H Jacobsen

Body dimensions and birth and organ weights of calves derived from in vitro produced embryos cultured with or without serum and oviduct epithelium cells.

Body dimensions, birth and organ weights of calves derived from embryos produced in 2 in vitro culture systems (modified SOFaa with 20% cattle serum and co-cultured with oviduct-epithelium cells [IVPserum, n=8], and modified SOFaa with 3 mg/mL PVA [IVPdefined, n=6]) were compared with calves originating from artificial insemination (AI, n=85). Three additional IVP calves were included which had been vitrified as mature oocytes by the open pulled straw (OPS) method, warmed, fertilized and cultured to the blastocyst stage in modified SOFaa with 5% cattle serum, then again OPS-vitrified and warmed prior to transfer (IVPops, n=3). At birth, gestation length and birth weights were registered for all calves. At 1 wk of age all 17 IVP and 7 of the AI calves were killed, and their body dimensions and organ weights recorded. Birth weight was higher for the IVPserum and IVPops calves than for AI control calves (kg +/- SEM: IVPserum 46.9+/-1.8, IVPops 50.6+/-2.4, AI 41.8+/-0.8; P < 0.002). There was no difference between IVP and AI calves regarding gestation length and no effect of culture conditions on body dimensions or organ weights, except for longer hind legs in IVPdefined calves compared with AI calves (cm +/- SEM: IVPdefined 93+/-2, AI 87+/-2; P < 0.04). The IVPops calves had an increased liver weight compared with AI and the other IVP calves (g +/- SEM: IVPops 1.457+/-59; AI 1,117+/-37; IVPserum 1,159+/-34, IVPdefined 1,073+/-39; P < 0.0003). It is concluded that in vitro culture of bovine embryos in the presence of serum and oviduct epithelium cells increased birth weight but not organ weight and body dimension in 1-wk-old calves. However, vitrification of the ova as oocyte and again as blastocysts increased birth weight and liver size. This possible effect of cryopreservation of oocytes on subsequent fetal development awaits further investigation.

EASE OF CALVING, BLOOD CHEMISTRY, INSULIN AND BOVINE GROWTH HORMONE OF NEWBORN CALVES DERIVED FROM EMBRYOS PRODUCED IN VITRO IN CULTURE SYSTEMS WITH SERUM AND CO-CULTURE OR WITH PVA

Blood chemistry (pH, pCO2, pO2, glucose, lactate) as well as plasma insulin and growth hormone of calves derived from embryos produced under 2 different in vitro culture systems (modified SOFaa with 20% serum and co-culture with bovine oviduct epithelial cells [IVP serum, n=8] or with 3 mg/mL PVA [IVPdefined, n=6]) were compared with those of calves derived from AI (n=5). Calvings were classified according to the ease (unassisted, light traction, heavy traction). Blood samples were taken from the jugular vein of calves at 5, 15, 30 and 60 min, and at 2, 3, 6, 12, 18 and 24 h after delivery, then daily for 6 d. At the second day of life after 4 feedings and a 4-h fasting period, a glucose tolerance test was performed to evaluate glucose metabolism and insulin secretion. Calves in the IVP serum group had higher birth weights than AI calves (LS mean +/- SEM, IVP serum: 45.2 +/- 1.4 kg vs AI: 40.4 +/- 1.7 kg; P < 0.05), while the birth weights of calves in the IVP defined group were in between (IVPdefined: 41.9 +/- 1.6 kg). More IVP serum calves (75%) needed assistance than IVP defined (33%) or AI (40%) calves. The effect of ease of calving vs the effect of embryo culture was compared in relation to blood parameters at birth. There was an effect of ease of calving but not of embryo culture conditions on blood pH, lactate and PCO2. Calves requiring heavy traction had lower pH during the first 3 h after calving, a higher lactate during the first 60 min after calving and a higher pCO2 the first 2 h after calving than calves born unassisted. Calves requiring heavy traction also had lower pH the first 2 h and higher lactate the first 3 h after calving than calves born by light traction. IVP defined calves had lower lactate than IVP serum calves the first 60 min after calving. At 6 h after delivery, all blood parameters had stabilized. There was no effect of either embryo culture or ease of calving on basal insulin and growth hormone level, or the ability of the calves to handle glucose postnatally and during a glucose tolerance test.

Full-term development of nuclear transfer calves produced from open-pulled straw (OPS) vitrified cytoplasts: Work in progress

Cryopreservation of cytoplasts would help to resolve the logistics of matching the availability of oocytes with embryo donors in nuclear transfer. Therefore, the developmental potential of nuclear transfer bovine embryos reconstructed using vitrified cytoplasts was investigated. In vitro matured oocytes were denuded, enucleated, activated with calcium ionophore (10 microM, 5 min) and cycloheximide (10 microg/mL, 6 h) and then vitrified by the open pulled straw (OPS) method. After immediate warming, the nuclear transfer embryos were reconstructed using blastomeres from nonvitrified,in vitro-produced embryo donors. Compared with control nuclear transfer embryos that were reconstructed using nonvitrified cytoplasts, fusion rates (% +/- SEM) were not affected (83.7+/-9.2 vs. 79.8+/-4.6; P>0.05), but cleavage (55.7+/-2.9 vs. 92.8+/-3.9; P = 0.0002) and blastocyst rates (7.2+/-5.0 vs. 32.6+/-7.8; P = 0.0025, vitrified vs. nonvitrified cytoplasts, respectively) per successful fusion were reduced. One nuclear transfer blastocyst reconstructed from a vitrified cytoplast was transferred to a synchronized recipient. After a normal length gestation (265 d), twin calves (21 and 26 kg) were delivered. Microsatellite analysis confirmed that the calves were homozygotic (the embryo split in utero), and were derived from the in vitro-produced embryo donor. The twins were dead at birth, but post-mortem analysis of the calves indicated no abnormalities or infections, suggesting that their death was related to the twin pregnancy and the known fragility of nuclear transfer calves. These data demonstrate that open pulled straw-vitrified cytoplasts are capable of supporting full-term development of nuclear transfer embryos.

Macromolecule absorption and cortisol secretion in newborn calves derived from in vitro produced embryos

Earlier reports indicate that calves derived from in vitro produced (IVP) embryos are more susceptible to neonatal disease than calves produced after artificial insemination (AI) or natural mating. The aims of the present study were to investigate whether calves born after IVP embryos show an altered macromolecule absorption (immunoglobulin G (IgG) and porcine serum albumin (PSA)) compared with AI calves and whether the macromolecule absorption could be related to the degree of acidosis or to the cortisol secretion around birth. Hence, IgG and PSA absorption in control AI calves (n=7) was compared with that in two groups of IVP calves (IVP-defined: SOFaa embryo culture with polyvinyl alcohol, n=6; IVP-serum: SOFaa embryo culture with serum and co-culture, n=8). The calves were fed colostrum (40ml/kg) at 2, 6 and 12h after birth. At 24h after birth, both AI and IVP calves had achieved a level of plasma IgG sufficient to provide passive immunization (>15mg/ml). When the values were adjusted for the varying colostral IgG contents and the degree of acidosis, the IVP-defined calves had significantly lower peak plasma IgG concentrations than the AI calves at 18-24h after birth (P<0.04). However, when the macromolecule marker (PSA), was fed to all calves at 2 and 12h after birth the resulting plasma PSA levels were significantly lower in the AI calves compared with the IVP calves during the whole observation period (P<0.0001). Calves with a moderate neonatal acidosis (mean pH<7.2 during the first 30min after birth) had reduced peak plasma IgG concentration at 18-24h after birth (P<0.02) compared to calves without acidosis. The basal and ACTH-stimulated cortisol levels were lower in the newborn IVP-defined calves than in the AI calves (P<0.05) and the IVP-serum calves (P<0.002). Cortisol levels shortly after birth correlated positively with birth weight (r=0.60, P<0.0001) and with gestation length (r=0.34, P<0.04). Since, the IVP calves absorbed sufficient amounts of IgG from colostrum to acquire sufficient passive immunity, we conclude that the lower viability described in IVP offspring probably is not caused by an impaired passive immunization. IVP-defined calves had significantly lower absorption efficiency of IgG compared with AI calves, whereas absorption of a non-Ig macromolecule (PSA) was higher for IVP than AI calves. This might indicate a more selective absorption in AI calves in favor of IgG. Acidosis around birth affected immunoglobulin absorption negatively. IVP-defined calves had significantly lower cortisol levels the first 3h after birth and during an ACTH-challenge and a lower IgG absorption efficiency, which might indicate a mild degree of organ dysmaturity in these calves.