Fetal programming in 2-year-old calving heifers: peri-conception and first trimester protein restriction alters fetal growth in a gender-specific manner

Additional keywords: beef, embryo, fetus, nutrition, oocyte.

Protein is the major limiting nutrient in range beef cattle in Australia. It has previously been established that protein restriction in early gestation may affect post-natal reproduction and production traits in progeny (Sullivan et al. 2009, 2010; Micke et al. 2010a, 2011; Mossa et al. 2013). Further, studies have also shown effects of maternal diet upon fetal growth to 123 days post-conception (dpc) after dietary intervention 30–123dpc (Long et al. 2009) although this effect was no longer apparent at birth (Long et al. 2012).

Critical windows exist during oocyte, embryo and fetal development where decreased maternal dietary protein may reset early cell lineages (Wu et al. 2006). Maternal nutrition affects both the number of oocytes that ovulate and their quality (Ashworth et al. 2009), which has then been associated with differences in embryo survival (Borowczyk et al. 2006). The gender of an embryo has also been shown to affect the susceptibility of the embryo to altered nutrition before mating (Vinsky et al. 2006). Gender-specific differences in embryo DNA methylation pattern (Dobbs et al. 2013), expression of key developmental genes (Kwong et al. 2006), fetal and placental perfusion (Prior et al. 2013) post-natal carcass traits (Micke et al. 2011) have all been reported. Intriguingly, neonatal and fetal death in cattle is increased if the fetus is male, as with most mammalian species.

The aim of this study is to further evaluate the critical windows during development where dietary protein may alter fetal development. By examining the effects of protein diets similar to those experienced in range conditions in supplemented and non-supplemented breeders during the period from oocyte development in the ovary to the end of the first trimester we aim to establish the effects of such restriction.

This paper focuses on the effects of protein restriction in early life upon conceptus growth and development. We hypothesise that low maternal protein during the peri-conception period and the first trimester will reduce fetal growth in a gender dependent manner.

This project was approved by the University of South Australia IMVS Animal Ethics Committee (Approval number 18/11) on 11 March 2011.

Three-hundred and sixty primiparous 12-month-old Santa Gertrudis (Bos taurus × Bos indicus) heifers were selected based on weight and age from SK Kidman herds at Glengyle and Morney Plains, south-western Queensland. All heifers were vaccinated against viral and bacterial diseases on two occasions 4 weeks apart before transport. The heifers were transported to Sedan, South Australia to purpose-built, shaded feedlot pens, acclimatised to diet, and trained to individual stall feeding.

At 12 months of age, 60 days before artificial insemination (AI), the heifers were randomly assigned to two equal groups stratified by weight. Each heifer was individually fed a high [14% crude protein (CP)] or low (7% CP) protein diet consisting of pellet diet fed in stalls and straw (5% CP) ad libitum in pens. The digestible energy content of the diet was as similar as possible in the ruminant and supplemented with a vitamin and mineral commercial preparation (Table 1).

Table 1.  Ingredients and nutrient content of heifer rations for peri-conception and during the first trimester of gestation

Heifers underwent an 8-day progesterone-based oestrous synchronisation program. On Day 0, heifers were AI sequentially with frozen semen from one low birthweight Santa Gertrudis bull, selected on the basis of estimated breeding value.

At 23dpc each high (HPERI) or low (LPERI) group was again split into high (HPOST) or low (LPOST) protein groups yielding four treatment groups [high high (HH), high low (HL), low high (LH), low low LL)] in a 2 × 2 factorial design. Detailed composition of the heifer rations is given in Table 1.

Pregnancy was positively diagnosed in 124 heifers (HH = 33; HL = 37; LH = 30; LL = 28) at 36dpc via transrectal ultrasound and non-pregnant heifers were removed from the trial. At 60dpc fetuses were sexed via ultrasonography enabling equal numbers of sex fetuses to be excised at slaughter from each of the four treatment groups. From the end of the first trimester of gestation (98dpc), the remaining pregnant heifers were individually fed at the NRC recommended ration for weight containing 12% CP until parturition.

Fetuses were measured using transrectal ultrasound (Sonosite M-Turbo; Sonosite Inc., Bothell, Washington, DC, USA) at 36, 60 and 95dpc. Fetal sex was determined at 60dpc by rectal ultrasound. Measurements of the conceptus were taken at the time of ultrasonsography (Micke et al. 2010b). All fetal body measurements were in centimetres. Crown–rump length (CRL) was measured from a lateral view of the fetus from the tip of nose to the base of the tail. Biparietal diameter (BPD) was measured from a dorso-ventral view of the cranium perpendicular to the sagittal crest at the widest span between the most lateral parts of the parietal bone.

A subset of heifers (HH = 12; HL = 15; LH = 10; LL = 9) were killed in a commercial abattoir at 98dpc and used for human consumption. Tissues for examination were immediately collected from the uterus and fetus after removal from the kill floor. The fetus was excised from the uterus, cord blood collected, weighed, measured and then dissected. Sex of fetus was recorded during dissection. Measures of BPD and CRL were obtained from fetuses using sliding calipers. The remaining heifers were taken to term.

At calving, heifers (n = 64; 19 female, 45 male) were monitored individually and assistance provided where necessary. Calves were collected within 15 min of birth, before sucking, and sex, birthweight and CRL recorded.

Multifactorial ANOVA using STATA SE version 11 (Stata Corporation, College Station, TX, USA) was used to interpret the main effects of peri- and post-conception diet, sex and their interaction terms on ultrasound measures of embryo and fetal body dimensions from 36dpc, fetal measures at 98dpc and live calf measures at term. Individual calf was considered the experimental unit, with gestation length as a co-variate for birth measures only. Males and females were analysed together and independently. A probability of 5% (P < 0.05) was accepted as the level of significance and trends reported as P < 0.1. Data presented as mean ± standard errors of the mean.

At 36dpc high protein (14% CP) preconception diets increased fetal crown–rump length (CRL) compared with low protein (7% CP) (P < 0.05) (Fig. 1) and by 60dpc BPD tended to be increased by diet post-conception (P < 0.1) though the greater effect upon BPD was still peri-conception diet (P < 0.05) (Fig. 1). In males, BPD was increased in those fetuses whose mothers were fed HPERI (P < 0.05). For females, maternal nutrition during PERI or POST did not affect BPD at 60dpc (P > 0.1).

Fig. 1.  The effects of peri-conception dietary protein; high (14% CP), low (7% CP) upon fetal growth at 36 and 60 gd (days gestation). Values are unadjusted means ± s.e.m.

Initial weight of the heifers at acclimatisation was 350 kg. The average daily gain of heifers was 0.4 kg/day during the high and 0.2 kg/day during the low protein treatment periods (Fig. 2).

Fig. 2.  Liveweight over time of heifers in each dietary treatment group. HH = high protein peri-conception and first trimester, LL = low protein peri-conception and first trimester, HL = high protein peri-conception and low protein first trimester, LH = low protein peri-conception and high protein first trimester.

In measures of the excised fetus at 98dpc, fetal weight was heavier (P < 0.01) in those whose mothers were fed HPOST diet (HH + LH = 326.3 ± 6.7 g) compared to their low counterparts (HL + LL = 301.2 ± 7.6 g). Male fetuses were heavier than female fetuses (P < 0.001). Fetal CRL at 98dpc was increased by HPERI diet compared to LPERI (P = < 0.05) and by this stage of development also tended to be increased by HPOST diet compared to LPOST (P < 0.1). Fetal BPD tended to be increased by HPERI diet (P < 0.1). In males, BPD tended to be increased in those fetuses whose mothers were fed the HPERI diet compared to LPERI (P < 0.1). For females, maternal nutrition during PERI or POST did not affect BPD at 98dpc (P > 0.1).

There was no effect of dietary treatment on birthweight, CRL or head measures (all P > 0.1). Gestation length did however affect birthweight (P < 0.05). Male calves were not larger or heavier at birth than female calves (P > 0.1).

The present study demonstrates a significant difference in bovine conceptus size as early as 36 days post-conception in response to maternal dietary protein restriction during peri-conception and first trimester. Further, this effect was gender specific there being a greater effect of early dietary intervention on measures of male fetal growth at least until the end of the first trimester.

The 360 heifers were individually fed for a total of 14 months (including acclimatisation) and inseminated via fixed time AI over 1.5 days. Thus, the nutritional regimens were instigated at exactly the same time points in gestation and were controlled with heifers being fed individually from 60 days before AI and throughout gestation until term. This is similar to previous fetal programming studies reported by (Sullivan et al. 2009; Micke et al. 2010a) but not others (Cafe et al. 2006; Greenwood and Cafe 2007; Hickson et al. 2008; Long et al. 2009, 2012). Furthermore, in these latter three studies, realimentation of the restricted heifers was applied so that all calved at similar weight. In this study the high protein diet group had average daily gain of 0.4 kg/day and low protein 0.2 kg/day over the treatment period. After this time they continued to be individually fed throughout gestation at positive weight gain [unlike studies with negative gain in the low treatment (Cafe et al. 2006)]; there being also no attempt, to attain similar liveweight at parturition. This difference in (1) nutritional window of dietary intervention and (2) severity and period of restriction applied, may explain the apparently conflicting results in fetal development reported from bovine fetal programming studies.

This period of dietary intervention is earlier than any previous studies investigating bovine fetal growth trajectory in utero. The sexual dimorphism observed, such that the effect peri-conception protein restriction effect was greater in the male, supports our previous findings in beef cattle (Micke et al. 2011, 2014). This suggests that the male embryo is more susceptible to early gestational intervention than the female, as seen in the in vitro embryo (Bermejo-Alvarez et al. 2011), and that this effect continues until at least 98dpc. The reason for this gender difference may be that the pattern of DNA methylation during embryo development in the two sexes is different over time making susceptibility to epigenetic change both gender and time dependent (Dobbs et al. 2013). We note however, that the expected gender effect on birthweight where the male is larger and heavier than the female was not observed in this study.

The finding that maternal peri-conception and first trimester nutrition did not affect birthweight at term may be an important dystocia management consideration when considering timing of supplementation to heifers. This is in agreement with previous reports following first trimester intervention (Micke et al. 2010b) and between 30 and 123dpc (Long et al. 2009). If restriction is applied during the second trimester (Micke et al. 2010b), however, or second and third trimester (Cafe et al. 2006) birthweight has reportedly been affected.

The lack of effect on gross morphology at birth, however, does not necessarily reflect the impact of early gestational intervention upon long-term growth, development and health in the offspring (Wu et al. 2006) as we have previously reported. In sheep, peri-conceptional undernutrition does not necessarily result in altered birthweight but has been associated with altered fetal growth trajectory and altered post-natal growth, metabolic and endocrine regulation (Jaquiery et al. 2011). The post-natal effects of similar periods of peri-conceptional undernutrition in the bovine are less well understood. Further studies of the post-natal development of the progeny are under way.