128 ADDING SERUM OF COWS SUPPLEMENTED WITH β-CAROTENE DURING BOVINE IN VITRO EMBRYO CULTURE HAS NO EFFECT ON EMBRYO DEVELOPMENT

Abstract

Elevated serum NEFA concentrations, typically present in negative energy balance (NEB) cows, are known to compromise bovine in vitro oocyte and embryo quality and developmental competence. These observations seem to be associated with oxidative stress. Therefore, antioxidant supplementation such as β-carotene (bC) can be a promising solution to ameliorate embryo quality and survival. However, little is known about the possible neutralizing effect of bC on NEB-compromised embryos. Accordingly, we hypothesise that bC can overcome the potential negative effects of metabolic conditions associated with NEB on embryo development. To investigate this we aimed to evaluate the effect of serum from bC-supplemented positive energy balance (PEB) or NEB cows on embryonic developmental competence. A total of 5 nonlactating Holstein-Friesian cows were subjected to 4 consecutive dietary treatments, 28 days each: 1) 1.2 × maintenance (M) (= PEB–bC), 2) 1.2 × M with daily 2000 mg of bC (Rovimix 10% bC, DSM) (= PEB+bC), 3) 0.6 × M + bC (= NEB+bC), and 4) after a 6 week acclimatization period 0.6 × M (= NEB–bC). Serum was collected 72 h after ovulation, pooled per dietary treatment, and heat inactivated during 30 min at 56°C. In total 1404 bovine slaughterhouse grade 1 cumulus-oocyte complexes were serum-free matured (4 repeats), routinely fertilized, and cultivated for 6.7 days with the addition of 10% serum of the 4 different treatments. Cleavage (48 h post-insemination), blastocyst rates (7.7 days post-insemination), and the rates of blastocysts from cleaved zygotes were calculated. Developmental competence data were compared between the 4 treatments using a binary logistic regression model taking replicate, treatment, and the interaction of both factors into account. The NEFA and bC data were analysed using a paired-samples t-test (IBM SPSS Statistics 20). Bonferroni correction was applied. Serum NEFA concentrations were significantly elevated in NEB compared to PEB (0.36 ± 0.18 mM v. 0.21 ± 0.11 mM; P = 0.011). β-Carotene supplementation drastically increased bC concentrations in serum in NEB (0.44 ± 0.18 μg mL^–1 v. 3.28 ± 0.78 μg mL^–1; P < 0.001) as well as in PEB (1.02 ± 0.91 μg mL^–1 v. 3.04 ± 1.28 μg mL^–1; P < 0.001). Unexpectedly no significant differences were found on cleavage rates (on average 81%), subsequent development until blastocyst stage (on average 29%), or blastocyst rates from cleaved zygotes (on average 36%). Briefly, our model was not able to indicate any negative effect of NEB serum on in vitro embryo development compared with PEB, and hence no extra beneficial effects of bC could be observed on the outcome. In conclusion, these data show that more research is needed to optimize this model to investigate the effect of specific dietary strategies on pre-implantation embryo quality.