Heifer nutrition during early- and mid-pregnancy alters fetal growth trajectory and birth weight

Animal Reproduction Science 117 (2010) 1–10

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Heifer nutrition during early- and mid-pregnancy alters fetal growth trajectory and birth weight G.C. Micke a , T.M. Sullivan a , R.J. Soares Magalhaes b , P.J. Rolls c , S.T. Norman d , V.E.A. Perry a,∗ a

School of Veterinary Science, The University of Queensland, St Lucia, QLD 4072, Australia School of Population Health, Public Health Building - Room 401A, The University of Queensland, Herston Road, QLD 4006, Australia c QLD Department of Primary Industries, Tick Fever Centre, Wacol, QLD 4076, Australia d Graham Centre for Agricultural Research, School of Animal and Veterinary Sciences, Charles Sturt University, Wagga Wagga, New South Wales 2650, Australia b

a r t i c l e

i n f o

Article history: Received 22 December 2008 Received in revised form 9 March 2009 Accepted 25 March 2009 Available online 1 April 2009 Keywords: Beef heifers Cattle pregnancy Development Fetal biometry Nutrition

a b s t r a c t Maternal nutrient intake during gestation can alter fetal growth. Whilst this has been studied extensively in the sheep, less is known about effects in the bovine. Composite-breed beef heifers were allocated to either a high (H/− = 76 MJ metabolisable energy (ME) and 1.4 kg crude protein (CP)) or low (L/− = 62 MJ ME and 0.4 kg CP daily) nutritional treatment at artificial insemination. Half of each nutritional group changed to an opposite nutritional group at the end of the first trimester (−/H = 82 MJ ME and 1.4 kg CP; −/L = 62 MJ ME and 0.4 kg CP daily), resulting in 4 treatment groups: HH (n = 16); HL (n = 19); LH (n = 17); LL (n = 19). During the third trimester all heifers were fed the same diets. Fetuses were measured at 4-weekly intervals beginning at day 39 of gestation. Calves were also measured at birth for physical body variables. Low maternal nutrient intake was associated with decreased crown-rump length at day 39 (P < 0.01) and increased thoracic diameter at day 95 (P < 0.01). Umbilical cord diameter was reduced in L/− fetuses in the first trimester (P < 0.05) but was greater in −/L fetuses in the second trimester compared to their respective H counterparts (P < 0.05). Calf birth weight was decreased in association with −/L maternal diets (P < 0.05). In conclusion, fetal development of cattle may be affected by maternal nutrition as early as day 39 of gestation. This may be followed by either compensatory fetal growth, or alternatively, preferential fetal tissue growth that is dependant upon maternal nutrition. Clearly, calf birth weight may be altered by maternal nutrition during mid-gestation. © 2009 Elsevier B.V. All rights reserved.

1. Introduction Maternal nutrient intake during gestation in various species has been shown to affect the development of both the placenta and the fetus (Redmer et al., 2004), resulting in long-term programming effects on postnatal growth and metabolism of the offspring (McMillen et al., 2001). The sheep has been widely used as a model for human fetal development and hence a vast amount of research has

∗ Corresponding author. Tel.: +61 7 4671 2417; fax: +61 7 4671 2781. E-mail address: [email protected] (V.E.A. Perry). 0378-4320/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.anireprosci.2009.03.010

occurred in this species, however there is less research on fetal development and its consequences in the bovine. Of the studies undertaken in cattle, both maternal diet and body condition during gestation have been shown to alter fetal growth trajectory, resulting in altered calf birth weight (Bellows and Short, 1978; Boyd et al., 1987; Café et al., 2006; Freetly et al., 2000; Tudor, 1972; Warrington et al., 1988). However, the results between studies have not been consistent. This may reflect differences in study design as nutritional regimens did not commence at the same stage of gestation as well as differing in the nutritional regimen imposed. In addition, the majority of bovine studies on the effects of maternal nutrition on fetal growth have not

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assessed relative tissue distribution. Redistribution of tissue mass may be important in relation to dystocia (Anthony et al., 1986; Laster, 1974) and as a potential indicator of long-term effects of the in utero environment on postnatal metabolism, endocrine function and growth characteristics including body composition (McMillen et al., 2001). Previous studies in other species, including ruminants, have demonstrated that fetal growth restriction due to an adverse intrauterine environment can be characterised by the preservation of vital organs such as the brain, despite an overall decrease in fetal body size (Greenwood et al., 1999a; Pond et al., 1992; Simmons et al., 1993). The aim of this experiment, therefore, was to examine the effects of heifer nutrient intake during earlyand mid-gestation on fetal development, calf weight and body morphology at birth. We hypothesised that increased maternal nutrition during early- and/or mid-gestation would result in increased fetal growth and calf birth weight. 2. Materials and methods All procedures were performed with the prior approval of The University of Queensland Animal Ethics Committee, approval number SVS/716/06/MLA/AACO. 2.1. Experimental animals One hundred and twenty 2-year-old heifers were obtained from Australian Agricultural Company’s herd at Springsure, Queensland (24◦ 12 S, 148◦ 09 E) and relocated to Goondiwindi, Queensland (28◦ 52 S, 150◦ 33 E). Heifers were a Bos taurus × Bos indicus composite of either 1/2 Senepol × 1/4 Brahman × 1/4 Charolais (CBX, n = 85) or 1/2 Senepol × 1/4 Brahman × 1/8 Charolais × 1/8 Red Angus (BeefX, n = 35). They were selected based on weight, temperament and functional reproductive tracts. All heifers were vaccinated on two occasions 4 weeks apart against viral and bacterial diseases (Websters Bovine Ephemeral Fever Vaccine (Living)® , Fort Dodge Australia Pty Limited, NSW; Pestigard Vaccine® , Pfizer Animal Health, NSW; Ultravac Botulinum Vaccine® , Pfizer Animal Health, West Ryde, NSW, Ultravac 7 in 1® , Pfizer Animal Health, West Ryde, NSW) and treated for cattle tick (Tixafly® , Coopers Animal Health, Baulkham Hills, NSW) prior to shipment from Springsure. Heifers were run as one mob during a 45-day acclimatisation period before undergoing a 10-day progesterone based estrous synchronization program. Intravaginal progesterone releasing devices were inserted on day −12 (progesterone 1.9 g, EAZI-BREEDTM CIDR® cattle device, Pfizer Animal Health, Australia) and heifers treated intramuscularly with 1 mg oestradiol benzoate (Ciderol® , Genetics Australia, Bacchus Marsh, Australia). On day −5 heifers were treated with 25 mg dinoprost trometamol intramuscularly (Lutalyse® , Pfizer Animal Health, Australia). Intravaginal devices were removed on day −2 and heifers were artificially inseminated (AI) with frozen semen from one Senepol bull on day 0 and again on day 1 for any heifers still showing signs of estrus (n = 6). Time between the first heifer AI on day 0 until the last heifer AI on day 1 was 24 h. Two heifers were removed due to

temperament-related problems, resulting in 118 heifers at commencement of the study. Pregnancy was positively diagnosed in 77 of the 118 heifers on day 39 via transrectal palpation with the aid of a 5 MHz linear rectal probe attached to a real time ultrasound scanner (model Aloka-500® , Aloka Inc., Tokyo, Japan). During the study, six spontaneous abortions occurred resulting in a total of 71 heifers that completed the study and gave birth. At the time of AI the heifer age range was 21.6–24.6 months. 2.2. Experimental design The study was a two-by-two factorial design. Heifers were divided into treatment groups on the first day of AI according to stratification by weight within each composite genotype. Half of each nutritional treatment group changed to the alternative nutritional treatment at the end of the first trimester of gestation (day 93) giving rise to four treatment groups: high/high (HH; n = 16), high/low (HL; n = 19), low/high (LH; n = 17), low/low (LL; n = 19). At the end of the second trimester of gestation (day 180) all heifers were run as one treatment group until parturition (see Fig. 1). 2.3. Nutritional treatments Diets were formulated using bambatsi hay (Panicum coloratum), barley straw (Hordeum spp.), cracked sorghum grain (Sorghum spp.), cotton seed meal (Gossypium spp.), ground limestone and a premix containing vitamins and minerals. The rations fed and their respective energy and protein contents are outlined in Table 1. Values for crude protein and metabolisable energy were measured by both biochemical and near infra red spectroscopy methods (CASCO Agritech, Toowoomba, Qld, Australia). Ground limestone was added to the rations to keep the calcium:phosphorous ratio near 2:1. The premix contained 17 g calcium, 9 g phosphorous, 2.91 g magnesium, 5 g sulphur, 27,200 IU vitamin A, 60 mg vitamin E, 70 mg iron, 150 mg zinc, 100 mg manganese, 55 mg copper, 0.5 mg selenium, 3.4 mg cobalt and 4.2 mg iodine per 100 g. Allocation of both the roughage and concentrate components of the rations was calculated on an average intake per head per day basis. The roughage component was fed on a group basis whilst the concentrate portion was fed individually to heifers daily, whilst they were in stanchions. The average liveweight of heifers at AI and at the end of each trimester of gestation is detailed in Table 2. 2.4. Fetal calf measurements Single fetuses (n = 71) were measured using trans-rectal ultrasonography on eight separate occasions at 4-weekly intervals between days 39 and 235 of gestation, resulting in a total of 568 measurement events. All images were recorded on VHS-video and digitalised. Measurements of the fetus were taken at the time of ultrasound and from digital images that were later reviewed by a single person. All fetal body measurements were in centimeters. Crown-rump length (CRL) was measured from a lateral view of the fetus from the tip of the nose to the base of the tail. Biparietal diameter (BPD) was measured from a dorso-

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Fig. 1. Experimental design illustrating nutritional treatment groups and number of heifers in each feed regime from artificial insemination until the end of gestation.

ventral view of the cranium. It was measured perpendicular to the sagital crest as the widest span between the most lateral parts of the parietal bone. Crown-nose length (CNL) was measured from a lateral view of the cranium as the distance from the planum nasale to the intercornual protuberance. Eye socket diameter was measured from a lateral view of the cranium in both the vertical (OV) and horizontal planes (OH). Abdominal diameter (AD) was measured from a transverse image at the point of insertion of the umbilical cord. Thoracic diameter (TD) was measured from a lateral image at the level of the twelfth rib. Umbilical cord diameter (UD) was measured at the point of insertion of the umbilical cord to the fetus. Limb cross-sectional diameter was measured on both the fore- and hind-limbs at the levels of the coronet and mid-cannon. Measurements were across a transverse plane as determined by manual palpation or concurrent visualisation of the hoof. The fetal body measurements were grouped into head (BPD, CNL, OH and OV), trunk (CRL, AD, TD and UD) and limb measurements.

2.5. Newborn calf measurements At calving, heifers were monitored individually and assistance provided where necessary. Calves were collected for measurement within 15 min of birth, prior to standing or sucking. The whole body and trunk measures that were recorded were calf birth weight (BW), CRL, and abdominal circumference (AC) at the level of the umbilical cord. Cranial measures recorded were BPD and CNL. Limb measures made on both fore- and hind-limbs were metacarpal and metatarsal length and diameter at their mid-points in both the mediolateral and craniocaudal planes, in addition to coronet diameter in both planes. Limb diameters and BPD were measured using sliding calipers and other measures were obtained using a flexible tape-measure. Trunk and cranial measures were measured to the nearest 0.5 cm, limb measures to the nearest 0.1 cm and birth weight to the nearest 0.1 kg. Gender was also recorded at birth.

Table 1 Components and nutritional content of diets fed to heifers during gestation. Ration as fed

Sorghum (kg) Cotton seed meal (kg) Bambatsi hay (kg) Barley straw (kg) Ground limestone (g) Premix (g) Dry matter (kg) Total energy (MJ ME) % of energy requirementa Total crude protein (kg) % of protein requirementa

Trimester 1

Trimester 2

Trimester 3

H

L

H

L

All

0.65 2.45 7.88 0.00 70 70 9.95 76.29 243 1.37 250

1.56 0.00 2.73 5.14 20 60 8.64 62.54 199 0.41 75

1.00 2.50 5.79 2.20 120 100 10.51 82.43 229 1.40 228

1.20 0.00 0.00 7.59 60 100 8.10 63.14 176 0.38 63

1.13 1.08 0.86 7.14 80 100 9.39 71.45 149 1.06 135

MJ ME = megajoules of metabolisable energy; H = high treatment group; L = low treatment group. a Dietary requirements were calculated using Nutrient Requirements of Beef Cattle Table Generator Software (1996), National Research Council (NRC), Washington. Input values used were: pregnant Brangus replacement heifer aged 23 months at breeding with a mature weight of 475 kg and a calf birth weight of 32 kg.

G.C. Micke et al. / Animal Reproduction Science 117 (2010) 1–10 179 399.2 ± 25.7

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2.6. Heifer measurements

−7 351.2 ± 25.0 −7 349.1 ± 17.2

95 383.9 ± 18.6

2.7. Statistical analyses

Values are unadjusted mean ± S.D. HH = high/high; HL = high/low; LH = low/high; LL = low/low.

−7 352.9 ± 22.9 95 428.7 ± 27.3

179 489.0 ± 27.9 −7 362.4 ± 26.5 Day of gestation Liveweight (kg)

95 419.2 ± 26.9

179 434.5 ± 23.5

LH (n = 17) HL (n = 19) HH (n = 16) Treatment group

Table 2 Liveweight of heifers by treatment group prior to artificial insemination and at the end of the first and second trimesters of pregnancy.

179 455.5 ± 23.1

LL (n = 19)

95 383.7 ± 26.6

Heifer liveweight was assessed at approximately monthly intervals throughout gestation and blood samples were collected at days −14, 28, 82, 179 and 271 day postAI to confirm the effect of maternal diets via blood urea nitrogen (BUN) measurement (Sullivan, T.M., unpublished data; see Fig. 2). Plasma BUN concentrations were measured in singlicate by enzymatic colorimetric analysis on a Hitachi autoanalyser, using commercially available kits (UREA/BUN, Roche Diagnostic Systems, Germany). Plasma samples were measured in 13 assay runs. The inter-assay CVs for a heifer QC plasma sample containing 2.64 mmol/L BUN was 3%.

All statistical analyses were performed using the statistical software, Stata SE Version 9.2 (Stata Corporation, College Station, TX). The analyses were undertaken in two stages: associations with fetal measurements during pregnancy followed by associations with calf measurements at birth. At each stage, the statistical analyses were performed in two steps using individual animal (calf ID) as the unit of analysis. Only animals that remained at calving were included in the analyses of fetal measurements. Firstly, all fixed effects were screened for univariate unconditional associations with the outcome. Fixed effects assessed at specific times points during gestation included fetal gender and dam nutritional treatment group. Gestation length was also included in models developed for measures obtained at birth and those developed to assess fetal growth over time. A univariate maximum-likelihood random-effect linear regression model, based on a liberal P-value of 0.20, using the likelihood-ratio test for significance with calf ID as a random effect was used in the analyses of unconditional associations with fetal measurements over time. The latter was used to take into account repeated measures on the same fetuses during the study. For univariate analysis of unconditional associations with calf measurements at birth and fetal measures at individual time points during gestation, a linear regression model based on a liberal P-value of 0.20 using the likelihood-ratio test for significance was used. Secondly, all explanatory variables significant in the screening phase were considered for inclusion in multivariate models through a backward step-wise variable selection process. For the effect of explanatory variables on successive measurements over time, a multivariate maximum likelihood random-effect model was used, whilst for measurements at specific time points, a multivariate linear regression model was used. For models developed for outcomes at birth, the criterion for removal of explanatory variables was based on statistical considerations using a likelihood-ratio statistic with a significance level of P > 0.05. In the multivariate models developed for repeated fetal measurements during pregnancy or from conception to birth, the criterion for selection or retention of each variable in the final model was based on the Akaike’s information criteria (AIC) statistic.

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Fig. 2. Pattern of maternal plasma blood urea nitrogen (BUN) by treatment group prior to and during gestation (mean ± S.D.).

Screening for the presence of confounding variables in the final model was performed by step-wise removal of variables which at some stage were not significant (P > 0.05), noting the impact on the coefficients of the remaining variables in the model. If a coefficient of another variable changed by more than 25%, the eliminated variable was likely to be a confounder and was reinstated in the model. Biologically meaningful first-order interaction terms were also tested for statistical significance. The goodness-of-fit of the final multivariable models was assessed and model fundamental assumptions were evaluated by analysis of standardised residuals, and testing for the presence of heteroscedasticity. The presence of influential and outlier observations was also assessed. 3. Results 3.1. Fetal measurement ability The ability to measure the fetus declined by day 179 to approximately half that of days 68–150 due to the position of the fetus within the abdominal cavity. By day 234, the frequency of successful fetal measurement increased (see Fig. 3). Head and limb measures were obtained throughout gestation, however trunk measures were not obtained beyond day 179. Fetal head measures were the most frequently obtained with at least one type of head measurement made on 60.4% (300/497) of all scanning events. Trunk and limb measures were similar in their frequency of success with at least one type of trunk or limb measure being obtained at 35.6% (177/497) and 36.8% (183/497) of all scanning events respectively. 3.2. First trimester fetal growth Between days 39 and 95 of gestation sufficient data for statistical analysis was available on five head (OH, OV, CNL, CRL, and BPD), three trunk (AD, TD and UD) and two limb (forelimb mid-cannon and coronet diameter) variables. At

day 39 of gestation, L fetuses had a significantly shorter CRL than H fetuses (P < 0.01, Table 3). At day 68 of gestation CNL of L fetuses had a tendency to be significantly longer that H fetuses (P < 0.10). At day 95 of gestation L fetuses had a significantly wider TD than H fetuses (P < 0.01) but had significantly smaller UD than H fetuses (P < 0.05). There were no other statistically significant treatment group differences for fetal measurements obtained during the first trimester of gestation. 3.3. Second and third trimester fetal growth Between days 123 and 234 of gestation sufficient data for statistical analysis was available for OH, OV, BPD and UD. At day 123 of gestation HL and LL fetuses had a significantly greater UD than HH and LH fetuses (P < 0.05, Table 4). There was no interaction between first and second trimester treatment group effects. At days 123 (P < 0.01) and 235 (P < 0.10) female fetuses had smaller OH than males (Table 5). There were no other statistically significant treatment group differences for fetal measurements obtained during the second and third trimesters of gestation. 3.4. Fetal growth over time Eye socket diameter in the horizontal plane was the only variable that had a sufficient number of consecutive measurements between days 68 and 234 of gestation to allow assessment of the effect of dam treatment group on head growth over time. There was no significant effect of first or second trimester treatment group, their interaction term or gestation length on this measure, however female fetuses had a significantly lower OH than males (Coef.: −0.063; 95% CI −0.127, −0.001; P < 0.05). Foreleg mid-cannon bone diameter and crown-nose length had sufficient numbers of consecutive measurements between day 68 and birth to allow the assessment of treatment group on fetal skeletal and head development

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Fig. 3. Frequency of fetal body part measurement at each scanning event during gestation across all treatment groups. Table 3 Fetal body measurements that were affected by maternal nutrition during the first trimester of gestation. Stage of gestation

Maternal nutritional group

Day 39 CRL (cm) Day 68 CNL (cm) Day 95 UD (cm) TD (cm)

P-value

n

High

n

Low

33

1.79 ± 0.05

33

1.55 ± 0.05

<0.01

23

3.06 ± 0.06

24

3.23 ± 0.06

<0.10

20 12

1.19 ± 0.03 4.04 ± 0.08

26 24

1.11 ± 0.02 4.35 ± 0.06

<0.05 <0.01

Values are predicted means ± S.E.M. from models described in Section 2.7. CRL = crown-rump length; CNL = crown-nose length; UD = umbilical cord diameter; TD = thoracic diameter. Table 4 Fetal body measurements that were affected by maternal nutrition during the second trimester of gestation. Stage of gestation

Maternal nutritional group

Day 123 UD (cm)

n 6

HH 1.72 ± 0.07

P-value n 10

HL 1.93 ± 0.5

n 10

LH 1.78 ± 0.05

n 10

LL 1.86 ± 0.05

<0.05a

Values are predicted means ± S.E.M. from models described in Section 2.7. HH = high/high; HL = high/low; LH = low/high; LL = low/low; UD = umbilical cord diameter. a −/H vs −/L.

over time. Univariate analysis indicated that progeny in the low protein treatment group in the second trimester of gestation had smaller foreleg cannon bone diameters from day 68 to birth than their high protein treatment group counterparts (Coef.: −0.584; 95% CI −0.858, −0.310; P < 0.01). This effect was not significant in a multivariate model that

also contained fetal gender. There was no significant effect of gestation length on foreleg mid-cannon bone diameter over time. There were no significant effects of either first or second trimester treatment group, their interaction term or gestation length on measures of crown-nose length over this time period.

Table 5 Means of fetal and calf body measurements during gestation and at birth that were affected by gender.

OH at day 123 (cm) OH at day 235 (cm) Birthweight (kg) CNL at birth (cm) CRL:BW

n

Male

25 22 32 33 32

1.64 2.14 33.37 20.48 2.55

± ± ± ± ±

0.02 0.04 0.64 0.26 0.04

n

Female

27 19 38 38 38

1.53 2.04 30.43 19.84 2.71

± ± ± ± ±

P-value 0.02 0.04 0.59 0.24 0.04

<0.01 <0.10 <0.01 <0.10 <0.01

Values are predicted means ± S.E.M. from models described in Section 2.7. OH = horizontal eye socket diameter; CNL = crown-nose length; CRL:BW = crownrump length:birthweight.

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Table 6 Calf body measurements at birth that were affected by maternal nutrition during gestation. Maternal nutritional group

BW (kg) AC (cm)

P-value

n

HH

n

HL

n

LH

n

LL

16 16

32.9 ± 0.9 74.4 ± 0.9

18 19

31.1 ± 0.9 75.8 ± 0.8

17 17

33.4 ± 0.9 77.4 ± 0.9

19 19

29.9 ± 0.8 74.4 ± 0.8

<0.05a , b , c <0.10d

Values are predicted means ± S.E.M. from models described in Section 2.7. HH = high/high; HL = high/low; LH = low/high; LL = low/low; BW = birth weight; AC = abdominal circumference. a −/H vs −/L. b HH vs LL. c LH vs LL. d LH vs HH.

3.5. Calf biometry at birth Low dam nutrition during the second trimester decreased BW (P < 0.05, Table 6). There was no interaction between first and second trimester treatment group effects. Female calves had significantly lower BW than male calves (P < 0.01, Table 5). Increased gestation length tended to be associated with an increased BW (Coef.: 0.234; 95% CI 0.019, 0.448; P = 0.053). Abdominal circumference tended to be greater in LH (P < 0.10, Table 6) than in HH calves. There was no interaction between first and second trimester treatment group effects. Both AC (Coef.: 0.24; 95% CI 0.0025, 0.487; P < 0.05) and CRL (Coef: 0.73; 95% CI 0.41, 1.05; P < 0.01) at birth were associated positively with length of gestation. There was a tendency for CNL to be shorter in female than male calves (P < 0.10, Table 5). There were no other significant effects on the other calf physical body variables at birth. 3.6. Indices of disproportionate growth The ratio between the BPD and the AD (BPD:AD) was used on days 68 and 95 of gestation to investigate the effect of maternal diet in the first trimester on proportional fetal growth. At days 68 and 95 of gestation there were no significant differences in BPD:AD due to maternal nutrition or fetal gender. At birth, there were no effects of first or second trimester treatment group, nor their interaction term, on either CRL:BW or CNL:CRL. Female calves had significantly higher CRL:BW ratios than male calves (P < 0.01, Table 5) and calves of longer gestation lengths had increased CRL:CNL (Coef.: 0.03; 95% CI 0.001, 0.052; P < 0.01). 3.7. Relationships between fetal measures during earlyand late-gestation and calf birth weight Univariate screening of the relationships between fetal measures of optic diameter and foreleg mid-cannon diameter obtained on days 68 and 95 with those obtained on days 207 and 234, revealed that foreleg mid-cannon diameter on day 95 was closely associated with its subsequent diameter on day 207 (Coef.: 0.13; 95% CI 0.09, 0.18; P < 0.01). There were no other significant associations between variable measurements obtained during early- and late-gestation. Univariate screening of fetal measurements obtained during early- and mid-gestation and calf birth weight

revealed that increased calf birth weight was associated with increased OV on day 95 (Coef.: 0.009; 95% CI: 0.001, 0.02; P < 0.05) and 123 (Coef.: 0.02; 95% CI 0.008, 0.02; P < 0.01). Calf birth weight tended to be increased in association with increased abdominal cross-section on day 65 (Coef.: 0.01; 95% CI −0.001, 0.02; P < 0.10). Similarly on day 123, increased biparietal diameter (Coef.: 0.03; 95% CI −0.003, 0.06; P < 0.10) and hind-limb mid-cannon diameter (Coef.: 0.03; 95% CI −0.004, 0.06, P < 0.10) tended to be associated with increased calf birth weight. There was no significant association between fetal-crown-rump length at day 39 and calf birth weight, nor any of the other fetal variable measurements obtained during early- and midgestation and calf birth weight.

4. Discussion The current study demonstrates that fetal growth in the bovine was enhanced by high maternal nutrient intake during early gestation. This enhancement appeared to be followed by a slowing in the growth trajectory of some fetal body parts that remained evident until early- to midgestation. In addition, differences in fetal growth trajectory due to gender differences were detectable at day 123 of gestation using trans-rectal ultrasonography. The present study also demonstrates that structures associated with maternal nutrient transfer to the fetus i.e., the size of the umbilical cord, may also be affected by altered maternal nutrient intake during the first half of gestation. These early perturbations to fetal growth culminated in an increased calf birth weight in association with increased maternal dietary protein during mid-gestation. Both maternal under- and over-nutrition during earlyand mid-gestation have been demonstrated to result in perturbations to fetal development that are proceeded by alterations in the post-natal growth pathway, metabolism and body composition of the offspring (Barker, 1992; Greenwood et al., 1998; McMillen et al., 2001; Redmer et al., 2004). These relationships however have not been explored in cattle. In south-western Queensland, Australia, rainfall occurs predominantly during the summer period and crude protein content of pasture during this time ranges between 12 and 15%. During the winter period however, the pasture is predominantly dry grass that has a very low crude protein content of approximately 5%. In some years, however winter rainfall occurs and medicago sp. predominate, producing pastures with an equivalent or higher crude protein content

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compared to summer grass pastures. The diets used in the current study, therefore, were designed to reflect the nutritive value of pastures in this grazing environment. The aim being to investigate the effects that these varying seasonal conditions may have on fetal development and furthermore may pre-empt long-term effects of maternal nutrient intake during gestation on the post-natal growth pathway, metabolic state and carcass composition of the offspring. In this study it was not possible to create an isocaloric diet because of the welfare considerations, feed stuffs available and the interaction between protein and energy in the ruminant diet. Thus, the observed fetal growth responses to maternal nutrient intake could be due to either the over-supply of maternal energy or protein or both, or the under-supply of maternal protein. The high group protein intake was between 228 and 250% of requirements whilst the low group protein intake was between 63 and 75% of requirements, a between treatment group difference of 3.5 times. Any effects of nutrient intake is therefore most likely to be associated with protein rather than energy or total dry matter intake as there was a difference in between group energy intake and dry matter intake between 1.15 and 1.3 times, with highs receiving 229–243% of energy requirements and lows 176–199%. It is plausible to suggest that the high energy intake and assumed increase in rumen volatile fatty acid production may have in part compensated for the reduced crude protein supply in the low treatment group heifers. Similarly, the different amino acid profile obtained from the cotton seed meal in the high diets cannot be ignored as a potentially significant contributor to any nutritional treatment group effects observed. Previous studies in sheep have demonstrated asynchronous organ and tissue development when fetuses are subjected to a growth restrictive intrauterine environment (Gatford et al., 2002; Heasman et al., 1999; McCrabb et al., 1992; Osgerby et al., 2002). It has been shown that the fetal response to growth restrictive environmental conditions is to partition nutrients towards the development of organs essential to life, such as the brain, at the expense of less vital organs and tissues (Owens et al., 1989; Quigley et al., 2008; Redmer et al., 2004). The repartitioning of nutrients to support fetal development has been demonstrated to have long-term effects on post-natal growth and metabolism, apparent as an increased propensity to develop insulin resistance, high blood pressure, more rapid weight gain and increased adiposity (Gluckman and Hanson, 2004; McMillen et al., 2001). Understanding the origins of these potential metabolic outcomes in the bovine may prove valuable to the cattle industry in the manipulation and management of adiposity and thus carcass composition and meat quality in the bovine. Therefore, in the current study; multiple measures of the head were made as an indirect measure of brain growth, trunk measures to assess non-preferential tissue growth and appendicular skeleton measures to assess the implications of fetal development on post-natal carcass composition, as suggested by Osgerby et al. (2002). The assessment of fetal growth over time using consecutive measures proved difficult due to the inability to measure the same body part on the same fetus at each scanning event using trans-rectal ultrasonography.

Trans-abdominal ultrasound and/or fetal recovery and measurement may have yielded more definitive measurements (Greenwood et al., 1999b, 2002). However, this is much more difficult in the bovine than the ovine due to the size of the animal and the distance of the bovine fetus from the abdominal wall. It is interesting to note that female fetuses had reduced horizontal eye socket diameters (the most consistently measured head variable) than males during gestation. Whilst it is expected that female fetuses would be smaller than male fetuses due to the well established relationship between gender and birth weight (Holland and Odde, 1992), an effect of gender on fetal measures has not been previously reported, to our knowledge, in the bovine. Previous ultrasonic studies of bovine fetal development have measured crown-rump length growth during early gestation at between 1.1 and 1.4 mm per day (Kahn, 1989; Pierson and Ginther, 1984) and have been able to ascertain differential growth rates due to the developmental environment (Bertolini et al., 2002). In the present study crown-rump length was increased by 2.4 mm at day 39 in fetuses whose dams were in the high nutrition treatment group. Despite the increase being small, it is representative of approximately 2 days of fetal growth based on previous reports of fetal growth rate at this stage of gestation. In the human, smaller than expected crown-rump length in the first trimester of gestation is related to an increased risk of low birth weight at term (Smith, 2004). This relationship was not found in the current study. Interestingly, at day 28 of gestation maternal blood urea nitrogen (BUN) was also increased in heifers in the high treatment group. Increased maternal BUN may be indicative of increased amino acid transfer to the fetus and thus offers a potential physiological mechanism underlying the observed increase in crown-rump length. Alternatively, exposure to a high urea environment may have re-programmed fetal development. A similar effect has been observed in sheep fed diets high in urea where despite additional dietary urea being associated with an increased rate of early embryonic mortality, those embryos that did survive went on to exhibit an increased rate of metabolism and growth compared with those from non-urea fed ewes (McEvoy et al., 1997). In the bovine, high maternal protein intake has also been associated with detrimental affects on early embryonic survival however an increase in the rate of embryonic development has not been observed (Butler, 1998; Gath et al., 1999). To confirm that the increase in CRL associated with increased maternal nutrient intake observed in the present study was not due to measurement error or positioning of the fetus, repeated measurements at more frequent intervals during the very early stages of gestation would be required. There was a weak tendency for crown-nose length of low group fetuses to be 1.6 mm longer than high group fetuses at day 68 of gestation. Whilst this may be attributable to measurement error rather than being of biological significance, it is interesting to note that thoracic diameter was also significantly increased in low group fetuses at day 95. Similarly in sheep under-nourished throughout gestation, fetal thoracic girth at the end of the first trimester of gestation was greater in fetuses from nutrient restricted ewes

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(Quigley et al., 2008). The findings of the current study of increased thoracic diameter and crown-nose length in the restricted group during early gestation may simply reflect preferential distribution of available nutrients to specific areas of need in the low group fetuses. The relative decrease in growth of the high group fetuses however, may indicate that that the level of maternal protein intake resulted in a concentration of urea in the intrauterine environment that was deleterious to fetal growth. The significant association between umbilical cord diameter and maternal nutrition is of interest. Umbilical cord diameter was increased in association with low maternal nutrition early in the second trimester despite being decreased in association with low maternal nutrition during the first trimester. Blood flow volume is a product of blood flow velocity and vessel diameter (Laurin et al., 1987). Umbilical cord diameter may be an indirect measure of umbilical vessel diameter, hence blood flow volume, and thus nutrients reaching the fetus. Whilst umbilical cord diameter represents a very crude measure of fetal nutrient supply, the current findings support previous findings of the dynamic and adaptive nature of bovine placental vascularity in response to maternal nutrient restriction (Vonnahme et al., 2007; Zhu et al., 2007). Repeated measures at a single measurement event and consecutive measures at closer time intervals throughout gestation in addition to measurement of umbilical cord vessel diameters and investigation of placental–fetal blood flow dynamics, would all be required to confirm this nutritional effect. This study shows that low dam nutrition in the second trimester reduces calf birth weight which is consistent with previous reports in the bovine (Café et al., 2006; Freetly et al., 2000; Hodge and Rowan, 1970; Warrington et al., 1988). However, in these previous studies significant maternal liveweight loss during gestation was necessary to achieve this results, a distinct contrast to the current study, which may relate, at least in part, to the timing of the nutritional treatments. Consistent with previous studies of maternal nutrient manipulation during gestation in cattle (Laster, 1974), but contrary to those in humans (Kramer, 1987), sheep (Mellor et al., 1977; Robinson et al., 1979) and rats (Desai et al., 1996), the fetal ultrasonic and physical measures of the calves at birth revealed no significant effect of maternal nutrient intake on the relative distribution of tissue mass of the calf. The only consistent relationship between fetal measurements obtained during early gestation and calf birth weight was that they were all positively related. Furthermore, there were no consistent relationships between fetal measures obtained during early- and late-gestation. These findings do not preclude any longterm effects of the prenatal environment on post-natal growth and metabolism from occurring, as such effects have been observed in offspring that were subjected to a restrictive in utero environment but were of symmetrical body shape and normal size at birth (Gluckman and Hanson, 2004). Prior studies in mature ewes that have restricted maternal nutrient intake during early- to mid-gestation (McCrabb et al., 1992; Osgerby et al., 2002), throughout gestation

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(Quigley et al., 2008) or used uterine carunculectomy to restrict the intrauterine environment (Gatford et al., 2002), report that measures of thoracic girth, crown-rump length and organs including the gut, kidneys, heart and liver, are reduced near term or a birth. As measurements of the calf were only obtained on the live calf at birth, it is not known if there were any effects of maternal nutrition during gestation on individual organ weights at birth. Determination of individual tissue and organ weights was not possible as the calves formed part of a larger study concerning the postnatal consequences of the in utero environment on growth and development. In the current study there was a tendency for abdominal circumference to be increased in low/high calves. This raises the possibility of increased abdominal organ size or fat mass compared with calves from the other maternal nutritional regimes. If the increase in abdominal circumference was indeed attributable to an increased visceral fat mass, it may have contributed to an improved chance of calf survival due to greater energy stores available for thermoregulation in the perinatal period. Similarly, an increased hepatic volume may also have had a positive effect on perinatal thermoregulation via an increased hepatic storage volume of glycogen for use in perinatal energy metabolism. In conclusion, the current study shows that fetal growth in the bovine may be affected by maternal nutrition as early as day 39 of gestation. This may be followed soon after by compensatory fetal growth, or alternatively, preferential fetal tissue growth, that is dependent upon maternal nutrition. Calf birth weight is reduced by maternal under-nutrition of protein during the second trimester of gestation, despite the maintenance of positive maternal liveweight gain throughout this period. Acknowledgements The authors thank the financial and in kind support of Meat and Livestock Australia, Australian Agricultural Co., Western Australian Cattle Industry Compensation Fund, Ridley AgriProducts and Milne AgriGroup. The study sponsors had no role in the collection, analysis or interpretation of the data nor in the writing of this manuscript. References Anthony, R.V., Bellows, R.A., Short, R.E., Staigmiller, R.B., Kaltenbach, C.C., Dunn, T.G., 1986. Fetal growth of beef calves. I. Effect of prepartum dietary crude protein on birth weight, blood metabolites and steroid hormone concentrations. J. Anim. Sci. 62, 1363–1374. Barker, D.J.P., 1992. The effect of nutrition of the fetus and neonate on cardiovascular disease in adult life. Proc. Nutr. Soc. 51, 135–144. Bellows, R.A., Short, R.E., 1978. Effects of precalving feed level on birth weight, calving difficulty and subsequent fertility. J. Anim. Sci. 46, 1522–1528. Bertolini, M., Mason, J.B., Beam, S.W., Carneiro, G.F., Sween, M.L., Kominek, D.J., Moyer, A.L., Famula, T.R., Sainz, R.D., Anderson, G.B., 2002. Morphology and morphometry of in vivo- and in vitro-produced bovine concepti from early pregnancy to term in association with high birth weights. Theriogenology 58, 973–994. Boyd, G.W., Kiser, T.E., Lowrey, R.S., 1987. Effects of prepartum energy intake on steroids during late gestation and on cow and calf performance. J. Anim. Sci. 64, 1703–1709. Butler, W.R., 1998. Symposium: optimizing protein nutrition for reproduction and lactation. J. Dairy Sci. 81, 2533–2539. Café, L.M., Hennessy, D.W., Hearnshaw, H., Morris, S.T., Greenwood, P.L., 2006. Influences of nutrition during pregnancy and lactation on birth-

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