Embrapa, Juiz de Fora, MG 36038-330, Brazil.
Reprod Biol Endocrinol. 2013 Aug 2;11:73. doi: 10.1186/1477-7827-11-73.
Most studies on granulosa cell (GC) function in cattle have been performed using GC and follicular fluid (FF) samples collected from slaughterhouse ovaries. Using this approach, the follicular developmental stage and functional status are unknown and indirectly inferred, limiting data interpretation. Ultrasound-guided follicle aspiration has previously been used to recover GC or FF samples, but this was mostly carried out in large follicles or pools of small follicles, without recording the efficiency of recovery. The present study was aimed at adapting and evaluating an ovum pick-up (OPU) system for the in vivo recovery of FF and GC from individual follicles of different diameters.
In the first trial, the losses of fluid inside the tubing system were calculated using a conventional or an adapted-OPU system. Blood plasma volumes equivalent to the amount of FF in follicles of different diameters were aspirated using a conventional OPU Teflon circuit. The OPU system was then adapted by connecting 0.25 mL straws to the circuit. A second trial evaluated the efficiency of FF recovery in vivo. Follicles ranging from 4.0 to 16.8 mm in diameter were aspirated individually using the conventional or adapted-OPU systems. A third trial assessed the in vivo recovery of GC and the subsequent amount of RNA obtained from the follicles of different diameters from Holstein and Gir cattle.
In Trial I, the plasma recovery efficiency was similar (P > 0.05) for the volumes expected for 12 and 10 mm follicles, but decreased (P < 0.05) for smaller follicles (45.7+/-4.0%, 12.4+/-4.3% and 0.0+/-0.0% for 8, 6, and 4 mm follicles, respectively). Using the adaptation, the losses intrinsic to the aspiration system were similar for all follicle diameters. In Trial II, the expected and recovered volumes of FF were correlated (r = 0.89) and the efficiency of recovery was similar among follicles <12 mm, while larger follicles had a progressive increase in FF losses that was not related to the tubing system. In Trial III, the number of GC and amount of RNA obtained were not affected (P > 0.05) by follicle size, but differed according to breed (615,054+/-58,122 vs 458,095+/-36,407 for Holstein and Gir, respectively; P < 0.05).
The adapted-OPU system can be successfully used for the in vivo collection of FF and GC from follicles of different diameters. This will enable further endocrine, cellular, and gene expression analyses.
大多数关于牛颗粒细胞（GC）功能的研究都是使用从屠宰场卵巢中采集的 GC 和卵泡液（FF）样本进行的。使用这种方法，卵泡的发育阶段和功能状态是未知的，只能间接推断，这限制了数据的解释。超声引导下的卵泡抽吸术以前曾用于回收 GC 或 FF 样本，但这主要是在大卵泡或小卵泡池中进行的，没有记录回收效率。本研究旨在适应和评估一种卵母细胞抽吸（OPU）系统，用于从不同直径的单个卵泡中体内回收 FF 和 GC。
在第一项试验中，使用传统或改良的 OPU 系统计算管内系统内液体的损失。使用传统的 OPU 聚四氟乙烯管回路从不同直径的卵泡中抽吸相当于 FF 量的血浆体积。然后通过将 0.25 mL 吸管连接到回路上来改进 OPU 系统。第二项试验评估了体内 FF 回收的效率。使用传统或改良的 OPU 系统分别抽吸直径为 4.0 至 16.8 毫米的卵泡。第三项试验评估了体内 GC 的回收以及随后从荷斯坦和吉尔牛的不同直径卵泡中获得的 RNA 量。
在试验 I 中，对于 12 和 10 毫米卵泡的预期体积，血浆回收效率相似（P>0.05），但对于较小的卵泡（8、6 和 4 毫米卵泡的 45.7+/-4.0%、12.4+/-4.3%和 0.0+/-0.0%）则降低（P<0.05）。使用改良方法，所有卵泡直径的抽吸系统固有的损失相似。在试验 II 中，预期和回收的 FF 体积呈正相关（r=0.89），<12 毫米的卵泡回收率相似，而较大的卵泡 FF 损失逐渐增加，与管腔系统无关。在试验 III 中，GC 的数量和获得的 RNA 量不受卵泡大小的影响（P>0.05），但根据品种而有所不同（荷斯坦牛和吉尔牛分别为 615,054+/-58,122 和 458,095+/-36,407；P<0.05）。
改良的 OPU 系统可成功用于从不同直径的卵泡中体内采集 FF 和 GC。这将能够进一步进行内分泌、细胞和基因表达分析。
