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a Department of Animal Science and Animal Health, Division of Nutrition, and
b Department of Clinical Studies, Division of Reproduction, Royal Veterinary and Agricultural University, DK-1870 Frederiksberg C, Denmark
c Department of Physiology, University of Cambridge, Cambridge CB2 3EG, United Kingdom
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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There is a large variation in the way and the extent to which IVP calves differ from normal calves. The majority of problems observed with IVP-derived calves occur at birth or in the immediate neonatal period. This suggests that the problems may originate from life in utero and reflect an inability of the IVP-derived calf to withstand the stresses of delivery and adaptation to life ex utero. However, little is known about the development of the IVP-derived calf in late gestation, when the maturational changes essential for neonatal survival normally occur.
The aim of the present study was to determine whether IVP and AI calves differ in blood gases and metabolite levels in the prenatal period, and whether IVP is associated with an altered response to the stress of preterm birth at about 90% gestation. This is the fetal age at which calves first become viable ex utero, and studies in organ function at this time were expected to maximize the likelihood of detecting differences in maturation between IVP and AI calves. In order to determine whether the physiological differences reflected enhanced or retarded organ maturation in IVP calves, both groups of premature animals were compared with a group of full-term AI calves in the immediate neonatal period.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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All the calves were produced with oocytes and semen that originated from the two main dairy breeds, Danish Red (RDM) and Holstein Friesian (SDM), which have almost identical phenotypes. The embryos were produced by standard methods, as described earlier [12, 13]. Ovaries were collected from a local abattoir, and cumulus-oocyte complexes were isolated by aspiration of antral follicles >2 mm in diameter. These complexes were matured in vitro by incubation for 22–24 h in Earle's tissue culture medium (TCM)-199 (Gibco, Copenhagen, Denmark) with 2.2 g L-1 sodium bicarbonate (Sigma, St. Louis, MO), 10% estrous cow serum, 0.2 mM sodium pyruvate, and 0.2 U ml-1 gonadotropins (Suigonan, a 2:1 mixture of eCG:hCG; Intervet Scandinavia, A/S Skovlunde, Denmark). The oocytes were fertilized for 20 h in medium containing 30 µg ml-1 heparin (170 USP mg-1; Sigma), 0.2 mM sodium pyruvate, 6 mg ml-1 BSA (fraction V; Sigma), and PHE (20 µM D-penicillamine, 10 µM hypotaurine, 1 µM epinephrine; Sigma). Frozen-thawed washed semen was added to a final concentration of 1.5–2 million ml-1. After fertilization, cumulus cells and excess spermatozoa were removed by vortex agitation for 2 min. Presumptive zygotes were cultured in Menezo-B2 medium (INRA Laboratoire, Paris, France) with 10% estrous cow serum and a 1:200 (v:v) suspension of bovine oviduct epithelial cells. Embryo development was assessed at Days 2, 5, 7, and 8 after insemination. From collection and throughout all of the procedures, the ovaries and oocytes were maintained at 30–34°C. Each incubation took place in humidified air with 5% CO2 at 38.8°C. Before transfer, the blastocysts were washed 10 times in Hepes-buffered TCM-199 medium supplemented with 5 mM bicarbonate, 0.4 mM L-glutamine, 0.2 mM pyruvate, and 50 µg ml-1 gentamycin. The embryos in the medium were loaded into 0.25-ml straws and were transported to the place of transfer in a portable incubator at 38.6°C. Twenty-one dairy heifers were synchronized with 2 injections of cloprostenol given 11 days apart (Estrumat Vet; Mallinckrodt Vet, Frederiksberg, Denmark). The embryos were transferred nonsurgically to the distal part of the uterine horn ipsilateral to the ovary with the corpus luteum. Pregnancy status was confirmed 5 and 8 wk after transfer by palpation per rectum. With a pregnancy rate of 67%, 13 pregnant IVP cows were used in the experiments, since one cow terminated pregnancy at Weeks 8–12 after embryo transfer (abortion rate = 4%).
For the pregnancies using AI, twenty-seven dairy heifers were synchronized and inseminated with semen originating from one of the two bulls used for the IVP pregnancies, and pregnancy was confirmed as above. A pregnancy rate of 68% resulted in a total group of 18 AI cows. Seven of these cows delivered at term without fetal catheterization, whereas fetal surgery was performed on eleven of the AI cows.
Fetal Surgery and Catheterization
The twenty-four cows used were operated on at 246–253 days of gestation, and the experimental procedures were approved by the Danish National Committee on Animal Experimentation. Food, but not water, was withheld for 24 h before surgery. All the cows received i.v. injections of antibiotics (2 mg kg-1 enrofloxacin, Baytril; Bayer, Leverkusen, Germany), and some of them (n = 17) were given 10 g paracetamol orally (Panodil; Smith Kline Beecham, Ballerup, Denmark) from 1 day before to 3 days after fetal surgery. Administration of paracetamol prevented hyperthermia (rectal temperatures >40°C without any sign of infection), which occurred in the first cows after surgery. Sodium propionate was given orally on the day before and the first day after surgery (250 g; Sigma) to reduce the risk of hypoglycemia and ketosis in association with surgery.
Anesthesia of the cows and their fetuses was induced by an i.v. infusion of thiopental into a jugular vein (11 mg kg-1 body weight, thiopenthal sodium; Abbott, North Chicago, IL). Each cow was intubated and subsequently ventilated with 2% fluothane in oxygen (Forene; Abott Scandinavia, Kista, Sweden). To improve cardiovascular function at surgery, 5 L Ringer's lactate (KVL, Copenhagen, Denmark) was administered i.v. Blood samples were taken from the jugular vein every 30 min throughout surgery to monitor the respiratory and metabolic state of the cow during anesthesia. In a some cases (n = 7), glucose was administered i.v. to the cow during surgery to maintain normoglycemia.
After surgical preparation of the cow in a lateral position, the uterus was exposed through a paramedian incision. A maternal vein (MV) and artery (MA) were catheterized via incisions on the surface of the uterus and advanced about 50 cm to reach the main vessels supplying and draining the uterine circulation. A fetal hind limb was identified by intra-abdominal palpation and moved so that the hoof was positioned in an inter-cotyledonary area of the uterus. An incision was made by cauterization through the uterine wall, and fetal membranes and the fetal leg were extended out of the uterus until the anterior surface of the hock joint was easily accessible. Care was taken to prevent bleeding and excessive loss of amniotic and allantoic fluids.
The anterior tibial artery and the saphenous vein of the fetus were catheterized with polyvinyl catheters (0.86 mm i.d. x 1.52 mm o.d. or 0.50 mm i.d. x 0.80 mm o.d., total lengths 4 m; Dural Plastics and Engineering, Silverwater, Australia), which were advanced 50–75 cm so as to lie with their tips in the fetal dorsal aorta (FA) and fetal caudal vena cava (FV), respectively [14, 15]. The catheters were sutured to the skin, and the leg was returned to the uterus and finally, the fetal membranes were tied. Catheters were then inserted 50–60 cm into the common umbilical vein (FuV) and artery (FuA) via cotyledonary branch vessels [14]. Blood samples were taken simultaneously from both sides of the placenta (FuV, FA, MV, and MA) before the catheters were sealed with a brass pin.
Before closure of the uterus, and on each of the days after surgery, antibiotics (2 mg kg-1; Baytril) were injected into the fetal vein. The uterus was closed with a row of continuous sutures through all fetal membranes and the uterine wall. A second row of continuous sutures was used to cover the first row. The abdominal incision was closed using standard procedures. Before the skin was sutured, all catheters were exteriorized at the most dorsal area of the sub-lumbar fossa, where they were kept in a waterproof bag sutured to the flank of the cow. After surgery, the skin incision was kept clean to prevent fetal contamination via the exteriorized catheters. The duration of surgery from induction of anesthesia to the final closure of the incision was 3 to 3.5 h. The catheters were flushed once the cow was standing and every 12 h for the first 2 days after surgery. A strong heparin solution (1000 IU ml-1 in 0.9% saline) was used for the first 2 days after surgery, and a more dilute solution (500 IU ml-1) thereafter. For the last 4 cows that were catheterized, the fetal and maternal catheters were infused continuously with a dilute heparin-saline solution (200 IU ml-1, 0.6 ml h-1). This reduced the problems with clotted catheters that was observed for a number of cows and their fetuses. The FuA catheter was used for sampling in stead of the FA catheter when the latter catheter ceased to flow.
Blood Sampling Protocol for Pregnant Cows and Their Fetuses
Daily samples At the end of surgery and daily (0900–1100 h) until the calves were delivered by cesarean section, simultaneous blood samples of 3 ml were taken, when possible, from all catheters using strict aseptic procedures. The blood samples were taken at 1–3 h after feeding of the cows and used for measurements of blood gases and metabolites.
Glucose tolerance test On Day 3 after surgery, the ability of the fetus to handle a bolus load of circulating glucose was investigated. Blood samples were drawn from the fetal arterial (FA) and maternal venous (MV) catheters immediately before and at intervals after (5, 15, 30, 60, 90, 120 min) injection of a 50% glucose solution into the fetal vena cava catheter (FV, 0.5 g kg-1). The fetal body weight was estimated at the time of surgery and generally taken to be 35 kg.
Blood flow measurements On Day 4 after surgery, blood flows through the umbilical and uterine circulations were measured by the steady-state diffusion technique using antipyrine [16]. Antipyrine (40% w:v in water; Sigma) was infused into the FV catheter at a rate of 0.4 ml h-1 kg-1 after a priming dose of 0.5 ml kg-1. Steady-state distribution of antipyrine across the umbilical and uterine circulations was obtained at 2 h from the start of infusion, and thereafter 5 sets of simultaneous blood samples were taken at 15-min intervals from the FuV, FA, MV, and MA catheters.
ACTH challenge test On Day 5 or 6, immediately before parturition was induced, the fetuses were subjected to an i.v. injection of synthetic ACTH to investigate the maturation of the fetal adrenal gland and its capacity to secrete cortisol. Blood samples were taken from the FV catheter before and at intervals after (15, 30, 60, 120, 150, 180 min) FV injection of 250 µg of ACTH1–25 (Synacthen; Ciba-Geigy, Basel, Switzerland) for measurements of cortisol in plasma.
Induction of Parturition and Cesarean Section
On Days 5–6 after surgery, fourteen catheterized calves were delivered by cesarean section 20 h after maternal induction of parturition with dexamethasone (25 mg i.m.; Boehringer Ingelheim, Copenhagen, Denmark). Cesarean section was applied to minimize the possible variation in physiological status of newborn calves induced by the potential variable degrees of calving complications not related to embryo treatment. Parturition was induced before cesarean delivery in order to study the cows and calves during the initial phases of the birth process.
After induction of parturition, blood samples were taken from mother and fetus at 10 h and at 20 h. Cesarean section was performed with the cow standing and with local infiltration anaesthesia of the flank (300 mg Lidocain; KVL). After cesarean delivery, the catheters in the hind limb of the calf (FA, FV) were secured to the loin of the calf by adhesive tape for blood sampling after birth. In seven remaining cows pregnant with uncatheterized AI calves, parturition was induced close to full term (at 274–280 days gestation), and the calves were delivered by cesarean section as above. These calves were used to evaluate the effects of premature birth, and blood samples were drawn from a catheter inserted into a jugular vein immediately after delivery.
Postnatal Calves
After birth, the calves were fed warmed bovine colostrum (40 ml kg-1) by stomach tube at 1, 6, 12, and 18 h. The same pool of frozen bovine colostrum was used for all calves. Blood samples were taken within 5 min of birth and at 1/2, 1, 2, 3, 6, 9, 12, 15, 18, 21, and 24 h thereafter. Rectal temperature, respiration rate, and heart rate were measured at the above time points for the first 15 h of life. After the last blood sample was taken, the calf was killed with an injection of sodium barbiturate (60 mg kg-1 body weight, i.v.), and measurements were taken of the lengths of head, crown to rump, and upper, middle and lower parts of fore and hind limbs. A number of organs were collected (heart, liver, abomasum, small intestine, large intestine, thymus, thyroid, lungs, adrenals, kidneys) and weighed, and the length of the intestines was measured.
Biochemical Analyses
Blood samples were collected in ice-chilled, heparinized syringes. Blood acidity (pH); partial pressure of oxygen (pO2); partial pressure of carbon-dioxide (pCO2); ionized sodium, potassium, chloride, and calcium (Na+, K+, Cl-, Ca2+, respectively); and glucose in whole blood were all analyzed using an automatic blood gas analyzer (NOVA Stat 5, Waltham, MA). Fetal samples were analyzed at a temperature that was 1.0°C higher than the maternal temperature. Blood hemoglobin (Hb), oxygen saturation (O2sat), and total oxygen content (O2ct) were measured by a hemoxymeter (OSM3; Radiometer, Copenhagen, Denmark). Cortisol concentrations in plasma were determined by RIA [17].
Lactate and glucose levels were determined in plasma using methods described previously [18]. The fraction of the fetal O2 uptake required to metabolize aerobically to CO2 the substrates acquired via the umbilical circulation was determined by calculating the molar substrate/O2 quotient [19]. Thus, the molar quotients for glucose and lactate are defined as 6 x ([Glucose]FuV - [Glucose]FA)/([O2]FuV - [O2]FA) and 3 x ([Lactate]FuV - [Lactate]FA)/([O2]FuV - [O2]FA), respectively.
Antipyrine concentrations for calculations of umbilical and uterine blood flows were measured in deproteinized whole blood [16] using a Technicon auto-analyzer (Pulse Instrumentation, Saskatoon, SK, Canada). The total blood flow was calculated as the antipyrine infusion rate divided by the mean A-V antipyrine concentration difference.
Data Analyses
The time-series of blood chemistry data for the premature fetal and neonatal calves and for the pregnant cows were analyzed by statistics for repeated measures as a split plot in time with treatment (IVP, AI), treatment-by-time interaction, sex, gestational age at birth, and father as the fixed effects [20]. The computations used the MIXED procedure of Statistical Analysis Systems (SAS) [21], which provided the adjusted means (least-squares means, LS means) ± SEM to test significant differences between the two embryo treatments (IVP and AI) across a series of sample points. The organ weight data were analyzed by standard General Linear Models procedures of SAS [21] with fixed effects as above, except that no time factor was present. For organ weight data and blood values at specific sample times the unadjusted means ± SEMs are given. Mean values for the seven term AI calves were compared with those in premature AI calves using Student's t-test for unpaired values. Throughout, the significance level is P < 0.05 unless otherwise stated.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Within five days after surgery, 6 IVP fetuses and 4 AI fetuses had died. Bacterial infection was associated with six fetal deaths (3 IVP, 3 AI), as indicated by the detection of either Arcanobacterium pyogenes, Escherichia coli, enterococci, or oral streptococci after bacteriological culture of fetal blood. The presence of maternal hyperthermia (rectal temperatures above 40°C) in response to surgery may have contributed to 2 deaths in the IVP group, since bacteriological examinations were sterile. In the remaining cows, the rectal temperature in the days after surgery was 38.7 ± 0.1°C and did not differ between IVP and AI cows. One sterile IVP calf died from a surgery-induced torsion of the uterus and another after a surgery-induced fetal hemorrhage. Compared with the 14 fetuses that survived until delivery, the 10 fetuses that died were more acidotic and hypercapnic than surviving fetuses at the time of surgery (pH and pCO2 means ± SEM in FA: 7.240 ± 0.022 vs. 7.292 ± 0.011 and 64.5 ± 2.6 vs. 55.9 ± 1.5 mm Hg). In addition, IVP (but not the AI) fetuses that died had significantly higher pO2 and Ca2+ levels at surgery than all the surviving fetuses (29.6 ± 2.5 vs. 22.5 ± 1.7 mm Hg and 1.40 ± 0.01 vs. 1.33 ± 0.02 mM, respectively). For the cows, there were no significant differences in blood chemistry values at surgery between those with fetuses that died and those with fetuses that survived for the 5 days after operation.
The values for some selected blood chemistry parameters are shown in Figs. 1–4 for the calves that survived the entire protocol. Values for the corresponding conscious pregnant cows before induction of parturition (Days 2–5) are shown in Table 1. Each sub-part of the experiment (conscious fetal calves, induction of parturition, neonatal period) is described in more detail below.
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Conscious Fetuses and Cows at 2–5 Days After Fetal Surgery
In conscious fetuses, before parturition was induced, the mean FA hemoglobin and oxygen contents were significantly higher (8.41 ± 0.28 vs. 7.52 ± 0.29% and 5.75 ± 0.41 vs. 3.79 ± 0.38%), while plasma lactate was significantly lower (1.89 ± 0.15 vs. 2.26 ± 0.13 mM) in IVP compared with AI fetuses (Fig. 1, B and C, and Fig. 3B). For the cows, Table 1 shows that the IVP group had significantly elevated blood O2 content and pCO2 and Ca2+ levels in MV blood, while lactate levels were lowered. Analyzed across the entire experiment, cortisol tended to be lower for IVP cows (Table 1, P < 0.08), but when cortisol had returned from high values at surgery to basal levels on Day 3, there was no longer any difference between IVP and AI cows (6.1 ± 1.4 vs. 7.6 ± 1.9 ng ml-1, day 3–5). Cortisol levels did not differ significantly between fetal AI and IVP calves. Only when the treatment comparison was made across all the sampling times before and after birth did IVP calves tend to have higher values than AI calves (28.7 ± 1.7 vs. 24.0 ± 1.9 mg ml-1, P = 0.07).
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Umbilical uptake of oxygen and nutrients The mean daily concentration differences in lactate, glucose, and oxygen across the umbilical and uterine vascular beds are shown in Table 2. For lactate, the only significant concentration difference was detected for the umbilical circulation in IVP fetuses. For glucose, significant concentration differences were present for both circulations and in both treatment groups. The difference tended to be lower for IVP calves than for AI calves in both the umbilical circulation and uterine circulations (P < 0.15). The IVP and AI calves had similar concentration differences in oxygen for both the umbilical and the uterine circulations. Calculation of the umbilical substrate/O2 quotient showed that the fraction of fetal oxygen uptake required to oxidize all umbilical uptake of glucose was significantly lower for IVP calves than for AI calves (0.40 ± 0.09 vs. 0.78 ± 0.06, means ± SEM, P < 0.01). For lactate, the trend was the opposite in that the mean substrate/O2 quotient was 0.47 ± 0.14 for IVP calves and 0.13 ± 0.23 for the AI calves (P = 0.20).
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Blood flow measurements Before Day 4 after surgery, one or more of the five catheters needed for blood flow measurements ceased to flow in a number of cows. Measurements of umbilical and uterine blood flows were therefore carried out for only some of the animals in each treatment group. These preliminary results provided no indication of a difference in umbilical or uterine blood flows between IVP and AI pregnancies (5.11 ± 0.39 vs. 5.41 ± 0.32 L min-1 and 11.52 ± 1.64 vs. 10.66 ± 0.85 L min-1, respectively, n = 2–4).
Glucose tolerance test Fetal blood glucose levels reached a peak within 5 min and had returned to basal levels by 90–120 min after the i.v. glucose injection (Fig. 4A).The peak glucose levels were significantly higher in IVP calves than in AI calves (6.3 ± 0.2 vs. 5.4 ± 0.3 mM, respectively). Maternal values also showed a small but significant increase for both treatments. Analyzed across all the sample times, there was no significant difference between fetal IVP and AI calves nor between IVP and AI cows.
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ACTH challenge test Basal plasma cortisol before injection of ACTH did not differ significantly between IVP and AI fetuses, and cortisol levels were similar in both groups of cows throughout the test (Fig. 4B). After ACTH injection, fetal cortisol levels increased rapidly and reached a plateau during the 30- to 120-min period, with no significant differences between the two treatments (41.7 ± 3.0 vs. 36.0 ± 2.8 ng ml-1 for IVP and AI calves, respectively, LS means ± SEM).
Induction of Parturition
Across the two treatments, induction of parturition was associated with more acidotic blood and higher hemoglobin, glucose, and lactate levels compared with values before induction (-0.03 pH unit, +0.6% hemoglobin, +1.9 mM glucose, +1.7 mM lactate, respectively; Fig. 1, A and B, and Fig. 3, A and B). After induction of parturition (at 10–20 h after dexamethasone injection), the FA O2ct and pCO2 levels were significantly higher in the IVP fetuses than in the AI fetuses (Fig. 1, C and D). The O2ct and O2 sat values decreased significantly during the final 10 h before delivery in the IVP fetuses (6.0 ± 0.2 to 4.2 ± 0.6% and 50.9 ± 7.0 to 34.3 ± 7.1%) while there were no response in AI fetuses (3.4 ± 0.4% and 38.7 ± 3.4%, respectively, at 0–20 h after induction). Despite the large drop in O2ct in IVP fetuses, blood pH tended to be less acidotic in these fetuses than in AI fetuses just before delivery (7.328 ± 0.010 vs. 7.305 ± 0.007, P = 0.09) (Fig. 1A).
Induction of parturition was followed by a significant increase in maternal blood glucose levels (3.8 ± 0.1 to 6.2 ± 0.6 mM at 20 h after induction), whereas there were no significant changes in the values for maternal blood pH, blood oxygenation, or blood ion levels shown in Table 1. Twenty hours after induction with the synthetic glucocorticoid, dexamethasone, endogenous cortisol levels decreased in all cows, but less in IVP than in AI cows (to 3.2 ± 0.5 vs. 1.2 ± 0.2 ng ml-1 just before cesarean section).
Postnatal Calves
Blood gases and metabolites Immediately after delivery, the 14 catheterized newborn calves showed some characteristics that reflected their premature condition when compared with the 7 AI calves delivered close to term. Particularly during the first 2 h after birth, blood pH and oxygenation values (pO2, O2sat, O2ct) in premature calves were low when compared with the values observed during the following 22 h or in the hours after birth in term calves (Fig. 1). Across the first 2 h of life, the 7 premature AI calves showed significantly lowered venous blood pH (7.203 ± 0.020 vs. 7.300 ± 0.021), O2ct (3.1 ± 0.9 vs. 6.2 ± 0.6%), glucose (1.83 ± 0.30 vs. 3.22 ± 0.35 mM), and rectal temperature (37.6 ± 0.3 vs. 38.7 ± 0.2°C); and significantly elevated blood pCO2 (65.0 ± 2.9 vs. 56.5 ± 3.8 mm Hg), Ca2+ (1.34 ± 0.01 vs. 1.25 ± 0.03 mM), and respiratory rate (53 ± 3 vs. 44 ± 3 inspirations min-1) when compared with the term AI calves (means ± SEM). The differences were also significant when analyzed across the entire 24-h period. The premature calves also differed from the term control calves in that they had poor sucking reflexes and had difficulty in standing. Furthermore, about half of the premature calves had distended guts and some degree of intestinal dysmotility and maldigestion at the end of the 24-h feeding period. The severity of the above clinical signs of prematurity showed no obvious relationship to embryo treatment (IVP or AI).
Analyzed across the 24-h period, the IVP calves, compared with the premature AI calves, had significantly elevated blood pH (7.294 ± 0.006 vs. 7.270 ± 0.006), hemoglobin (9.06 ± 0.13 vs. 8.25 ± 0.14%), O2ct (6.33 ± 0.23 vs. 4.64 ± 0.28%), and K+ (4.89 ± 0.05 vs. 4.56 ± 0.05) (Fig. 1, A–C, and Fig. 2D); and significantly lowered blood Na+ (139.9 ± 0.4 vs. 141.0 ± 0.4 mM), Cl- (100.2 ± 0.3 vs. 103.1 ± 0.3 mM), and glucose levels (2.86 ± 0.09 vs. 3.11 ± 0.09; Fig. 2, B and C, and Fig. 3A). While Ca2+ levels were stable during the first 24 h in all three groups, Na+ and K+ levels showed marked changes, especially for the IVP calves (Fig. 2). Three hours after birth, blood Na+ and Cl- levels started to fall while K+ levels started to increase in the IVP calves. Compared with the term control calves, the IVP calves (and to a lesser extent also the AI calves) became significantly hyponatremic (139.7 ± 0.5 vs. 142.4 ± 0.3 mM) and hyperkalemic (5.20 ± 0.06 vs 4.48 ± 0.06 mM) during the 6- to 24-h period after birth.
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Respiratory frequency was significantly elevated in the IVP calves (66 ± 2 vs. 56 ± 2 inspirations min-1), while the mean heart rate was the same in the two treatment groups (136 ± 3 beats min-1). Rectal temperature decreased from birth to 2 h after birth in both premature IVP and AI calves (from 39.3 ± 0.1 to 37.0 ± 0.2°C) and in mature AI calves (from 39.3 ± 0.1 to 38.3 ± 0.2°C) (Fig. 3D). Thereafter, rectal temperatures increased rapidly in all premature calves, but most rapidly in the premature IVP calves, in which the temperature was significantly elevated during the 2- to 6-h period compared with the premature AI calves (38.9 ± 0.1 vs. 37.4 ± 0.1°C). Analyzed across the first 15 h after birth, rectal temperatures also tended to be higher for premature IVP calves vs. premature AI calves (38.04 ± 0.06 vs. 37.90 ± 0.07°C, P = 0.14).
Calf size and organ weights Body weight at 24 h did not differ from that at birth for any of the calves, and the mean weight was identical in the two groups of premature calves, being 74% of that for the 7 term AI calves (Table 3). Comparison of organ weights for premature and term AI calves showed that the mean weight of organs such as brain, lungs, heart, kidneys, spleen, adrenals, pancreas, abomasum, small intestine, and caecum-colon increased during the last month of gestation, while the mean weights for liver, thyroid, thymus, and brown fat either remained unchanged or decreased during this period. When organ weights were expressed relative to body weight, only the large intestinal weight was significantly higher in term AI calves than in premature AI calves. The relative weights of liver, thyroid, thymus, and brown fat were significantly lower in term AI calves than in premature AI calves (Table 3).
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The front and hind legs were slightly but significantly longer in the premature IVP group than in the premature AI group. Measurements of the lengths of each of 3 leg segments (lower, middle, upper segment; data not shown) showed that for both legs the increase in total length mainly arose from increases in the length of the middle and upper segments. There were no significant differences between the IVP and AI groups in the absolute or relative weights of the internal organs indicated in Table 3.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The increase in fetal and neonatal O2 content in the IVP calves was due primarily to the rise in hemoglobin, as arterial oxygen saturation was unaffected by the method of embryo production. The explanation for the elevated hemoglobin concentrations remains obscure but may involve activation of the sympatico-adrenal system or an increase in hemopoietic tissue in the bone marrow or in the fetal liver, which produces erythrocytes in utero. In catheterized fetal pigs, cortisol levels are highly correlated with fetal hemoglobin levels [22], but such a relation was not present in fetal calves. Basal cortisol levels were similar in newborn IVP and AI calves, and no differences in the basal and ACTH-stimulated cortisol levels were present before birth. In most species investigated, including cattle, basal cortisol level and adrenal sensitivity to ACTH increase concomitantly in late gestation [23], and these endocrinological changes play a pivotal role in the normal maturation of many essential organs in the fetus and neonate [24]. In the present study, IVP did not appear to change the normal organ allometry during the last month of gestation although earlier studies have indicated increased weights of organs such as liver and heart in IVP calves [2, 7]. However, a tendency to lowered relative thymus weight in IVP calves (P = 0.14) would lend support to the hypothesis that the IVP calves were slightly more mature than the corresponding control AI calves.
The increase in arterial O2 content in the IVP calves did not appear to be associated with a change in fetal oxygen consumption, as umbilical blood flow and the venous-arterial difference in O2 content across the umbilical circulation were similar in the two groups of calves. The observed preliminary values for umbilical and uterine blood flows were in the low range of values found in Jersey cows close to term [14] but in the high range of values found for Charolais, Hereford, or Brahman pregnant cows in the second trimester of gestation [25, 26]. There were, however, differences in the supply of oxidative substrates between IVP and AI fetal calves. In the IVP fetuses, there was a significant venous-arterial concentration difference in blood lactate across the umbilical circulation, which was not observed in the AI fetuses. On the other hand, the umbilical venous-arterial concentration difference in blood glucose tended to be lower in IVP than in AI fetal calves. These observations suggest that umbilical lactate uptake was greater, and umbilical glucose uptake lower, in IVP than in AI fetuses. The difference in fetal substrate/oxygen quotients between IVP and AI calves indicates that IVP animals rely more on lactate and less on glucose as oxidative substrates than AI calves.
The IVP glucose quotient was low not only compared with that in the AI calves, but also compared with values for Hereford fetuses at 180 days gestation (0.74 ± 0.11) [25] and for Jersey fetuses at a similar stage in gestation (0.57 ± 0.04) [14]. The third major oxidative substrate in fetal calves, amino acids, was not measured in the present study, but these are known to contribute less than glucose and lactate to total substrate oxidation in utero [14, 25]. As the umbilical uptake of glucose normally decreases while that of lactate increases towards term [14, 17, 25, 26], our glucose/lactate data indicate that IVP has advanced the normal gestational changes in the nutritional demands of the calf fetus. Alternatively, there may have been changes in the transfer capacity and/or nutrient requirements of the placenta that account for the apparent differences in umbilical uptakes of glucose and lactate between the IVP and AI calves.
Differences were also observed in the blood chemistry of the pregnant cows carrying IVP and AI calves (blood gas, ions and hemoglobin levels), and these changes may have consequences for uterine oxygen delivery and for placental and myometrial function during late gestation in IVP calves. It is also noteworthy that an altered placenta morphology [2, 27] and elevated incidence of abnormal fetal membranes have been reported for IVP pregnancies [1, 3, 28]. Nevertheless, it remains that disturbances at the materno-fetal interface were associated with only limited effects on the fetus in the present study.
Contrary to earlier reports, IVP offspring in the present study did not appear to have a decreased capacity to adapt to the stress of birth and neonatal life. Although the induction of birth was associated with a drop in O2ct values only in the IVP calves, there were no differences in blood chemistry between the IVP and AI calves just before delivery. Both groups also showed similar signs of prematurity compared with term AI calves. The slightly lowered levels of glucose, sodium, and chloride in the first 24 h after birth of the IVP calves suggest that glucose homeostasis and renal function were delayed in the IVP calves at birth. On the other hand, the slightly better ability to restore body temperature, blood pH, and blood oxygenation after birth indicates that thermogenetic capacity and respiratory function were better in the IVP calves.
The observation that IVP increased the length of limbs is consistent with the longer limbs observed recently in 7-day-old IVP calves [29] and the disproportional skeletal growth in IVP calf fetuses [2, 30]. The cause of these effects remains unknown, but may involve both bone growth metabolites (e.g., Ca2+) and endocrinological factors (e.g., insulin-like growth factor [IGF]-I, IGF-II). In relation to the limbs, it is noteworthy that the premature IVP calves showed more disturbance in blood ion levels after birth (hyponatremic + hyperkalemic). From studies in other species, it is known that a period of hyperkalemia will potentiate the inhibitory effect of low extracellular sodium levels on skeletal muscle force development [31, 32]. A clinical assessment of skeletal muscle function could not be done in the present study since none of the premature calves were able stand or walk independently during the first 24 h after birth.
Embryo cloning and IVP techniques with extreme culture or freezing conditions appear to induce the most pronounced increases in birth weight and incidence of malformations [1, 4, 7–9, 28, 33–36]. More moderate effects upon pregnancy rates, perinatal morbidity, and mortality are reported when offspring are produced with conventional in vitro fertilization techniques [5, 6, 37]. Whether IVP alters fetal development continuously, across the animal population, or just in a few individuals remains unclear. The majority of abnormal IVP embryos may be lost early in pregnancy as the pregnancy loss and abortion rate are highest in early gestation and higher after transfer of IVP embryos than after transfer of normally fertilized embryos [3, 6, 35, 37, 38]. The physiological differences observed in the IVP calves late in gestation in the present study may therefore reflect the developmental changes in IVP calves with the least pronounced abnormalities, which have survived implantation, placentation, and the early stages of fetal life.
In conclusion, all the effects of IVP identified in the present study were relatively small and unlikely to account for the widely reported reduced perinatal survival of IVP calves. In fact, a number of the parameters indicated enhanced rather than retarded maturation. Further studies on IVP calves exhibiting a more typical "large offspring syndrome" are needed to determine the physiological basis for the growth disturbances and poor viability associated with certain IVP techniques in this species.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by the Danish Biotechnology Program and by the Danish Agricultural and Veterinary Research Council. 
2 Correspondence: M. Schmidt, Division of Animal Reproduction, Royal Veterinary and Agricultural University, Dyrlægevej 68, DK-1870 Frederiksberg C, Denmark. FAX: 45 35 28 29 72; e-mail: mhs@kvl.dk
Accepted: December 22, 1999.
Received: August 17, 1999.
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