Effect of dosage and application mode of l‐carnitine on plasma lipid and egg‐yolk cholesterol of turkeys,hatchability of eggs and post‐hatch growth of their offsprings

The effect of dosage and application mode of l ‐carnitine on plasma lipid and egg‐yolk cholesterol of breeder turkeys, hatchability of eggs and post‐hatch growth response was investigated using 180 breeder hens. The hens were assigned to six dietary treatments in a 2 × 3 factorial arrangements of two application modes of l ‐carnitine (diet and drinking water) supplemented at 0, 50 and 100 ppm (mg/kg or mg/l) levels, respectively. Each treatment was replicated five times with six hens per replicate. Dietary inclusion of 50 ppm l ‐carnitine showed the lowest (p < 0.01) plasma total cholesterol (TC) and low‐density lipoprotein concentration (LDL). Breeder hens offered 50 ppm l ‐carnitine with no regard to application mode recorded the highest (p < 0.01) plasma high‐density lipoprotein (HDL). Hens offered 50 and 100 ppm l ‐carnitine irrespective of application mode also showed reduced (p < 0.01) egg‐yolk TC concentration at 32 weeks of age. Dietary supplementation of 50 ppm l ‐carnitine for breeder turkeys recorded the lowest (p < 0.01) egg‐yolk triglyceride (TG) at 40 weeks of age. Hens offered 50 ppm l ‐carnitine irrespective of application mode recorded the highest (p < 0.05) hen‐day egg production. Incidence of dead‐in‐shell also reduced (p < 0.05) with increasing dosage of l ‐carnitine. Dietary supplementation of 50 ppm and oral application in drinking water of 100 ppm l ‐carnitine for breeder turkeys resulted in highest (p < 0.05) egg fertility. Offsprings from breeder hens fed diets supplemented with l ‐carnitine recorded no post‐hatch mortality. Highest (p < 0.05) post‐hatch final live weight and weight gain was obtained with poults obtained from hens fed diet supplemented with 50 ppm l ‐carnitine. In conclusion, dietary supplementation of 50 ppm l ‐carnitine for turkey hens showed improved serum lipid profile, egg fertility, reduced dead‐in‐shell, egg‐yolk cholesterol and resulted in improved post‐hatch growth performance.     

Developmental regulation and modulation of apoptotic genes expression in sheep oocytes and embryos cultured in vitro with L‐carnitine

The objective of this study was to find out the impact of L‐carnitine (10 mM) on developmental regulation of preimplantation sheep embryos cultured in vitro when supplemented in maturation medium and post‐fertilization medium separately. Subsequent objective was to observe the L‐carnitine‐mediated alteration in expression of apoptotic genes (Bcl2, Bax, Casp3 and PCNA) in sheep oocytes and developing embryos produced in vitro. Oocytes matured with L‐carnitine showed significantly (p < .05) higher cleavage (67.23% vs 43.12%), morula (47.65% vs 28.58%) and blastocysts (32.12% vs 13.24%) percentage as compared to presumptive zygotes cultured with L‐carnitine during post‐fertilization period. So it is suggested to use L‐carnitine during maturation than post‐fertilization period. Antiapoptotic and proliferative effects of L‐carnitine were confirmed by inducing culture medium with actinomycin D (apoptotic agent) and TNFα (antiproliferative agent), respectively, with and without L‐carnitine. Oocytes and embryos cultured with actinomycin D and TNFα showed developmental arrest with significant (p < .05) decrease in morula and blastocysts percentage but s upplementation of L‐carnitine to actinomycin D and TNFα induced culture medium showed similar result as that of control . L‐carnitine supplementation during IVM significantly (p < .05) upregulated the expression of Bcl2 and PCNA genes in majority of the developmental stages. Although L‐carnitine upregulated the expression of Bax in initial developmental stages but downregulated at latter part, whereas the expression of Casp3 was upregulated upto 16‐cell stage but after that there was no difference in expression. Expression of GAPDH gene was not affected by L‐carnitine supplementation. In conclusion, L‐carnitine acted as an antiapoptotic and proliferative compound during embryo development and supplementation of L‐carnitine during IVM altered the expression of apoptotic genes in the developmental stages of embryos.     

Effect of Sericin Supplementation During In Vitro Maturation on the Maturation,Fertilization and Development of Porcine Oocytes

This study aimed to examine the effects of sericin supplementation during in vitro oocyte maturation on the nuclear maturation, fertilization and development of porcine oocytes. Cumulus‐oocyte complexes (COCs) were cultured in maturation medium supplemented with 0 (control), 0.1, 0.5, 1.0, 2.5 or 5.0% sericin and were then subjected to in vitro fertilization and embryo culture. More COCs matured with 1.0% sericin underwent germinal vesicle breakdown and reached metaphase II compared with the control COCs matured without sericin (p < 0.01). The proportions of oocytes with DNA‐fragmented nuclei did not differ between the groups, regardless of the sericin level. The total fertilization rate of oocytes matured with 1.0% sericin was higher (p < 0.05) than that of oocytes matured with 0.1%, 2.5% and 5.0% sericin. Supplementation with more than 1.0% sericin decreased the DNA fragmentation index of the blastocysts compared with the control group (p < 0.05). However, the supplementation of the maturation medium with sericin had no beneficial effects on the cleavage, development to the blastocyst stage and the total cell number of the embryos. Our findings indicate that supplementation with 1.0% sericin during maturation culture may improve the nuclear maturation and the quality of the embryos but does not affect blastocyst formation.     

Melatonin Supplementation During In Vitro Maturation and Development Supports the Development of Porcine Embryos

Melatonin has been reported to improve the in vitro development of embryos in some species. This study was conducted to investigate the effect of melatonin supplementation during in vitro maturation (IVM) and development culture on the development and quality of porcine embryos. In the first experiment, when the in vitro fertilized embryos were cultured with different concentrations of melatonin (0, 10, 25 and 50 ng/ml) for 8 days, the blastocyst formation rate of embryos cultured with 25 ng/ml melatonin (10.7%) was significantly increased (p < 0.05) compared to the control embryos cultured without melatonin (4.2%). The proportion of DNA‐fragmented nuclei in blastocysts derived from embryos cultured with 50 ng/ml melatonin was significantly lower (p < 0.05) than that of embryos cultured without melatonin (2.1% vs 7.2%). In the second experiment, when oocytes were cultured in the maturation medium supplemented with different concentrations of melatonin (0, 10, 25 and 50 ng/ml), fertilized and then cultured with 25 ng/ml melatonin for 8 days, there were no significant differences in the rates of cleavage and blastocyst formation among the groups. However, the proportions (2.7–5.4%) of DNA‐fragmented nuclei in blastocysts derived from oocytes matured with melatonin were significantly decreased (p < 0.05) compared to those (8.9%) from oocytes matured without melatonin, irrespective of the concentration of melatonin. Our results suggest that supplementation of the culture media with melatonin (25 ng/ml) during IVM and development has beneficial effects on the developmental competence and quality of porcine embryos.     

Chlorogenic acid supplementation during in vitro maturation improves maturation,fertilization and developmental competence of porcine oocytes

Chlorogenic acid (CGA) is a quinic acid conjugate of caffeic acid, and a phytochemical found in many fruits and beverages that acts as an antioxidant. The present study investigated the effects of CGA supplementation during in vitro maturation (IVM), on in vitro development of porcine oocytes, to improve the porcine in vitro production (IVP) system. Oocytes were matured either without (control) or with CGA (10, 50, 100 and 200 μM). Subsequently, the matured oocytes were fertilized and cultured in vitro for 7 day. The rates of maturation, fertilization and blastocyst formation of oocytes matured with 50 μM CGA were significantly (p < .05) higher than those of the control oocytes. Hydrogen peroxide (H₂O₂) is one of the reactive oxygen species and induces DNA damage in porcine oocytes. When oocytes were matured with 1 mM H₂O₂ to assess the protective effect of CGA, 50 μM CGA supplementation improved the maturation rate and the proportion of DNA‐fragmented nuclei in oocytes compared with control oocytes matured without CGA. Moreover, when oocytes were matured with either 50 μM CGA (control) or caffeic acid (10, 50 and 100 μM), the rates of maturation, fertilization and the blastocyst formation of oocytes matured with 50 μM CGA were similar to those of oocytes matured with 10 and 50 μM caffeic acid. Our results suggest that CGA has comparable effects to caffeic acid, and IVM with 50 μM CGA is particularly beneficial to IVP of porcine embryos and protects oocytes from DNA damage induced by oxidative stress. Supplementation of CGA to the maturation medium has a potential to improve porcine IVP system.     

Effects of supplementation of medium with different antioxidants during in vitro maturation of bovine oocytes on subsequent embryo production

The aim of this study was to assess the effects of different antioxidants on the levels of reactive oxygen species (ROS) and glutathione (GSH) in oocytes during in vitro maturation (IVM), as well as on the production of embryos. Oocyte of slaughterhouse‐derived cattle ovaries were placed in IVM with different antioxidants: quercetin (2 μM), cysteamine (100 μM), carnitine (0.5 mg/ml), vitamin C (50 μg/ml) or resveratrol (2 μM). Oocytes matured without any antioxidant supplementation were used as control. The oocytes were assessed for maturation rates and for ROS and GSH levels by fluorescence staining in 2′,7′‐dichlorodihydrofluorescein diacetate and Cell Tracker Blue, respectively. Embryo production was assessed in terms of cleavage, blastocysts and hatching rates and embryo cell numbers. The results expressed in arbitrary fluorescence units showed ROS reduction (p < .05) in the groups with quercetin (27.5 ± 3.4), vitamin C (27.1 ± 3.0) or resveratrol (28.1 ± 4.7), in comparison with those with cysteamine (34.9 ± 4.5), carnitine (34.6 ± 3.8) or to the control group (36.5 ± 5.2). GSH levels increased (p < .05) in cysteamine (63.5 ± 5.5) or carnitine (60.8 ± 4.4) groups in comparison with quercetin (52.7 ± 5.1), vitamin C (53.0 ± 3.8), resveratrol (53.1 ± 4.4) or to the control (49.6 ± 4.5). Nuclear maturation cleavage and hatched blastocysts rates did not differ (p > .05) between groups. However, blastocyst rates after in vitro fertilization in quercetin (53.5 ± 3.9%), vitamin C (52.1 ± 3.1%) resveratrol (54.2 ± 4.0%), cysteamine (52.4 ± 2.7%) or carnitine (54.2 ± 3.1%) groups were higher (p < .05) than in the control (47.2 ± 2.7%). Total cell numbers in embryos from the vitamin C, resveratrol, cysteamine or carnitine groups were higher than in quercetin and control groups, which were similar to each other. The results suggest that using antioxidants during IVM may reduce oxidative stress either by decreasing ROS levels directly or by increasing GSH levels in oocytes, depending on the type of antioxidant used. Overall, oxidative stress control during IVM with the antioxidants examined here improved blastocyst development with similar efficacy.     

Effect of Follicle Size on In Vitro Maturation of Pre‐Pubertal Porcine Cumulus Oocyte Complexes

Very small follicles (<3.0 mm diameter) are over‐represented on the surface of ovaries of non‐cycling pigs, and the oocytes collected from these follicles generally have reduced developmental competence in vitro. This study examined the effect of follicle size on the nuclear maturation (n = 608), the potential of parthenogenetic activation (n = 243) and the cyclic AMP (cAMP) content of pre‐pubertal porcine oocytes (n = 480). In addition, the influence of follicle size on steroid hormone synthesis was analysed. Cumulus oocyte complexes (COCs) flushed from small (2.5–4.0 mm) or large (4.5–6.0 mm) ovarian follicles were cultured for 0, 28 and 46 h. After 46 h of IVM, a greater proportion of oocytes from 4.5‐ to 6.0‐mm follicles reach metaphase II (MII) compared with those from follicles with 2.5–4.0 mm of diameter (96.1 vs 77.0%, respectively; p < 0.001). Parthenogenetic activation of oocytes from large follicles produced higher developmental rates than oocytes from large follicles (p < 0.05). At 28 h, the IVM medium with oocytes from large follicles contained significantly more 17ß‐oestradiol (E₂) than the medium with oocytes from small follicles (5.55 vs 3.45 ng/ml, respectively; p < 0.05) and at 46 h, the medium with oocytes from small follicles contained significantly more progesterone (P₄) than the medium with oocytes from large follicles (276.7 vs 108.2 ng/ml, respectively, p < 0.05). Porcine oocytes from large follicles have higher nuclear and cytoplasmic maturation capacities, but the differences did not appear to be cAMP‐mediated. Our findings also suggest that COCs from small follicles undergo more intensive luteinization than COCs from large follicles. The results show that oocytes from follicles with a diameter greater than 4.0 mm are more suitable for in vitro studies.     

Effect of L‐carnitine on maturation,cryo‐tolerance and embryo developmental competence of bovine oocytes

In this study, the effects of the addition of L‐carnitine in in vitro maturation (IVM) medium for bovine oocytes on their nuclear maturation and cryopreservation were investigated; they were matured in IVM medium supplemented with 0.0, 0.3, 0.6 and 1.2 mg/mL of L‐carnitine (control, 0.3, 0.6 and 1.2 groups, respectively) and some of them were vitrified by Cryotop. Moreover, the effects of L‐carnitine during in vitro fertilization (IVF) and in vitro culture (IVC) on the developmental potential and quality of IVF embryos were also examined. A significantly higher maturation rate of oocytes was obtained for 0.3 and 0.6 mg/mL groups compared with the control (P < 0.05). The blastocyst formation rate in the 0.6 group was significantly improved, whereas the rate in the 1.2 group was significantly decreased when compared with the control group (P < 0.05). No significant difference was found in embryo development between the control and the L‐carnitine group after oocyte vitrification. Supplementation of IVF and IVC media with L‐carnitine had no effect on development to the blastocyst stage of IVM oocytes treated with 0.6 mg/mL L‐carnitine. In conclusion, the supplementation of L‐carnitine during IVM of bovine oocytes improved their nuclear maturation and subsequent embryo development after IVF, but when they were vitrified the improving effects were neutralized.     

The effects of canthaxanthin on porcine oocyte maturation and embryo development in vitro after parthenogenetic activation and somatic cell nuclear transfer

The objective of this study was to examine the effects of canthaxanthin (Cx) treatment during in vitro maturation (IVM) of porcine oocytes on embryonic development after parthenogenetic activation (PA) and somatic cell nuclear transfer (SCNT), on intracellular glutathione (GSH) and reactive oxygen species (ROS) levels in mature oocytes, and on gene expression in both PA‐ and SCNT‐derived blastocysts. To determine the optimal effective concentration of Cx, porcine oocytes were cultured in IVM medium supplemented with various concentrations (0, 20, 40 and 80 μM) of Cx for 22 hr. Compared to other groups, supplementation with 40 μM Cx significantly improved blastocyst formation rates after PA (p < .05), but no significant differences were observed among groups in total blastocyst cell numbers. Subsequently, oocytes were cultured in IVM medium supplemented with or without 40 μM Cx. Oocytes treated with 40 μM Cx showed significantly increased cleavage and blastocyst formation rates after SCNT compared to the control group (p < .05). Moreover, significantly increased intracellular GSH and reduced ROS levels were observed in the Cx‐treated group (p < .05). In addition, both PA‐ and SCNT‐derived blastocysts from the 40 μM Cx‐treated group showed significantly increased mRNA expression of Bcl2 and Oct4 and decreased Caspase3 expression level (p < .05), when compared with the control group. PA‐derived blastocysts from the 40 μM Cx‐treated group also exhibited significantly decreased expression of Bax (p < .05). Our results demonstrated that treatment with 40 μM Cx during IVM improves the developmental competence of PA and SCNT embryos. Improvement of embryo development by Cx is most likely due to increased intracellular GSH synthesis, which reduces ROS levels in oocytes, and it may also positively regulate apoptosis‐ and development‐related genes.     

Effects of chlorogenic acid (CGA) supplementation during in vitro maturation culture on the development and quality of porcine embryos with electroporation treatment after in vitro fertilization

Electroporation is the technique of choice to introduce an exogenous gene into embryos for transgenic animal production. Although this technique is practical and effective, embryonic damage caused by electroporation treatment remains a major problem. This study was conducted to evaluate the optimal culture system for electroporation‐treated porcine embryos by supplementation of chlorogenic acid (CGA), a potent antioxidant, during in vitro oocyte maturation. The oocytes were treated with various concentrations of CGA (0, 10, 50, and 100 μmol/L) through the duration of maturation for 44 hr. The treated oocytes were then fertilized, electroporated at 30 V/mm with five 1 msec unipolar pulses, and subsequently cultured in vitro until development into the blastocyst stage. Without electroporation, the treatment with 50 μmol/L CGA had useful effects on the maturation rate of oocytes, the total cell number, and the apoptotic nucleus indices of blastocysts. When the oocytes were electroporated after in vitro fertilization, the treatment with 50 μmol/L CGA supplementation significantly improved the rate of oocytes that developed into blastocysts and reduced the apoptotic nucleus indices (4.7% and 7.6, respectively) compared with those of the untreated group (1.4% and 13.0, respectively). These results suggested that supplementation with 50 μmol/L CGA during maturation improves porcine embryonic development and quality of electroporation‐treated embryos.     

Effects of resveratrol on in vitro maturation of porcine oocytes and subsequent early embryonic development following somatic cell nuclear transfer

As a natural plant‐derived antitoxin, resveratrol possesses several pharmacological activities. This study aimed to evaluate the effects of resveratrol addition on nuclear maturation, oocyte quality during in vitro maturation (IVM) of porcine oocytes and subsequent early embryonic development following somatic cell nuclear transfer (SCNT). Our experiments showed that the treatment of porcine oocytes with 5 µM resveratrol during IVM resulted in the highest rate of the first polar body extrusion. Treatment of oocytes with resveratrol had no influence on cytoskeletal dynamics, whereas it significantly increased glucose uptake ability compared to the control oocytes. Oocytes matured with 5 μM resveratrol displayed significantly lower intracellular reactive oxygen species (ROS) levels and higher relative mRNA expression levels of the genes encoding such antioxidant enzymes as catalase (CAT) and superoxide dismutase 1 (SOD1). In addition, resveratrol also prevented onset and progression of programmed cell death in porcine oocytes, which was confirmed by significant upregulation of the anti‐apoptotic B‐cell lymphoma 2 (BCL‐2) gene and significant downregulation of the pro‐apoptotic BCL2‐associated X (BAX) gene. Furthermore, the blastocyst rates and the blastocyst cell numbers in cloned embryos derived from the oocytes that had matured in the presence of 5 μM resveratrol were significantly increased. In conclusion, supplementation of IVM medium with 5 μM resveratrol improves the quality of porcine oocytes by protecting them from oxidative damage and apoptosis, which leads to the production of meiotically matured oocytes exhibiting enhanced developmental potential following SCNT.     

Hanging Drop Monoculture for Selection of Optimal Antioxidants During In Vitro Maturation of Porcine Oocytes

We analysed the effect of three antioxidants that have different functional mechanisms on the in vitro maturation (IVM) of porcine oocytes. Single oocyte monoculture using the hanging drop (HD) system has some advantages such as improving analysis efficiency brought by the smaller number of samples than the number of oocytes cultured in one drop. Direct effects of ligands on single oocytes could also be detected without considering the effects of paracrine factors from other oocytes. After 22 h of pre‐culture, denuded oocytes were cultured for 22 h with 0.01 and 0.1 μg/ml of L‐carnitine (LC), lactoferrin (LF) or sulforaphane (SF) in the presence/non‐presence of oxidant stress induced by H₂O₂ supplementation to evaluate the reducing effects against oxidative stress on nuclear maturation. As a result, compared with LC and SF, LF showed effective reduction in oxidative stress at a lower concentration (0.01 μg/ml), suggesting that LF is a more effective antioxidant in porcine oocyte IVM. Additionally, LF also increased maturation rate even in culture without H₂O₂. Our results clearly suggest that the HD monoculture system is useful for screening the substances that affect porcine oocyte culture.     

Lipid Droplet Analysis Using In Vitro Bovine Oocytes and Embryos

The aim of this study was to quantify the content of lipid droplets in bovine oocytes and embryos from Bos indicus (Bi), Bos taurus (Bt) and Bos indicus × Bos taurus (Bi × Bt). Oocytes were aspirated post‐mortem and subjected to in vitro maturation, in vitro fertilization and in vitro development; the medium employed at each stage (TCM‐199, TALP, SOF) was supplemented with (i) serum replacement (SR), (ii) foetal calf serum (FCS) or (iii) oestrous cow serum (ECS). The structure and distribution of the lipid droplets were established using electron microscopy, but were quantified using an optical microscope on semi‐fine toluidine blue‐stained sections. The highest percentage of embryos corresponded to those produced with FCS and ECS, which differed from embryos generated with SR (p < 0.05). The highest percentage of morulae and the lowest percentage of blastocysts were obtained with the SR supplement (p < 0.05). The oocytes cultured in FCS demonstrated a higher number of lipid droplets compared to those cultured in SR and ECS (p < 0.05). Less accumulation of lipids was observed in embryos supplemented with SR. The lowest and highest numbers of lipid droplets in oocytes corresponded to the Bi and Bt strain, respectively. The lowest amount of lipid droplets in embryos was observed in Bi (p < 0.05). In conclusion, supplementation of the in vitro development culture medium (synthetic oviduct fluid) with a synthetic substitute serum produced similar results in terms of embryo development compared to those obtained with FCS, but a decreased degree of lipid droplet accumulation was observed in the in vitro‐cultured embryos.     

l‐carnitine and fat type in the maternal diet during gestation and lactation modify the fatty acid composition and expression of lipid metabolism‐related genes in piglets

This study examined the influence of adding different amounts of maternal dietary l ‐carnitine and two fat types on fatty acid (FA) composition and the expression of lipid metabolism‐related genes in piglets. The experiment was designed as a 2 × 2 factorial with two fat types (3.5% soyabean oil, SO, and 3.5% fish oil, FO) and two levels of l ‐carnitine (0 and 100 mg/kg) added to the sows' diets. A higher proportion of n‐3 polyunsaturated fatty acids (PUFA) and a lower ratio of n‐6/n‐3 PUFA in sow milk and piglet tissues were observed in the FO groups than in the SO groups. Adding l ‐carnitine increased the proportion of C16:1 in sow milk and decreased n‐3 PUFA in piglet subcutaneous fat. Hepatic peroxisome proliferator‐activated receptor α (PPAR‐α) was more abundantly expressed in piglets from the FO groups than from the SO groups (p < 0.05), whereas stearoyl‐CoA‐desaturase (SCD), sterol regulatory element binding protein‐1 (SREBP1) and ?6‐desaturase (D6D) genes were less expressed in the FO groups compared with piglets from the SO groups. The expression of fatty acid synthase (FAS) genes was decreased in the SO groups with l ‐carnitine compared to that of the other dietary treatments. No differences among dietary treatments were observed with regard to the expression of acetyl‐CoA carboxylase (ACC). In conclusion, FO and l ‐carnitine supplementation in sows affect FA composition and hepatic gene expression in piglets.     

Effects of different dl‐selenomethionine and sodium selenite levels on growth performance,immune functions and serum thyroid hormones concentrations in broilers

This trial was conducted in a 2 × 3 + 1 factorial arrangement based on a completely randomized design to evaluate the effects of different dl ‐selenomethionine (dl ‐Se‐Met) and sodium selenite (SS) levels on growth performance, immune functions and serum thyroid hormones concentrations in broilers. A total of 840 Ross 308 broilers (7 days old) were allocated by body weight to seven treatments (three replicates of 40 birds each treatment) including (1) basal diet (containing 0.04 mg of selenium (Se)/kg; control) without supplementary Se; (2, 3 and 4) basal diet + 0.05, 0.15 or 0.25 mg/kg Se as SS; (5, 6 and 7) basal diet + 0.05, 0.15 or 0.25 mg/kg Se as dl ‐Se‐Met. The experiment lasted 42 days. The results revealed that dietary Se supplementation improved (p < 0.05) average daily gain, feed efficiency, immune organ index, serum immunoglobulin A (IgA), immunoglobulin G (IgG), immunoglobulin M (IgM) and triiodothyronine (T₃) concentrations and decreased (p < 0.01) thyroxine (T₄)/T₃ ratio in serum compared with the control. Broilers receiving the dl ‐Se‐Met‐supplemented diets had higher (p < 0.05) feed efficiency, thymus index, the amounts of IgA, IgG, IgM and T₃ as well as lower (p < 0.05) serum T₄ concentrations and T₄/T₃ ratio than those consuming the SS‐supplemented diets. Serum IgA and IgM levels of broilers fed 0.15 mg Se/kg were significantly higher (p < 0.05) than those of broilers fed 0.05 or 0.25 mg Se/kg. In summary, we concluded that dl ‐Se‐Met is more effective than SS in increasing immunity and promoting conversion of T₄ to T₃, thus providing an effective way to improve the growth performance of broilers. Besides, based on a consideration of all experiment indices, 0.15 mg Se/kg was suggested to be the optimal level of Se supplementation under the conditions of this study.     

Effect of Temporary Meiotic Attenuation of Oocytes with Butyrolactone I and Roscovitine in Resistance to Bovine Embryos on Vitrification

This study aimed to produce in vitro bovine embryos by the addition of two drugs, which is responsible for oocyte meiosis inhibition: roscovitine (ROS) and butyrolactone I (BL‐I). Oocytes were recovered from slaughtered cows and matured in a commercial medium and maintained in a 5% CO₂ atmosphere. Oocytes were maintained for 6 h in an in vitro maturation (IVM) medium containing ROS (12.5 μm ), BL‐I (50 μm ) and association of drugs (ROS 6.25 μm and BL‐I 25 μm ). Oocytes were cultured for 18 h in an agent‐free medium for the resumption of meiosis. After 24 h of maturation, oocytes were inseminated in the commercial in vitro fertilization (IVF) medium. Presumptive zygotes were cultured in SOFaa medium in a 5% CO₂ atmosphere. On day 3, rate of cleavage was evaluated and on days 6 and 7, rate of blastocyst formation. BL‐I and its association with the ROS increased the rates of cleavage and blastocyst formation (p < 0.05). The ROS alone was inefficient, impairing embryonic development, with low rates of blastocyst formation when compared to the control group and other treatments (p < 0.05). The embryos from BL‐I and ROS+BL‐I groups presented higher number of cells and lower rates of cellular apoptosis compared to other groups, either for the fresh or for post‐thawing embryos. Embryos from ROS+BL‐I group showed to be more resistant to the vitrification process, presenting a higher rate of embryonic re‐expansion (p < 0.05). In conclusion, block of meiosis using BL‐I or its association with ROS increased the rate of blastocyst formation, and the association of ROS+BL‐I resulted in a better resistance to the embryo cryopreservation process.     

L‐carnitine improves hydrogen peroxide‐induced impairment of nuclear maturation in porcine oocytes

We investigated the effect of oxidative stress induced by hydrogen peroxide (H₂O₂) on lipid peroxide (LPO) level and nuclear maturation in porcine oocytes cultured with or without cumulus cells. After 22 h of pre‐culture, oocytes with attached cumulus cells (COC group) or denuded oocytes (DO group) were cultured with H₂O₂, and intra‐oocyte H₂O₂ and LPO levels were quantitatively analyzed using immunofluorescence. This is the first report evaluating LPO levels in porcine oocytes. After H₂O₂ supplementation, the DO group showed severe accumulation of H₂O₂ and LPO in the oocytes. Similarly, while inhibition of progression of nuclear maturation was observed in both groups, the effect was more severe in the DO group. These results demonstrate that cumulus cells reduce the accumulation of H₂O₂ stress in oocytes. Furthermore, we attempted to reduce the oxidative stress by H₂O₂ with L‐carnitine, a H₂O₂ scavenger. L‐carnitine decreased H₂O₂ and LPO levels in the oocytes in both groups, and improvement in the progression of impaired nuclear maturation was observed. These effects were different by the presence of cumulus cells. Our results provide that L‐carnitine is useful for alleviating H₂O₂‐induced oxidative stress by reducing LPO levels and improving the progression of nuclear maturation.