Acosta D A V, Denicol A C, Tribulo P, Rivelli M I, Skenandore C, Zhou Z, Luchini D, Corrêa M N, Hansen P J, Cardoso F C
Department of Animal Sciences, University of Illinois, Urbana, Illinois, USA; Faculty of Veterinary Medicine, Department of Clinics, Universidade Federal de Pelotas, Pelotas, Rio Grande do Sul, Brazil.
Department of Biology, Northeastern University, Boston, Massachussets, USA; Department of Animal Science, University of Florida, Gainesville, Florida, USA.
Theriogenology. 2016 Jun;85(9):1669-1679. doi: 10.1016/j.theriogenology.2016.01.024. Epub 2016 Feb 4.
Our objective was to determine the effects of supplementing methionine and choline during the prepartum and postpartum periods on preimplantation embryos of Holstein cows. Multiparous cows were assigned in a randomized complete-block design into four treatments from 21 days before calving to 30 days in milk (DIM). Treatments (TRT) were MET (n = 9, fed the basal diet + rumen-protected methionine at a rate of 0.08% [w:w] of the dry matter [DM], Smartamine M), CHO (n = 8, fed the basal diet + choline 60 g/d, Reashure), MIX (n = 11, fed the basal diet + Smartamine M and 60 g/d Reashure), and CON (n = 8, no supplementation, fed the close-up and fresh cow diets). Cows were randomly reassigned to two new groups (GRP) to receive the following diets from 31 to 72 DIM; control (CNT, n = 16, fed a basal diet) and SMT (n = 20, fed the basal diet + 0.08% [w:w] of the dry matter intake as methionine). An progesterone intravaginal insert (CIDR) device was inserted in all cows after follicular aspiration (60 DIM) and superovulation began at Day 61.5 using FSH in eight decreasing doses at 12-hour intervals over a 4-day period. On Days 63 and 64, all cows received two injections of PGF2α, and CIDR was removed on Day 65. Twenty-four hours after CIDR removal, ovulation was induced with GnRH. Cows received artificial insemination at 12 hours and 24 hours after GnRH. Embryos were flushed 6.5 days after artificial insemination. Global methylation of the embryos was assessed by immunofluorescent labeling of 5-methylcytosine, whereas lipid content was assessed by staining with Nile red. Nuclear staining was used to count the total number of cells per embryo. There was no difference between TRT, GRP, or their interaction (P > 0.05) for embryo recovery, embryos recovered, embryo quality, embryo stage, or cells per embryo. Methylation of the DNA had a TRT by GRP interaction (P = 0.01). Embryos from cows in CON-CNT had greater (P = 0.04) methylation (0.87 ± 0.09 arbitrary units [AU]) than embryos from cows in MET-CNT (0.44 ± 0.07 AU). The cytoplasmic lipid content was not affected (P > 0.05) by TRT or their interaction, but lipid content was greater (P = 0.04) for SMT (7.02 ± 1.03 AU) than that in CNT (3.61 ± 1.20 AU). In conclusion, cows in MET-CNT had embryos with lower methylation, and SMT cows had a higher lipid content than CNT. Methionine supplementation seems to impact the preimplantation embryo in a way that enhances its capacity for survival because there is strong evidence that endogenous lipid reserves serve as an energy substrate.
我们的目标是确定在产前和产后期间补充蛋氨酸和胆碱对荷斯坦奶牛植入前胚胎的影响。经产奶牛采用随机完全区组设计,从产犊前21天至产奶30天(DIM)分为四种处理。处理方式(TRT)分别为:MET组(n = 9,饲喂基础日粮 + 瘤胃保护蛋氨酸,添加量为干物质(DM)的0.08% [w:w],即Smartamine M)、CHO组(n = 8,饲喂基础日粮 + 胆碱60 g/d,即Reashure)、MIX组(n = 11,饲喂基础日粮 + Smartamine M和60 g/d Reashure)以及CON组(n = 8,不添加,饲喂围产后期和新产母牛日粮)。奶牛在产奶31至72天被随机重新分配到两个新组(GRP),分别接受以下日粮;对照组(CNT,n = 16,饲喂基础日粮)和SMT组(n = 20,饲喂基础日粮 + 占干物质摄入量0.08% [w:w]的蛋氨酸)。在所有奶牛卵泡抽吸后(60 DIM)插入孕酮阴道栓(CIDR),并在第61.5天开始使用促卵泡素(FSH)进行超数排卵,在4天内以12小时间隔分八次递减剂量注射。在第63天和第64天,所有奶牛接受两次前列腺素F2α注射,CIDR在第65天取出。CIDR取出24小时后,用促性腺激素释放激素(GnRH)诱导排卵。奶牛在GnRH注射后12小时和24小时进行人工授精。人工授精6.5天后冲洗胚胎。通过对5 - 甲基胞嘧啶进行免疫荧光标记评估胚胎的整体甲基化水平,而脂质含量通过尼罗红染色进行评估。使用细胞核染色来计算每个胚胎的细胞总数。在胚胎回收率、回收的胚胎数、胚胎质量、胚胎阶段或每个胚胎的细胞数方面,TRT、GRP或它们的交互作用之间没有差异(P > 0.05)。DNA甲基化存在TRT与GRP的交互作用(P = 0.01)。CON - CNT组奶牛的胚胎甲基化水平(0.87 ± 0.09任意单位[AU])高于MET - CNT组奶牛的胚胎(0.44 ± 0.07 AU)(P = 0.04)。细胞质脂质含量不受TRT或它们的交互作用影响(P > 0.05),但SMT组(7.02 ± 1.03 AU)的脂质含量高于CNT组(3.61 ± 1.20 AU)(P = 0.04)。总之,MET - CNT组奶牛的胚胎甲基化水平较低,SMT组奶牛的脂质含量高于CNT组。补充蛋氨酸似乎以一种增强其生存能力的方式影响植入前胚胎,因为有强有力的证据表明内源性脂质储备作为能量底物。