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Effects of diet type on establishment of pregnancy and embryo development in beef heifers
Animal Reproduction Science 133 (2012) 139–145
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Animal Reproduction Science journal homepage: www.elsevier.com/locate/anireprosci
Effects of diet type on establishment of pregnancy and embryo development in beef heifers V.P. Gath a , M.A. Crowe a,∗ , D. O’Callaghan a,1 , M.P. Boland b , P. Duffy a,b , P. Lonergan b , F.J. Mulligan a a b
UCD School of Veterinary Medicine, University College Dublin, Belfield, Dublin 4, Ireland UCD School of Agriculture and Food Science, University College Dublin, Belfield, Dublin 4, Ireland
a r t i c l e
i n f o
Article history: Received 8 November 2011 Received in revised form 20 June 2012 Accepted 22 June 2012 Available online 30 June 2012
Keywords: Cattle Urea Energy Superovulation Embryo development
a b s t r a c t The objectives were to determine the effects of elevated blood urea concentrations on: (i) the response to superovulation, fertilisation rate, and early embryonic development in beef heifers, and (ii) embryo survival from days 7 to 35 of gestation. In Experiment 1, heifers (18–24 months) were allocated at random (n = 20 per treatment) to one of the following diets: (i) ad libitum grass silage plus 5 kg commercial beef concentrates per day (controls); (ii) ad libitum grass silage plus 5 kg concentrates and 250 g feed grade urea per day (HE/HU); or (iii) ad libitum wheaten straw plus 250 g feed grade urea and 50 g vitamin/mineral mix per day (LE/HU). Serum urea concentrations were monitored throughout the experiment. Oestrus in heifers was synchronised using an intravaginal releasing device (CIDR® , InterAg, New Zealand). Oestrus was detected and in vitro produced blastocysts (day 7, morphological grades 1 and 2) were transferred to the heifers 7 days later (19 days after start of treatment diets). The heifers were maintained on the dietary treatments for a further 28 days, when pregnancy status was determined by transrectal ultrasonography. Detected pregnancies were terminated using 15 mg luprostiol and recycled for Experiment 2. In Experiment 2, following a 14-day dietary rest period, the heifers were re-allocated at random to the three dietary treatments above. Heifers were treated with a CIDR for 8 days and 15 mg luprostiol was given 12 h before pessary withdrawal. They received 144 mg pFSH (Folltropin® -V, Vetrepharm, Canada) given as 8 injections over 4 days commencing on day 6 of CIDR/dietary treatment. Heifers were artificially inseminated 48 h after progesterone pessary withdrawal using commercial semen of proven fertility by a competent inseminator. The heifers were maintained on their diets until slaughter, 3 days post insemination when corpora lutea numbers were determined and embryos were recovered and cell numbers determined visually. Serum urea concentrations were greater in heifers on LE/HU than in those on HE/HU diets, which in turn were greater than controls (7.1 ± 0.5, 4.9 ± 0.3 and 3.2 ± 0.1 mmol/L, respectively; P < 0.05). There was no effect of diet type on pregnancy rate at day 35 (42%, 47% and 46%) and on the number of corpora lutea following superovulation (5.2 ± 0.8, 5.8 ± 1.5 and 6.8 ± 1.1) for heifers on control, HE/HU and LE/HU diets, respectively. The total number of embryos recovered per heifer was not different between the three groups (2.7 ± 0.6, 3.4 ± 1.1 and 4.8 ± 0.8 for heifers on control, HE/HU and LE/HU diets, respectively; P > 0.05), but the number of embryos with 8 or more cells at recovery was greater in heifers on LE/HU than on control diets (3.4 ± 0.8 compared with 1.0 ± 0.3; P < 0.05). However the percentage of embryos recovered with 8 or more cells was not different between groups (70.0 ± 13.3, 86.9 ± 7.2 and 76.5 ± 7.9%, for heifers on
∗ Corresponding author. Tel.: +353 1 7166255; fax: +353 1 7166253. E-mail address: [email protected] (M.A. Crowe). 1 Present address: Institute of Technology Blanchardstown, Blanchardstown Road North, Dublin 15, Ireland. 0378-4320/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.anireprosci.2012.06.019
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control, HE/HU and LE/HU diets respectively). Fertilisation rate, expressed as the proportion of embryos with more than one cell at recovery relative to the total number of embryos recovered, was less in the heifers on the control diet than in the other two dietary treatments (61.3 ± 11.8, 92.0 ± 3.5 and 86.8 ± 5.4% for heifers on control, HE/HU and LE/HU diets, respectively; P < 0.05). Deleterious effects of urea on reproduction were not found, suggesting that adverse effects of urea are likely to take place at the early oocyte development stage prior to ovulation or fertilisation following an increase in protein intake. © 2012 Elsevier B.V. All rights reserved.
1. Introduction
2. Materials and methods
The association between amounts of dietary protein, milk production and serum urea concentrations, and reduced fertility in dairy cows has been reported previously (Jordan et al., 1983; Kaim et al., 1983; Ropstad and Refsdal, 1987; Canfield et al., 1990; Elrod and Butler, 1993; Butler et al., 1996). In most cases, as dietary crude protein increases, services per conception and days not pregnant also increase. However, the physiological mechanism(s) by which this reduction of fertility occurs remains unclear. Urea is a relatively small molecule (molar mass = 60.06 g/mol) that has the ability to move freely through cell membranes. It is, therefore, logical that when blood urea concentrations are high, urea will diffuse from the blood into other organs of the body, for example, the reproductive tract and mammary gland. Elevated plasma urea concentrations resulting from excess rumen degradable protein or dietary urea can decrease uterine luminal pH (Elrod and Butler, 1993) and pregnancy rate in cows (Butler et al., 1996). Reduced fertility and embryonic loss can occur when dietary urea is in excess (McEvoy et al., 1997) or when rumen degradable protein is increased (Blanchard et al., 1990). In sheep, the deleterious effects of urea on fertility are likely to occur before day 4 of pregnancy (Fahey et al., 2001). Alternatively, imbalances between the nitrogenous substrates available to rumen microbes for anabolism to microbial protein, and the necessary energy available, leads to increased serum urea concentrations. The efficient utilisation of dietary urea depends on the availability of dietary energy to the microbial flora to support greater microbial protein synthesis (Erdman et al., 1986). Excess dietary protein intake may influence reproduction via direct effects on the uterine environment. Toxic by-products of nitrogen metabolism, including ammonia (or more specifically ammonium ions) from the rumen may affect sperm transport and capacitation, oocyte maturation, fertilisation and early embryo survival. Such effects may be mediated via changes in uterine pH (Elrod et al., 1993). The aim of this study was to investigate the effects of a high dietary crude protein, offered as feed grade urea, in combination with high and low metabolisable energy diets on (i) embryo survival in the uterus from day 7 to day 35 of pregnancy and (ii) embryo development and quality following superovulation, in heifers. Beef heifers were used, as crude protein can be manipulated in a model devoid of the complications associated with postpartum insults (as occurs in dairy cows), so that the specific effects of dietary crude protein on embryo development and survival could be assessed.
This study was divided into two parts. The first part studied the effects of elevated blood urea concentrations on development of embryos within the uterus from days 7 to 35 of pregnancy. This was achieved by transferring good quality embryos, produced using in vitro embryo production procedures, into urea treated cattle. The second part studied the effects of urea on (i) the ovarian response to superovulation, (ii) embryo recovery rate following non-surgical flushing, (iii) fertilisation rate, and (iv) early embryo development to day 3 of pregnancy. 2.1. Experiment 1 2.1.1. Animals and treatments Sixty Charolais crossbred beef heifers of similar weight, aged 18–24 months were used. All animal use was licenced under the Cruelty to Animals act (Ireland; 1876) in accordance with European Union directive 86-609-EC. They were then randomly allocated to one of three dietary treatments. Each treatment group consisted of 20 heifers penned in two groups of 10 in an indoor slatted housing unit. Each group was allocated one of the following diets: (i) high energy (control) diet, (ii) high energy/high urea (HE/HU) diet and (iii) low energy/high urea (LE/HU) diet. The control diet consisted of ad libitum grass silage plus 5 kg of commercial beef concentrate feedstuff per heifer per day (nutritional analysis in Table 1). The HE/HU diet consisted of ad libitum grass silage plus 5 kg of commercial beef concentrate feedstuff plus 250 g of feed grade urea (ICI, Ireland) per heifer per day. The LE/HU diet consisted of ad libitum wheaten straw plus 50 g of a commercial vitamin/mineral mix plus 250 g of feed grade urea per heifer Table 1 Nutritional analysis of the grass silage, supplementary concentrates and wheaten straw. Grass silage Dry matter (%) pH Crude protein (g/kg DM) Crude fibre (g/kg DM) NDF (g/kg DM) Ash (g/kg DM) Oil (g/kg DM) Volatile N (% of total N) Metabolisable energy (MJ/kg DM)
20.8 3.94 136 318.9 548.8 79.9 30.4 8.96 10.9
NDF: neutral detergent fibre; N = nitrogen.
Concentrates 87.0
Wheaten straw 86.0
160
35 420
70 25
71 15
12.3
7.2
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Fig. 1. Diagram of experimental protocol (Experiment 1) showing times of oestrous synchronisation, embryo transfer, blood sampling, and pregnancy diagnosis. Oestrous synchronisation commenced 10 days after commencement of dietary feeding with urea using a 10-day CIDR and prostaglandin on day 8 of the CIDR protocol.
per day. The concentrates were offered once per day at the rate of 50 kg to a pen of 10 heifers. There was adequate trough space for all heifers to feed simultaneously. The feed grade urea and concentrates were mixed thoroughly with the silage to the HE/HU group. When adding the feed grade urea and vitamin/mineral mix to the LE/HU group, for each pen of 10 heifers, 2.5 kg of urea was mixed with water to form a thick paste and 500 g of vitamin/mineral mix was also mixed to form a thick paste prior to mixing thoroughly with the straw. This ensured the urea and vitamin/mineral mix adhered to the straw and did not sift to the bottom of the trough. The addition of the vitamin/mineral mix ensured that the heifers on this diet would be supplied with their required amounts of vitamins and minerals, similar to the other groups, thus preventing any deficiency effects on fertility (Table 1). At the start of the experiment, heifers were health checked and the reproductive tract of each animal was examined using transrectal ultrasonography to ensure each heifer was oestrous cyclic, not pregnant, and free from abnormalities. The dietary urea was gradually introduced in daily increments of 25 g over 10 days to allow the rumen microflora adapt to high dietary non-protein nitrogen concentrations. 2.1.2. Live weight determination The body weight (kg) of each heifer was determined using an accurate walk-on electronic weighing scales at the start of the experiment and at pregnancy diagnosis for replicate 1 of the experiment. 2.1.3. Oestrous synchronisation and embryo transfer The oestrous cycles of the heifers were synchronised using an intravaginal releasing device containing 1.9 g progesterone (CIDR® , InterAg, New Zealand) for 10 days, coupled with a prostaglandin analogue injection, 15 mg luprostiol (Prosolvin® , Intervet, UK) 2 days before pessary withdrawal (Fig. 1). The synchronisation treatment started 10 days after the start of dietary acclimatisation,
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Fig. 2. Diagram of experimental protocol (Experiment 2) showing oestrous synchronisation, superovulation, insemination, and embryo recovery. Oestrous synchronisation commenced on day 10 after commencement of dietary feeding with urea using an 8-day CIDR, with superovulation commencing on day 6 of the CIDR and prostaglandin to ensure luteolysis of any CL present 12 h before CIDR removal.
i.e., once the 250 g/day of urea intake was achieved in the urea treatment groups (Fig. 1). The heifers were observed for signs of oestrus on a non-slip surface for 30 min every 6 h from 24 to 76 h post device withdrawal. Tail paint was used as an aid to oestrous detection. One in vitro produced blastocyst (day 7, morphological grade 1 or 2) was transferred to each heifer, 7 days after detected oestrus (19 days after urea treated heifers were supplemented with 250 g urea/day). Embryos were only transferred to heifers that were detected in oestrus during the expected time frame. The heifers were maintained on the experimental diets for a further 28 days, at which time each heifer was checked for pregnancy using transrectal ultrasonography (Aloka SSD500 v ultrasonic scanner with a 7.5 MHz linear transducer rectal probe; Aloka Ltd., Toyko, Japan). A positive pregnancy diagnosis was based on the presence of an apparently viable foetus with a visible heartbeat and clear amniotic fluid. Detected pregnancies were terminated using a 15 mg luprostiol injection. All heifers were then allocated to a normal production diet of ad libitum grass silage and 2 kg of concentrates per heifer per day for 14 days. They were then re-allocated at random to the three experimental diets and Experiment 1 repeated to increase the numbers for statistical power. 2.2. Experiment 2 In the second part of the study, the heifers were reallocated at random to the dietary treatments, as previously described, following a 14-day period of feeding a normal production diet of ad libitum grass silage and 2 kg of concentrates per heifer per day. 2.2.1. Oestrous synchronisation and superovulation Stage of oestrous cycles were synchronised using an intravaginal CIDR device for 8 days and a 15 mg luprostiol injection was administered 12 h before pessary withdrawal (Fig. 2). Heifers were superovulated using 144 mg pFSH (Folltropin® -V, Vetrepharm, Canada) administered as 8 injections over 4 days commencing on day 6 of CIDR treatment. They were artificially inseminated using semen from a single bull of proven fertility, by a competent inseminator 1.5 and 2 days after CIDR device removal. The heifers
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were maintained on the experimental diets until slaughter, 3 days post second insemination. Ovaries and reproductive tracts were recovered at slaughter and the number of corpora lutea quantified. Embryos and/or unfertilised ova were recovered and cell numbers determined visually. 2.2.2. Blood sampling Blood samples were collected into 10 ml plain vacuum tubes (Vacutainer® , Becton Dikinson, England) via aseptic jugular venepuncture using an 18 gauge, 1 in. needle. Samples were collected from each heifer on the days of insemination, embryo transfer, and pregnancy diagnosis. Blood samples were labelled and immediately stored at room temperature for 1 h and then at 4 ◦ C for 18–24 h. Samples were then centrifuged at 1600 × g for 20 min. Serum was decanted into plastic tubes, labelled and stored at −20 ◦ C until assayed. 2.2.3. Serum urea concentration determination Serum urea concentrations were determined using an enzymatic colorimetric test kit (Urea S, MPR 3 777510, Boehringer Mannheim, Germany) following the manufacturers instructions. This test kit utilised the hydrolysis of urea catalysed by urease to produce ammonium ions that react with salicylate and hypochlorite to yield a green dye via the Berthelot colour reaction (Fawcett and Scott, 1960). The light absorbency of this dye was measured using a spectrophotometer at 600 nm wavelength and adjusted against a reagent blank. The concentration of urea in serum was then calculated using this absorbance value and a standard value as follows: Serum urea concentration (mmol/L) = 5 ×
Asample Astandard
Quality control of the test was carried out using commercial reference sera (Precinorm® U, Precipath® U and Precinorm® UPX, Boehringer Mannheim, Germany). 2.2.4. Embryo collection and cell number determination The reproductive tracts (uterus and ovaries) were collected at slaughter, labelled with the heifer’s identity, and immediately returned to the laboratory for corpora lutea counting and embryo recovery. Embryos were recovered by retrograde flushing of each oviduct with 20 ml of phosphate buffered saline containing 10% foetal calf serum into 50 ml labelled conical tubes. The contents were then transferred to a searching dish and searched using a stereoscopic light microscope. The number of embryos recovered per heifer was recorded and also embryo cell numbers were quantified visually under stereoscopic microscopy by a single experienced operator. Normal day 3 embryos would be expected to have undergone cell cleavage three times resulting in eight or more cells per embryo. Embryos with two or more cells were deemed to be fertilised embryos, whereas any single cell embryos were deemed unfertilised. The numbers of corpora lutea on the ovaries of each heifer were also counted to determine any differences in the response to the superovulation treatment.
Table 2 Live weights of heifers (kg) at the start and end of Experiment 1 (mean ± S.E.). Dietary treatment
Mean live weight at start (kg)
Mean live weight at end (kg)
Control (n = 20) HE/HU (n = 20) LE/HU (n = 20)
487 ± 23a,b 471 ± 18a,b 483 ± 21a,b
496 ± 21a 490 ± 19a 448 ± 17b
a,b
Means with different superscripts are different (P < 0.05).
2.3. Statistical analyses In Experiment 1, all animals responded to synchronisation treatment and were included in analyses, and with the two replicates resulted in a final n = 40 per treatment group. In Experiment 2, only 18, 19 and 19 heifers (in control, HE/HU and LE/HU groups, respectively) were successfully synchronised, superovulated and flushed and final data analysis are based on these. Data for serum urea concentration, number of corpora lutea, number, and grade of embryos were analysed using analysis of variance (ANOVA), using the mixed model procedure (procMIXED, SAS, 1998). Fishers Exact Chi-square analysis as described by Snedecor and Cochran (1989) was used for pregnancy rates. Results are presented as mean ± S.E. 3. Results 3.1. Body weights Heifers allocated to the LE/HU dietary treatment had a lesser weight than those allocated to the HE/HU and control diets after 62 days (P < 0.05; Table 2). 3.2. Serum urea concentrations Serum urea concentrations were greater in animals on LE/HU diet than in those on HE/HU diet, which in turn were greater than those on control diets (P < 0.05; Table 3). 3.3. Pregnancy rate The overall pregnancy rate using in vitro produced embryos was 45% (51/113). There was no effect of diet type on pregnancy rate at day 35 when transferring grade 1 and 2 in vitro produced embryos (42%, 47%, and 46%) for controls, HE/HU, and LE/HU, respectively (Fig. 3).
Table 3 Serum urea concentrations (mmol/L) at embryo transfer and at pregnancy diagnosis (mean ± S.E.). Dietary treatment
Serum urea (mmol/L)
Control (n = 40) HE/HU (n = 40) LE/HU (n = 40)
3.2 ± 0.1a 4.9 ± 0.3b 7.1 ± 0.5c
a,b,c Means with different superscripts within the same column are different (P < 0.05).
Percentage pregnant
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4. Discussion
75%
50%
18/38
17/37
16/38
25%
HE / HU
Control
LE / HU
Dietary treatment Fig. 3. Percentage heifers pregnant in each group following transfer of good quality embryos. Table 4 Number of corpora lutea and number of embryos/ova recovered per treatment group (mean ± s.e). Dietary treatment
Number of corpora lutea
Number of embryos/ova recovered
Control (n = 18) HE/HU (n = 19) LE/HU (n = 19)
5.2 ± 0.8 5.8 ± 1.5 6.8 ± 0.8
2.7 ± 0.6 3.4 ± 1.1 4.8 ± 0.8
Differences were not significant (P > 0.05).
3.4. Number of corpora lutea and recovered embryos/ova The number of corpora lutea following superovulation was not affected by dietary treatment. The total number of embryos/ova recovered was not significantly different between the three groups (Table 4). 3.5. Number of fertilised embryos and embryo cell numbers The number of embryos with eight or more cells at recovery was greater in animals on LE/HU than in controls (3.4 ± 0.8 compared with 1.0 ± 0.3; P < 0.05). However the percentage of embryos recovered with eight or more cells was not different between groups (70.0 ± 13.3, 86.9 ± 7.2 and 76.5 ± 7.9%, respectively; Table 5). Fertilisation rate, expressed as the proportion of embryos with more than one cell at recovery relative to the total number of embryos recovered, was less in the controls than in the other two dietary treatments (61.3 ± 11.8, 92.0 ± 3.5 and 86.8 ± 5.4% for controls, HE/HU and LE/HU, respectively; P < 0.05). Table 5 Number of fertilised embryos recovered per treatment group (mean ± s.e.) and % of these with greater or equal to eight cells. Dietary treatment
Number of fertilised embryos recovered
% of embryos with ≥8 cells at day 3 post insemination
Control (n = 18) HE/HU (n = 19) LE/HU (n = 19)
1.4 ± 0.5 2.9 ± 1.0 4.4 ± 0.9
70.0 ± 13.3 86.9 ± 7.2 76.5 ± 7.9
The supplementation of heifers with 250 g of feed grade urea per day resulted in greater serum urea concentrations than in un-supplemented heifers. This is in agreement with Canfield et al. (1990) and Kenny et al. (2002a) who found that excess dietary crude protein, or direct supplementation of the diet with urea as a source of nitrogen, can result in increased plasma urea concentrations. However, following supplementation of 250 g feed grade urea per animal to two groups of heifers, one on a high plane of nutrition and the other on a low plane of nutrition, the resulting serum urea concentrations were greater in the heifers on a low plane of nutrition. These heifers were in negative energy balance, so this difference may be explained by the fact that there is less fermentable energy available to the rumen microbes to utilise the greater amounts of non-protein nitrogen (urea) for microbial protein synthesis. Urea which is not utilised by microbes in the rumen is quickly hydrolysed to ammonium ions which quickly diffuse from the rumen into the circulatory system. These toxic ammonium ions are in turn converted to less toxic urea by the liver. This urea returns to the circulatory system where excess is removed by the kidneys and excreted via urine. A period of gradual dietary acclimatisation is required for detoxification of high concentrations of ammonium ions to urea by the liver to become fully effective. A sudden increase in dietary urea intake would lead to ammonium toxicity which is often fatal. Animals in negative energy balance will also mobilise fat reserves to generate metabolic energy. Non-esterified fatty acids are released from the adipose tissue and then extracted from the circulation by the liver. Excess fat mobilisation leads to increased hepatic triacylglycerol content that can also decrease liver function. Liver function, in particular, is important to prevent ammonium toxicity. Cows that lose a lot of body condition in early lactation (Snijders et al., 2000) and those with high serum triacylglycerol content (Kruip et al., 2001) produce lesser quality oocytes based on their ability to develop in vitro. Cows in negative energy balance also have low blood glucose and blood insulin levels. Low blood insulin concentrations are responsible for low IGF-1 production from the liver (Butler et al., 2003), which can reduce the responsiveness of the ovary to gonadotrophins. Thus feeding 250 g urea with straw to the heifers in the LE/HU group should create physiological conditions similar to the high yielding dairy cow in negative energy balance on a high protein diet. It would, therefore, be expected that this group of heifers would have lesser fertility. However, the overall metabolic rate of a high yielding dairy cow would be greater than a beef heifer on a straw diet. The liver in the lactating dairy cow therefore, would be highly metabolically active due to the increased portal circulation from the digestive system as a consequence of high dietary intake. The ability to detoxify ammonium ions may, therefore, be compromised. The serum urea concentrations of the heifers on the LE/HU diet, although within the accepted physiological range (2.8–8.8 mmol/L), were similar to the concentrations found in cows experiencing infertility due to excess feeding of crude protein (Canfield et al., 1990; Elrod and Butler,
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1993; Butler et al., 1996; Larson et al., 1997; Rajala-Schultz et al., 2001). But in the present experiment, when good quality in vitro produced embryos were transferred into these heifers plus the control heifers, no difference in pregnancy rate at day 35 was observed despite the different serum urea concentrations. This suggests that the uterine environment, between days 7 to 35 post fertilisation, is not the sole contributor to early embryo loss when high urea diets are offered, even in a situation of relative energy deficit (which is common in the lactating dairy cow). Good quality in vitro produced embryos are insensitive to high urea concentrations after day 7. This is supported by Fahey et al. (2001) who concluded that, in ewes, the effects of high urea on embryo quality are likely to be due to alterations in the oviduct environment or deleterious changes in the follicle rather than changes in the uterine environment. Early embryonic death may be as a result of a single effect of raised blood urea concentrations, but is more likely to be a result of the cumulative effects starting in the pre-ovulatory follicle to the in-utero expanded blastocyst. In vitro studies using oocytes of cattle matured in medium containing 0, 5.0, 7.5, or 10.0 mmol/L urea found that the cleavage rate was not reduced by any concentration of urea, but the proportion of oocytes developing to the blastocyst stage at day 8 after insemination was reduced by 7.5 mmol/L urea (Ocon and Hansen, 2003). De Wit et al. (2001) found that the addition of 6.0 mmol/L urea to maturation medium hastened completion of metaphase I, inhibited completion of metaphase II, reduced fertilisation rate, and decreased the proportion of oocytes that became blastocysts. There was no effect of urea exposure during maturation on development of cleaved embryos to the blastocyst stage. Thus Ocon and Hansen (2003) demonstrated an effect of urea between cleavage and blastocyst stage whereas De Wit et al. (2001) demonstrated an effect due to reduced fertilisation and cleavage rate. This difference may be attributed to different embryo culture media, De Wit et al. (2001) used a complex medium, and included serum, epidermal growth factor, insulin and other constituents not present in the modified potassium simplex optimised medium (KSOM) used by Ocon and Hansen (2003). Dawuda et al. (2002) investigated the effects of supplementing urea to superovulated cows from either 10 days prior to insemination or the day of insemination with a control diet. The supplementation of urea from 10 days before insemination had no effect on either the quality or number of embryos recovered 7 days later. However, significantly fewer embryos of poorer quality were recovered from the cows supplemented with urea from the day of insemination than the other two treatments. This suggests that cows may adapt to high concentrations of dietary nitrogen over a period of less than 10 days. This may explain that, in Experiment 1, the cows had already adapted to their different dietary intakes of energy and urea before receiving the in vitro produced embryos. As there were no changes in the diets from embryo transfer to pregnancy diagnosis, there was no effect on pregnancy rate. The second experiment looked at the effects of diet type on the response to superovulation and fertilisation rate during the period of superovulation treatment and up
to day 3 post insemination. The high urea concentrations did not affect the response to superovulation, fertilisation, and embryo recovery or embryo development to day 3. This suggests that an extended period of exposure to elevated urea concentrations is necessary and/or that the adverse effects on embryo development are gradual, and not obvious until later during embryonic development. Rooke et al. (2004), demonstrated that excess rumen degradable protein lead to increased concentrations of ammonia in follicular fluid and Hammon et al. (2005), indicated the high plasma urea concentrations were associated with raised ammonia and urea concentrations in preovulatory follicular fluids on the day of ovulation and in the uterine fluids during the luteal phase of the oestrous cycle during early lactation in dairy cows. However, in vitro data of Ocon and Hansen (2003) showed that the cleavage rate of embryos, matured in various media with differing urea concentrations up to 10 mM, was not reduced, but there was an effect on the proportion of oocytes developing to day 8 blastocysts. Even though these heifers would have acclimatised to the different diets before insemination and fertilisation, when oocytes were collected from these follicles and fertilised and cultured in vitro, there was an associated reduction in the number of blastocysts produced. They concluded that it was likely that this was caused by deleterious effects on the granulosa cells that support the oocyte. Also Jorritsma et al. (2004) reported that raised non-esterified fatty acids, often due to negative dietary energy balance, reduced the in vitro proliferation of granulosa cells, delayed oocyte maturation, and impaired blastocyst production. These fatty acids are processed by the liver into various metabolic pathways: (a) very low density lipoproteins, (b) esterified as triglycerides and stored in the liver, (c) oxidised to CO2 , and (d) partially oxidised to acetate or ketone bodies. Amounts of non-esterified fatty acids and ketone bodies in the blood were greater in cows that failed to ovulate the first postpartum dominant follicle in comparison with cows that had ovulatory follicles (Marr et al., 2002). Experiment 2 also indicates that there were no adverse effects of raised serum urea or negative energy balance on the capacity of sperm cells to fertilise the oocytes, even though they must travel through the uterus to reach the oviduct for fertilisation to take place. This is in agreement with Kenny et al. (2002b) who supplemented beef heifers with 240 g feed grade urea from approximately 8 days before insemination and found no effect on the survival of embryos 30 and 40 days after insemination. They concluded that any link between poor fertility and serum urea concentrations in dairy cattle may be due to the interaction between a high protein intake and a severe negative energy balance rather than a direct effect of increased nitrogen intake itself. The mobilisation of protein body reserves in the high yielding dairy cow in severe negative energy balance will also contribute to raised blood urea concentrations (Tamminga, 2006). In high yielding dairy cows the excessive negative energy balance and raised blood urea concentrations associated with the early post-partum period, coincides with the selection and development of the cohort of follicles from which the first ovulated oocyte will
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emerge. Leese (2002) postulated that dietary effects during oocyte development causes cellular stress which up regulates subsequent embryo metabolism which in turn is not conducive to optimum embryo survival preimplantation. Rhoads et al. (2006) recovered day 7 embryos from lactating dairy cows with moderate and high plasma urea levels. These embryos were then frozen and transferred to synchronised recipient maiden heifers on two different diets to result in high and low plasma urea concentrations. They reported that pregnancy rate was not affected by the recipient diet, but embryos from cows with moderate plasma urea levels resulted in a greater pregnancy rate than the embryos from the high plasma urea cows. They concluded that high plasma urea concentrations in lactating dairy cows decrease embryo viability through effects exerted on the oocyte or embryo before day 7 post insemination. Although alterations to the uterine environment, such as pH changes, were not directly measured in this experiment, the concentration of dietary urea was designed to be in excess of nitrogen requirements and this resulted in mean serum urea concentrations that were significantly higher than the accepted concentration, above which fertility is generally impaired. The results of these two experiments imply that any adverse effects of high urea are not in fact around ovulation, fertilisation or in the uterus post fertilisation, but may be at earlier stages (i.e. during oocyte development). Acknowledgements The authors acknowledge the UCD Lyons Research Farm for provision of research animals and the farm staff for assistance with the animal studies. They also acknowledge Kevin Thornton for assistance with Urea assays. References Blanchard, T., Ferguson, J.D., Love, L., Takeda, T., Henderson, B., Hasler, J., Chalupa, W., 1990. Effect of dietary crude protein type on fertilisation and embryo quality in dairy cattle. Am. J. Vet. Res. 51, 905–908. Butler, W.R., Calaman, J.J., Beam, S.W., 1996. Plasma and milk urea nitrogen in relation to pregnancy rate in lactating dairy cattle. J. Anim. Sci. 74, 858–865. Butler, S.T., Marr, A.L., Pelton, S.H., Radcliff, R.P., Lucy, M.C., Butler, W.R., 2003. Insulin restores GH responsiveness during lactation-induced negative energy balance in dairy cattle: effects on expression of IGF-1 and GH receptor 1A. J. Endocrinol. 176, 205–207. Canfield, R.W., Sniffen, C.J., Butler, W.R., 1990. Effects of excess degradable protein on postpartum reproduction and energy balance in dairy cattle. J. Dairy Sci. 73, 2342–2349. Dawuda, P.M., Scaramuzzi, R.J., Leese, H.J., Hall, C.J., Peters, A.R., Drew, S.B., Wathes, D.C., 2002. Effect of timing of urea feeding on the yield and quality of embryos in lactating dairy cows. Theriogenology 58, 1443–1455. De Wit, A.A.C., Cesar, M.L.F., Kruip, T.A.M., 2001. Effect of urea during in vitro maturation on nuclear maturation and embryo development of bovine cumulus-oocyte-complexes. J. Dairy Sci. 84, 1800–1804. Elrod, C.C., Butler, W.R., 1993. Reduction of fertility and alteration of uterine pH in heifers fed excess ruminally degradable protein. J. Anim. Sci. 71, 694–701. Elrod, C.C., Van Amburgh, M., Butler, W.R., 1993. Alterations of pH in response to increased dietary protein in cattle are unique to the uterus. J. Anim. Sci. 71, 702–706.
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Erdman, R.A., Proctor, G.H., Vandersall, J.H., 1986. Effect of rumen ammonia concentration on in situ rates and extent of digestion of feedstuffs. J. Dairy Sci. 69, 2312–2321. Fahey, J., Boland, M.P., O’Callaghan, D., 2001. Effects of dietary urea on embryo development in superovulated donor ewes and on embryo survival following transfer in recipient ewes. Anim. Sci. 72, 395–400. Fawcett, J.K., Scott, J.E., 1960. A rapid and precise method for the determination of urea. J. Clin. Pathol. 13, 156–159. Hammon, D.S., Holyoak, G.R., Dhiman, T.R., 2005. Association between blood plasma urea nitrogen levels and reproductive fluid urea nitrogen and ammonia concentrations in early lactation dairy cows. Anim. Reprod. Sci. 86, 195–204. Jordan, E.R., Chapman, T.E., Holtan, D.W., Swanson, L.V., 1983. Relationship of dietary crude protein to composition of uterine secretions and blood in high-producing postpartum dairy cows. J. Dairy Sci. 66, 1854–1862. Jorritsma, R., César, M.L., Hermans, J.T., Krintwagen, C.L.J.J., Vos, P.L.A.M., Kruip, T.A.M., 2004. Effects of non-esterified fatty acids on bovine granulosa cells and developmental potential of oocytes in vitro. Anim. Reprod. Sci. 81, 225–235. Kaim, M., Folman, Y., Neumark, H., 1983. The effect of protein intake and lactation number on post-partum body weight loss and reproductive performance of dairy cows. Anim. Prod. 37, 229–235. Kenny, D.A., Boland, M.P., Diskin, M.G., Sreenan, J.M., 2002b. Effect of rumen degradable protein with or without fermentable carbohydrate supplementation on blood metabolites and embryo survival in cattle. Anim. Sci. 74, 529–537. Kenny, D.A., Humpherson, P.G., Lees, H.J., Morris, D.G., Tomos, A.D., Diskin, M.G., Sreenan, J.M., 2002a. Effect of elevated systemic concentrations of ammonia and urea on the metabolite and ionic composition of oviductal fluid in cattle. Biol. Reprod. 66, 1797–1804. Kruip, T.A.M., Wensing, T., Vos, P.L.A.M., 2001. Characteristics of abnormal puerperium in dairy cattle and the rationale for common treatments. In: Diskin, M.G. (Ed.), Fertility in the High Producing Dairy Cow. Occasional publication no. 26. British Society of Animal Science, Edinburgh, pp. 63–79. Larson, S.F., Butler, W.R., Currie, W.B., 1997. Reduced fertility associated with low progesterone post breeding and increased milk urea nitrogen in lactating cows. J. Dairy Sci. 80, 1288–1295. Leese, H.J., 2002. Quiet please, do not disturb: a hypothesis of embryo metabolism and viability. BioEssays 24, 845–849. Marr, A.L., Piepenbrink, M.S., Overton, T.R., Lucy, M.C., Butler, W.R., 2002. The somatotrophic axis and lipid metabolism in transition dairy cows in relation to timing of first postpartum ovulation. J. Anim. Sci. 80 (Suppl. 1), 66 (Abstr. 263). McEvoy, T., Robinson, J., Aitken, R., Findlay, P., Robertson, I., 1997. Dietary excesses of urea influence the viability and metabolism of preimplantation sheep embryos and may affect fetal growth among the survivors. Anim. Reprod. Sci. 47, 71–90. Ocon, O.M., Hansen, P.J., 2003. Disruption of bovine oocytes and preimplantation embryos by urea and acidic pH. J. Dairy Sci. 86, 1194–1200. Rajala-Schultz, P.J., Saville, W.J.A., Frazer, G.S., Wittum, T.E., 2001. Association between milk urea nitrogen and fertility in Ohio dairy cows. J. Dairy Sci. 84, 482–489. Rhoads, M.L., Rhoads, R.P., Gilbert, R.O., Toole, R., Butler, W.R., 2006. Detrimental effects of high plasma urea nitrogen levels on viability of embryos from lactating dairy cows. Anim. Reprod. Sci. 91, 1–10. Rooke, J.A., Ewen, M., Makie, K., Staines, M.E., McEvoy, T.G., Sinclair, K.D., 2004. Effect of ammonium chloride on the growth of bovine ovarian granulosa cells and the development of ovine oocytes matures in the presence of bovine granulosa cells and the development of ovine oocytes matured in the presence of bovine granulosa cells previously exposed to ammonium chloride. Anim. Reprod. Sci. 84, 53–71. Ropstad, E., Refsdal, A.O., 1987. Herd reproduction performance related to urea concentration in bulk milk. Acta Vet. Scand. 28, 55–63. SAS, 1998. SAS/STAT Users Guide, release 8.1 edition. Statistical Analysis Systems Institute, Cary, NC. Snedecor, G.W., Cochran, W.G., 1989. Statistical Methods, eighth edition. Iowa State University Press. Snijders, S.E.M., Dillon, P., O’Callaghan, D., Boland, M.P., 2000. Effect of genetic merit, milk yield, body condition and lactation number on in vitro oocytes development in dairy cows. Theriogenology 53, 981–989. Tamminga, S., 2006. The effect of the supply of rumen degradable protein and metabolisable protein on negative energy balance and fertility in dairy cows. Anim. Reprod. Sci. 96, 227–239.
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Animal Reproduction Science journal homepage: www.elsevier.com/locate/anireprosci
Effects of diet type on establishment of pregnancy and embryo development in beef heifers V.P. Gath a , M.A. Crowe a,∗ , D. O’Callaghan a,1 , M.P. Boland b , P. Duffy a,b , P. Lonergan b , F.J. Mulligan a a b
UCD School of Veterinary Medicine, University College Dublin, Belfield, Dublin 4, Ireland UCD School of Agriculture and Food Science, University College Dublin, Belfield, Dublin 4, Ireland
a r t i c l e
i n f o
Article history: Received 8 November 2011 Received in revised form 20 June 2012 Accepted 22 June 2012 Available online 30 June 2012
Keywords: Cattle Urea Energy Superovulation Embryo development
a b s t r a c t The objectives were to determine the effects of elevated blood urea concentrations on: (i) the response to superovulation, fertilisation rate, and early embryonic development in beef heifers, and (ii) embryo survival from days 7 to 35 of gestation. In Experiment 1, heifers (18–24 months) were allocated at random (n = 20 per treatment) to one of the following diets: (i) ad libitum grass silage plus 5 kg commercial beef concentrates per day (controls); (ii) ad libitum grass silage plus 5 kg concentrates and 250 g feed grade urea per day (HE/HU); or (iii) ad libitum wheaten straw plus 250 g feed grade urea and 50 g vitamin/mineral mix per day (LE/HU). Serum urea concentrations were monitored throughout the experiment. Oestrus in heifers was synchronised using an intravaginal releasing device (CIDR® , InterAg, New Zealand). Oestrus was detected and in vitro produced blastocysts (day 7, morphological grades 1 and 2) were transferred to the heifers 7 days later (19 days after start of treatment diets). The heifers were maintained on the dietary treatments for a further 28 days, when pregnancy status was determined by transrectal ultrasonography. Detected pregnancies were terminated using 15 mg luprostiol and recycled for Experiment 2. In Experiment 2, following a 14-day dietary rest period, the heifers were re-allocated at random to the three dietary treatments above. Heifers were treated with a CIDR for 8 days and 15 mg luprostiol was given 12 h before pessary withdrawal. They received 144 mg pFSH (Folltropin® -V, Vetrepharm, Canada) given as 8 injections over 4 days commencing on day 6 of CIDR/dietary treatment. Heifers were artificially inseminated 48 h after progesterone pessary withdrawal using commercial semen of proven fertility by a competent inseminator. The heifers were maintained on their diets until slaughter, 3 days post insemination when corpora lutea numbers were determined and embryos were recovered and cell numbers determined visually. Serum urea concentrations were greater in heifers on LE/HU than in those on HE/HU diets, which in turn were greater than controls (7.1 ± 0.5, 4.9 ± 0.3 and 3.2 ± 0.1 mmol/L, respectively; P < 0.05). There was no effect of diet type on pregnancy rate at day 35 (42%, 47% and 46%) and on the number of corpora lutea following superovulation (5.2 ± 0.8, 5.8 ± 1.5 and 6.8 ± 1.1) for heifers on control, HE/HU and LE/HU diets, respectively. The total number of embryos recovered per heifer was not different between the three groups (2.7 ± 0.6, 3.4 ± 1.1 and 4.8 ± 0.8 for heifers on control, HE/HU and LE/HU diets, respectively; P > 0.05), but the number of embryos with 8 or more cells at recovery was greater in heifers on LE/HU than on control diets (3.4 ± 0.8 compared with 1.0 ± 0.3; P < 0.05). However the percentage of embryos recovered with 8 or more cells was not different between groups (70.0 ± 13.3, 86.9 ± 7.2 and 76.5 ± 7.9%, for heifers on
∗ Corresponding author. Tel.: +353 1 7166255; fax: +353 1 7166253. E-mail address: [email protected] (M.A. Crowe). 1 Present address: Institute of Technology Blanchardstown, Blanchardstown Road North, Dublin 15, Ireland. 0378-4320/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.anireprosci.2012.06.019
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control, HE/HU and LE/HU diets respectively). Fertilisation rate, expressed as the proportion of embryos with more than one cell at recovery relative to the total number of embryos recovered, was less in the heifers on the control diet than in the other two dietary treatments (61.3 ± 11.8, 92.0 ± 3.5 and 86.8 ± 5.4% for heifers on control, HE/HU and LE/HU diets, respectively; P < 0.05). Deleterious effects of urea on reproduction were not found, suggesting that adverse effects of urea are likely to take place at the early oocyte development stage prior to ovulation or fertilisation following an increase in protein intake. © 2012 Elsevier B.V. All rights reserved.
1. Introduction
2. Materials and methods
The association between amounts of dietary protein, milk production and serum urea concentrations, and reduced fertility in dairy cows has been reported previously (Jordan et al., 1983; Kaim et al., 1983; Ropstad and Refsdal, 1987; Canfield et al., 1990; Elrod and Butler, 1993; Butler et al., 1996). In most cases, as dietary crude protein increases, services per conception and days not pregnant also increase. However, the physiological mechanism(s) by which this reduction of fertility occurs remains unclear. Urea is a relatively small molecule (molar mass = 60.06 g/mol) that has the ability to move freely through cell membranes. It is, therefore, logical that when blood urea concentrations are high, urea will diffuse from the blood into other organs of the body, for example, the reproductive tract and mammary gland. Elevated plasma urea concentrations resulting from excess rumen degradable protein or dietary urea can decrease uterine luminal pH (Elrod and Butler, 1993) and pregnancy rate in cows (Butler et al., 1996). Reduced fertility and embryonic loss can occur when dietary urea is in excess (McEvoy et al., 1997) or when rumen degradable protein is increased (Blanchard et al., 1990). In sheep, the deleterious effects of urea on fertility are likely to occur before day 4 of pregnancy (Fahey et al., 2001). Alternatively, imbalances between the nitrogenous substrates available to rumen microbes for anabolism to microbial protein, and the necessary energy available, leads to increased serum urea concentrations. The efficient utilisation of dietary urea depends on the availability of dietary energy to the microbial flora to support greater microbial protein synthesis (Erdman et al., 1986). Excess dietary protein intake may influence reproduction via direct effects on the uterine environment. Toxic by-products of nitrogen metabolism, including ammonia (or more specifically ammonium ions) from the rumen may affect sperm transport and capacitation, oocyte maturation, fertilisation and early embryo survival. Such effects may be mediated via changes in uterine pH (Elrod et al., 1993). The aim of this study was to investigate the effects of a high dietary crude protein, offered as feed grade urea, in combination with high and low metabolisable energy diets on (i) embryo survival in the uterus from day 7 to day 35 of pregnancy and (ii) embryo development and quality following superovulation, in heifers. Beef heifers were used, as crude protein can be manipulated in a model devoid of the complications associated with postpartum insults (as occurs in dairy cows), so that the specific effects of dietary crude protein on embryo development and survival could be assessed.
This study was divided into two parts. The first part studied the effects of elevated blood urea concentrations on development of embryos within the uterus from days 7 to 35 of pregnancy. This was achieved by transferring good quality embryos, produced using in vitro embryo production procedures, into urea treated cattle. The second part studied the effects of urea on (i) the ovarian response to superovulation, (ii) embryo recovery rate following non-surgical flushing, (iii) fertilisation rate, and (iv) early embryo development to day 3 of pregnancy. 2.1. Experiment 1 2.1.1. Animals and treatments Sixty Charolais crossbred beef heifers of similar weight, aged 18–24 months were used. All animal use was licenced under the Cruelty to Animals act (Ireland; 1876) in accordance with European Union directive 86-609-EC. They were then randomly allocated to one of three dietary treatments. Each treatment group consisted of 20 heifers penned in two groups of 10 in an indoor slatted housing unit. Each group was allocated one of the following diets: (i) high energy (control) diet, (ii) high energy/high urea (HE/HU) diet and (iii) low energy/high urea (LE/HU) diet. The control diet consisted of ad libitum grass silage plus 5 kg of commercial beef concentrate feedstuff per heifer per day (nutritional analysis in Table 1). The HE/HU diet consisted of ad libitum grass silage plus 5 kg of commercial beef concentrate feedstuff plus 250 g of feed grade urea (ICI, Ireland) per heifer per day. The LE/HU diet consisted of ad libitum wheaten straw plus 50 g of a commercial vitamin/mineral mix plus 250 g of feed grade urea per heifer Table 1 Nutritional analysis of the grass silage, supplementary concentrates and wheaten straw. Grass silage Dry matter (%) pH Crude protein (g/kg DM) Crude fibre (g/kg DM) NDF (g/kg DM) Ash (g/kg DM) Oil (g/kg DM) Volatile N (% of total N) Metabolisable energy (MJ/kg DM)
20.8 3.94 136 318.9 548.8 79.9 30.4 8.96 10.9
NDF: neutral detergent fibre; N = nitrogen.
Concentrates 87.0
Wheaten straw 86.0
160
35 420
70 25
71 15
12.3
7.2
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Fig. 1. Diagram of experimental protocol (Experiment 1) showing times of oestrous synchronisation, embryo transfer, blood sampling, and pregnancy diagnosis. Oestrous synchronisation commenced 10 days after commencement of dietary feeding with urea using a 10-day CIDR and prostaglandin on day 8 of the CIDR protocol.
per day. The concentrates were offered once per day at the rate of 50 kg to a pen of 10 heifers. There was adequate trough space for all heifers to feed simultaneously. The feed grade urea and concentrates were mixed thoroughly with the silage to the HE/HU group. When adding the feed grade urea and vitamin/mineral mix to the LE/HU group, for each pen of 10 heifers, 2.5 kg of urea was mixed with water to form a thick paste and 500 g of vitamin/mineral mix was also mixed to form a thick paste prior to mixing thoroughly with the straw. This ensured the urea and vitamin/mineral mix adhered to the straw and did not sift to the bottom of the trough. The addition of the vitamin/mineral mix ensured that the heifers on this diet would be supplied with their required amounts of vitamins and minerals, similar to the other groups, thus preventing any deficiency effects on fertility (Table 1). At the start of the experiment, heifers were health checked and the reproductive tract of each animal was examined using transrectal ultrasonography to ensure each heifer was oestrous cyclic, not pregnant, and free from abnormalities. The dietary urea was gradually introduced in daily increments of 25 g over 10 days to allow the rumen microflora adapt to high dietary non-protein nitrogen concentrations. 2.1.2. Live weight determination The body weight (kg) of each heifer was determined using an accurate walk-on electronic weighing scales at the start of the experiment and at pregnancy diagnosis for replicate 1 of the experiment. 2.1.3. Oestrous synchronisation and embryo transfer The oestrous cycles of the heifers were synchronised using an intravaginal releasing device containing 1.9 g progesterone (CIDR® , InterAg, New Zealand) for 10 days, coupled with a prostaglandin analogue injection, 15 mg luprostiol (Prosolvin® , Intervet, UK) 2 days before pessary withdrawal (Fig. 1). The synchronisation treatment started 10 days after the start of dietary acclimatisation,
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Fig. 2. Diagram of experimental protocol (Experiment 2) showing oestrous synchronisation, superovulation, insemination, and embryo recovery. Oestrous synchronisation commenced on day 10 after commencement of dietary feeding with urea using an 8-day CIDR, with superovulation commencing on day 6 of the CIDR and prostaglandin to ensure luteolysis of any CL present 12 h before CIDR removal.
i.e., once the 250 g/day of urea intake was achieved in the urea treatment groups (Fig. 1). The heifers were observed for signs of oestrus on a non-slip surface for 30 min every 6 h from 24 to 76 h post device withdrawal. Tail paint was used as an aid to oestrous detection. One in vitro produced blastocyst (day 7, morphological grade 1 or 2) was transferred to each heifer, 7 days after detected oestrus (19 days after urea treated heifers were supplemented with 250 g urea/day). Embryos were only transferred to heifers that were detected in oestrus during the expected time frame. The heifers were maintained on the experimental diets for a further 28 days, at which time each heifer was checked for pregnancy using transrectal ultrasonography (Aloka SSD500 v ultrasonic scanner with a 7.5 MHz linear transducer rectal probe; Aloka Ltd., Toyko, Japan). A positive pregnancy diagnosis was based on the presence of an apparently viable foetus with a visible heartbeat and clear amniotic fluid. Detected pregnancies were terminated using a 15 mg luprostiol injection. All heifers were then allocated to a normal production diet of ad libitum grass silage and 2 kg of concentrates per heifer per day for 14 days. They were then re-allocated at random to the three experimental diets and Experiment 1 repeated to increase the numbers for statistical power. 2.2. Experiment 2 In the second part of the study, the heifers were reallocated at random to the dietary treatments, as previously described, following a 14-day period of feeding a normal production diet of ad libitum grass silage and 2 kg of concentrates per heifer per day. 2.2.1. Oestrous synchronisation and superovulation Stage of oestrous cycles were synchronised using an intravaginal CIDR device for 8 days and a 15 mg luprostiol injection was administered 12 h before pessary withdrawal (Fig. 2). Heifers were superovulated using 144 mg pFSH (Folltropin® -V, Vetrepharm, Canada) administered as 8 injections over 4 days commencing on day 6 of CIDR treatment. They were artificially inseminated using semen from a single bull of proven fertility, by a competent inseminator 1.5 and 2 days after CIDR device removal. The heifers
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were maintained on the experimental diets until slaughter, 3 days post second insemination. Ovaries and reproductive tracts were recovered at slaughter and the number of corpora lutea quantified. Embryos and/or unfertilised ova were recovered and cell numbers determined visually. 2.2.2. Blood sampling Blood samples were collected into 10 ml plain vacuum tubes (Vacutainer® , Becton Dikinson, England) via aseptic jugular venepuncture using an 18 gauge, 1 in. needle. Samples were collected from each heifer on the days of insemination, embryo transfer, and pregnancy diagnosis. Blood samples were labelled and immediately stored at room temperature for 1 h and then at 4 ◦ C for 18–24 h. Samples were then centrifuged at 1600 × g for 20 min. Serum was decanted into plastic tubes, labelled and stored at −20 ◦ C until assayed. 2.2.3. Serum urea concentration determination Serum urea concentrations were determined using an enzymatic colorimetric test kit (Urea S, MPR 3 777510, Boehringer Mannheim, Germany) following the manufacturers instructions. This test kit utilised the hydrolysis of urea catalysed by urease to produce ammonium ions that react with salicylate and hypochlorite to yield a green dye via the Berthelot colour reaction (Fawcett and Scott, 1960). The light absorbency of this dye was measured using a spectrophotometer at 600 nm wavelength and adjusted against a reagent blank. The concentration of urea in serum was then calculated using this absorbance value and a standard value as follows: Serum urea concentration (mmol/L) = 5 ×
Asample Astandard
Quality control of the test was carried out using commercial reference sera (Precinorm® U, Precipath® U and Precinorm® UPX, Boehringer Mannheim, Germany). 2.2.4. Embryo collection and cell number determination The reproductive tracts (uterus and ovaries) were collected at slaughter, labelled with the heifer’s identity, and immediately returned to the laboratory for corpora lutea counting and embryo recovery. Embryos were recovered by retrograde flushing of each oviduct with 20 ml of phosphate buffered saline containing 10% foetal calf serum into 50 ml labelled conical tubes. The contents were then transferred to a searching dish and searched using a stereoscopic light microscope. The number of embryos recovered per heifer was recorded and also embryo cell numbers were quantified visually under stereoscopic microscopy by a single experienced operator. Normal day 3 embryos would be expected to have undergone cell cleavage three times resulting in eight or more cells per embryo. Embryos with two or more cells were deemed to be fertilised embryos, whereas any single cell embryos were deemed unfertilised. The numbers of corpora lutea on the ovaries of each heifer were also counted to determine any differences in the response to the superovulation treatment.
Table 2 Live weights of heifers (kg) at the start and end of Experiment 1 (mean ± S.E.). Dietary treatment
Mean live weight at start (kg)
Mean live weight at end (kg)
Control (n = 20) HE/HU (n = 20) LE/HU (n = 20)
487 ± 23a,b 471 ± 18a,b 483 ± 21a,b
496 ± 21a 490 ± 19a 448 ± 17b
a,b
Means with different superscripts are different (P < 0.05).
2.3. Statistical analyses In Experiment 1, all animals responded to synchronisation treatment and were included in analyses, and with the two replicates resulted in a final n = 40 per treatment group. In Experiment 2, only 18, 19 and 19 heifers (in control, HE/HU and LE/HU groups, respectively) were successfully synchronised, superovulated and flushed and final data analysis are based on these. Data for serum urea concentration, number of corpora lutea, number, and grade of embryos were analysed using analysis of variance (ANOVA), using the mixed model procedure (procMIXED, SAS, 1998). Fishers Exact Chi-square analysis as described by Snedecor and Cochran (1989) was used for pregnancy rates. Results are presented as mean ± S.E. 3. Results 3.1. Body weights Heifers allocated to the LE/HU dietary treatment had a lesser weight than those allocated to the HE/HU and control diets after 62 days (P < 0.05; Table 2). 3.2. Serum urea concentrations Serum urea concentrations were greater in animals on LE/HU diet than in those on HE/HU diet, which in turn were greater than those on control diets (P < 0.05; Table 3). 3.3. Pregnancy rate The overall pregnancy rate using in vitro produced embryos was 45% (51/113). There was no effect of diet type on pregnancy rate at day 35 when transferring grade 1 and 2 in vitro produced embryos (42%, 47%, and 46%) for controls, HE/HU, and LE/HU, respectively (Fig. 3).
Table 3 Serum urea concentrations (mmol/L) at embryo transfer and at pregnancy diagnosis (mean ± S.E.). Dietary treatment
Serum urea (mmol/L)
Control (n = 40) HE/HU (n = 40) LE/HU (n = 40)
3.2 ± 0.1a 4.9 ± 0.3b 7.1 ± 0.5c
a,b,c Means with different superscripts within the same column are different (P < 0.05).
Percentage pregnant
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4. Discussion
75%
50%
18/38
17/37
16/38
25%
HE / HU
Control
LE / HU
Dietary treatment Fig. 3. Percentage heifers pregnant in each group following transfer of good quality embryos. Table 4 Number of corpora lutea and number of embryos/ova recovered per treatment group (mean ± s.e). Dietary treatment
Number of corpora lutea
Number of embryos/ova recovered
Control (n = 18) HE/HU (n = 19) LE/HU (n = 19)
5.2 ± 0.8 5.8 ± 1.5 6.8 ± 0.8
2.7 ± 0.6 3.4 ± 1.1 4.8 ± 0.8
Differences were not significant (P > 0.05).
3.4. Number of corpora lutea and recovered embryos/ova The number of corpora lutea following superovulation was not affected by dietary treatment. The total number of embryos/ova recovered was not significantly different between the three groups (Table 4). 3.5. Number of fertilised embryos and embryo cell numbers The number of embryos with eight or more cells at recovery was greater in animals on LE/HU than in controls (3.4 ± 0.8 compared with 1.0 ± 0.3; P < 0.05). However the percentage of embryos recovered with eight or more cells was not different between groups (70.0 ± 13.3, 86.9 ± 7.2 and 76.5 ± 7.9%, respectively; Table 5). Fertilisation rate, expressed as the proportion of embryos with more than one cell at recovery relative to the total number of embryos recovered, was less in the controls than in the other two dietary treatments (61.3 ± 11.8, 92.0 ± 3.5 and 86.8 ± 5.4% for controls, HE/HU and LE/HU, respectively; P < 0.05). Table 5 Number of fertilised embryos recovered per treatment group (mean ± s.e.) and % of these with greater or equal to eight cells. Dietary treatment
Number of fertilised embryos recovered
% of embryos with ≥8 cells at day 3 post insemination
Control (n = 18) HE/HU (n = 19) LE/HU (n = 19)
1.4 ± 0.5 2.9 ± 1.0 4.4 ± 0.9
70.0 ± 13.3 86.9 ± 7.2 76.5 ± 7.9
The supplementation of heifers with 250 g of feed grade urea per day resulted in greater serum urea concentrations than in un-supplemented heifers. This is in agreement with Canfield et al. (1990) and Kenny et al. (2002a) who found that excess dietary crude protein, or direct supplementation of the diet with urea as a source of nitrogen, can result in increased plasma urea concentrations. However, following supplementation of 250 g feed grade urea per animal to two groups of heifers, one on a high plane of nutrition and the other on a low plane of nutrition, the resulting serum urea concentrations were greater in the heifers on a low plane of nutrition. These heifers were in negative energy balance, so this difference may be explained by the fact that there is less fermentable energy available to the rumen microbes to utilise the greater amounts of non-protein nitrogen (urea) for microbial protein synthesis. Urea which is not utilised by microbes in the rumen is quickly hydrolysed to ammonium ions which quickly diffuse from the rumen into the circulatory system. These toxic ammonium ions are in turn converted to less toxic urea by the liver. This urea returns to the circulatory system where excess is removed by the kidneys and excreted via urine. A period of gradual dietary acclimatisation is required for detoxification of high concentrations of ammonium ions to urea by the liver to become fully effective. A sudden increase in dietary urea intake would lead to ammonium toxicity which is often fatal. Animals in negative energy balance will also mobilise fat reserves to generate metabolic energy. Non-esterified fatty acids are released from the adipose tissue and then extracted from the circulation by the liver. Excess fat mobilisation leads to increased hepatic triacylglycerol content that can also decrease liver function. Liver function, in particular, is important to prevent ammonium toxicity. Cows that lose a lot of body condition in early lactation (Snijders et al., 2000) and those with high serum triacylglycerol content (Kruip et al., 2001) produce lesser quality oocytes based on their ability to develop in vitro. Cows in negative energy balance also have low blood glucose and blood insulin levels. Low blood insulin concentrations are responsible for low IGF-1 production from the liver (Butler et al., 2003), which can reduce the responsiveness of the ovary to gonadotrophins. Thus feeding 250 g urea with straw to the heifers in the LE/HU group should create physiological conditions similar to the high yielding dairy cow in negative energy balance on a high protein diet. It would, therefore, be expected that this group of heifers would have lesser fertility. However, the overall metabolic rate of a high yielding dairy cow would be greater than a beef heifer on a straw diet. The liver in the lactating dairy cow therefore, would be highly metabolically active due to the increased portal circulation from the digestive system as a consequence of high dietary intake. The ability to detoxify ammonium ions may, therefore, be compromised. The serum urea concentrations of the heifers on the LE/HU diet, although within the accepted physiological range (2.8–8.8 mmol/L), were similar to the concentrations found in cows experiencing infertility due to excess feeding of crude protein (Canfield et al., 1990; Elrod and Butler,
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1993; Butler et al., 1996; Larson et al., 1997; Rajala-Schultz et al., 2001). But in the present experiment, when good quality in vitro produced embryos were transferred into these heifers plus the control heifers, no difference in pregnancy rate at day 35 was observed despite the different serum urea concentrations. This suggests that the uterine environment, between days 7 to 35 post fertilisation, is not the sole contributor to early embryo loss when high urea diets are offered, even in a situation of relative energy deficit (which is common in the lactating dairy cow). Good quality in vitro produced embryos are insensitive to high urea concentrations after day 7. This is supported by Fahey et al. (2001) who concluded that, in ewes, the effects of high urea on embryo quality are likely to be due to alterations in the oviduct environment or deleterious changes in the follicle rather than changes in the uterine environment. Early embryonic death may be as a result of a single effect of raised blood urea concentrations, but is more likely to be a result of the cumulative effects starting in the pre-ovulatory follicle to the in-utero expanded blastocyst. In vitro studies using oocytes of cattle matured in medium containing 0, 5.0, 7.5, or 10.0 mmol/L urea found that the cleavage rate was not reduced by any concentration of urea, but the proportion of oocytes developing to the blastocyst stage at day 8 after insemination was reduced by 7.5 mmol/L urea (Ocon and Hansen, 2003). De Wit et al. (2001) found that the addition of 6.0 mmol/L urea to maturation medium hastened completion of metaphase I, inhibited completion of metaphase II, reduced fertilisation rate, and decreased the proportion of oocytes that became blastocysts. There was no effect of urea exposure during maturation on development of cleaved embryos to the blastocyst stage. Thus Ocon and Hansen (2003) demonstrated an effect of urea between cleavage and blastocyst stage whereas De Wit et al. (2001) demonstrated an effect due to reduced fertilisation and cleavage rate. This difference may be attributed to different embryo culture media, De Wit et al. (2001) used a complex medium, and included serum, epidermal growth factor, insulin and other constituents not present in the modified potassium simplex optimised medium (KSOM) used by Ocon and Hansen (2003). Dawuda et al. (2002) investigated the effects of supplementing urea to superovulated cows from either 10 days prior to insemination or the day of insemination with a control diet. The supplementation of urea from 10 days before insemination had no effect on either the quality or number of embryos recovered 7 days later. However, significantly fewer embryos of poorer quality were recovered from the cows supplemented with urea from the day of insemination than the other two treatments. This suggests that cows may adapt to high concentrations of dietary nitrogen over a period of less than 10 days. This may explain that, in Experiment 1, the cows had already adapted to their different dietary intakes of energy and urea before receiving the in vitro produced embryos. As there were no changes in the diets from embryo transfer to pregnancy diagnosis, there was no effect on pregnancy rate. The second experiment looked at the effects of diet type on the response to superovulation and fertilisation rate during the period of superovulation treatment and up
to day 3 post insemination. The high urea concentrations did not affect the response to superovulation, fertilisation, and embryo recovery or embryo development to day 3. This suggests that an extended period of exposure to elevated urea concentrations is necessary and/or that the adverse effects on embryo development are gradual, and not obvious until later during embryonic development. Rooke et al. (2004), demonstrated that excess rumen degradable protein lead to increased concentrations of ammonia in follicular fluid and Hammon et al. (2005), indicated the high plasma urea concentrations were associated with raised ammonia and urea concentrations in preovulatory follicular fluids on the day of ovulation and in the uterine fluids during the luteal phase of the oestrous cycle during early lactation in dairy cows. However, in vitro data of Ocon and Hansen (2003) showed that the cleavage rate of embryos, matured in various media with differing urea concentrations up to 10 mM, was not reduced, but there was an effect on the proportion of oocytes developing to day 8 blastocysts. Even though these heifers would have acclimatised to the different diets before insemination and fertilisation, when oocytes were collected from these follicles and fertilised and cultured in vitro, there was an associated reduction in the number of blastocysts produced. They concluded that it was likely that this was caused by deleterious effects on the granulosa cells that support the oocyte. Also Jorritsma et al. (2004) reported that raised non-esterified fatty acids, often due to negative dietary energy balance, reduced the in vitro proliferation of granulosa cells, delayed oocyte maturation, and impaired blastocyst production. These fatty acids are processed by the liver into various metabolic pathways: (a) very low density lipoproteins, (b) esterified as triglycerides and stored in the liver, (c) oxidised to CO2 , and (d) partially oxidised to acetate or ketone bodies. Amounts of non-esterified fatty acids and ketone bodies in the blood were greater in cows that failed to ovulate the first postpartum dominant follicle in comparison with cows that had ovulatory follicles (Marr et al., 2002). Experiment 2 also indicates that there were no adverse effects of raised serum urea or negative energy balance on the capacity of sperm cells to fertilise the oocytes, even though they must travel through the uterus to reach the oviduct for fertilisation to take place. This is in agreement with Kenny et al. (2002b) who supplemented beef heifers with 240 g feed grade urea from approximately 8 days before insemination and found no effect on the survival of embryos 30 and 40 days after insemination. They concluded that any link between poor fertility and serum urea concentrations in dairy cattle may be due to the interaction between a high protein intake and a severe negative energy balance rather than a direct effect of increased nitrogen intake itself. The mobilisation of protein body reserves in the high yielding dairy cow in severe negative energy balance will also contribute to raised blood urea concentrations (Tamminga, 2006). In high yielding dairy cows the excessive negative energy balance and raised blood urea concentrations associated with the early post-partum period, coincides with the selection and development of the cohort of follicles from which the first ovulated oocyte will
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emerge. Leese (2002) postulated that dietary effects during oocyte development causes cellular stress which up regulates subsequent embryo metabolism which in turn is not conducive to optimum embryo survival preimplantation. Rhoads et al. (2006) recovered day 7 embryos from lactating dairy cows with moderate and high plasma urea levels. These embryos were then frozen and transferred to synchronised recipient maiden heifers on two different diets to result in high and low plasma urea concentrations. They reported that pregnancy rate was not affected by the recipient diet, but embryos from cows with moderate plasma urea levels resulted in a greater pregnancy rate than the embryos from the high plasma urea cows. They concluded that high plasma urea concentrations in lactating dairy cows decrease embryo viability through effects exerted on the oocyte or embryo before day 7 post insemination. Although alterations to the uterine environment, such as pH changes, were not directly measured in this experiment, the concentration of dietary urea was designed to be in excess of nitrogen requirements and this resulted in mean serum urea concentrations that were significantly higher than the accepted concentration, above which fertility is generally impaired. The results of these two experiments imply that any adverse effects of high urea are not in fact around ovulation, fertilisation or in the uterus post fertilisation, but may be at earlier stages (i.e. during oocyte development). Acknowledgements The authors acknowledge the UCD Lyons Research Farm for provision of research animals and the farm staff for assistance with the animal studies. They also acknowledge Kevin Thornton for assistance with Urea assays. References Blanchard, T., Ferguson, J.D., Love, L., Takeda, T., Henderson, B., Hasler, J., Chalupa, W., 1990. Effect of dietary crude protein type on fertilisation and embryo quality in dairy cattle. Am. J. Vet. Res. 51, 905–908. Butler, W.R., Calaman, J.J., Beam, S.W., 1996. Plasma and milk urea nitrogen in relation to pregnancy rate in lactating dairy cattle. J. Anim. Sci. 74, 858–865. Butler, S.T., Marr, A.L., Pelton, S.H., Radcliff, R.P., Lucy, M.C., Butler, W.R., 2003. Insulin restores GH responsiveness during lactation-induced negative energy balance in dairy cattle: effects on expression of IGF-1 and GH receptor 1A. J. Endocrinol. 176, 205–207. Canfield, R.W., Sniffen, C.J., Butler, W.R., 1990. Effects of excess degradable protein on postpartum reproduction and energy balance in dairy cattle. J. Dairy Sci. 73, 2342–2349. Dawuda, P.M., Scaramuzzi, R.J., Leese, H.J., Hall, C.J., Peters, A.R., Drew, S.B., Wathes, D.C., 2002. Effect of timing of urea feeding on the yield and quality of embryos in lactating dairy cows. Theriogenology 58, 1443–1455. De Wit, A.A.C., Cesar, M.L.F., Kruip, T.A.M., 2001. Effect of urea during in vitro maturation on nuclear maturation and embryo development of bovine cumulus-oocyte-complexes. J. Dairy Sci. 84, 1800–1804. Elrod, C.C., Butler, W.R., 1993. Reduction of fertility and alteration of uterine pH in heifers fed excess ruminally degradable protein. J. Anim. Sci. 71, 694–701. Elrod, C.C., Van Amburgh, M., Butler, W.R., 1993. Alterations of pH in response to increased dietary protein in cattle are unique to the uterus. J. Anim. Sci. 71, 702–706.
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