (P)

Multiparous (M)

Breeding Period

ACCEPTED MANUSCRIPT

Figure 5

Cows (-3 to 12 weeks post-calving) Group*Week: P<0.05 WK: P<0.01

130

RI PT

B

260

M vs. N: P<0.05 P vs. N: P<0.05 0

SC

Tota-Cholesterol (mg/dL)

A

Cows (-3 to 12 weeks post-calving) Week: P<0.01

M AN U

C-HDL (mg/dL)

60

C

30

Group*Week: P<0.001 M vs. N: P<0.05 P vs. N P<0.05

0

C-VLDL (mg/dL)

TE D

Cows (-3 to 12 weeks post-calving) Week: P<0.01

EP

110

0 7

M vs. N: P<0.05; P vs. N: P<0.05

AC C

C-LDL (mg/dL)

220

D

Schuermann et al

Cows (-3 to 12 weeks post-calving) Week: P<0.01

3.5

M vs. N: P<0.05 M vs. P: P<0.05 0

-3

-2

-1

0

1

2

3

4

5

6

7

8

Weeks Relative to Calving Nulliparous (N)

Primiparous (P)

Multiparous (M)

9

10

11

12

Pre-Breeding Weeks Breeding Period

ACCEPTED MANUSCRIPT

Figure 6

Schuermann et al

RI PT

A

SC

1

M AN U

GSH (µmol/L)

1.5

TE D

0.5

B0.3

EP

0.2

AC C

FRAP (mmol/L)

Cows (-3 to 12 weeks post-calving) Week: P<0.01

P vs. N: P<0.05 M vs. N: P<0.05

0.1 -3

-2

-1

0

1

2

3

4

5

6

7

8

9

10

11

12

Weeks Relative to Calving Pre-Breeding Weeks Nulliparous (N)

Primiparous (P)

Multiparous (M)

Breeding Period

ACCEPTED MANUSCRIPT

Schuermann et al

M AN U

SC

RI PT

Table 1

Group

Group P-value

Nulliparous Heifers

Primiparous Cows

Multiparous Cows

# of animals1

13

5

7

Pregnancy rates (%) Calving interval (days)

75.99 ± 6.84A

44.24 ± 11.05B

48.16 ± 9.33B

0.02

N/A

414.45 ± 26.76

433.39 ± 31.74

0.66

TE D

Item

AC C

EP

All results are expressed as mean ± SEM 1Only animals that remained in the herd and were pregnant by breeding were included in the table (one cow was removed due to illness in mid-lactation and three animals were pregnant by embryo transfer) Values within a row with different superscript letters have a tendency to differ P<0.1 (A&B)

ACCEPTED MANUSCRIPT

Highlights: Different metabolic profiles during the pre-breeding period in heifers and cows

•

Higher total, HDL and LDL cholesterol, bile acids and FRAP in cows than heifers

•

Lower levels of glucose and VLDL in lactating cows than heifers

AC C

EP

TE D

M AN U

SC

RI PT

•