Department of Large Animal Clinical Sciences, School of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, TX, USA.
Department of Large Animal Clinical Sciences, School of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, TX, USA.
Theriogenology. 2023 Jan 1;195:85-92. doi: 10.1016/j.theriogenology.2022.10.014. Epub 2022 Oct 17.
Intracytoplasmic Sperm Injection (ICSI) using frozen/thawed sperm is a common procedure to obtain embryos from fertile or subfertile mares and stallions. Stallion-associated factors that impact the efficiency of ICSI have been studied less than those associated with the mare. Three experiments were conducted: Experiment 1: the effect of freezing extender composition and cryoprotectant; Experiment 2: the effect of sperm exposure to seminal plasma prior to freezing (ejaculated vs. epididymal sperm; two-freeze/thaw cycles each); and Experiment 3: the effect of sperm morphologic feature used for fertilization (normal vs. cytoplasmic droplet vs. bent tail); on the blastocyst rate after ICSI. In Experiment 1, stallion sperm was cryopreserved using commercially available extenders containing: a) 2% egg-yolk + milk + 4% glycerol (MFR5); b) 2% egg-yolk + milk + 2% glycerol + 3% methyl formamide (CMMFR5); c) 20% egg-yolk + 4.75% glycerol (LE); or d) 20% egg-yolk + 2% glycerol + 3% methyl formamide (CMLE). Sperm from each of the treatment groups were used for Piezo-driven ICSI on in vitro-matured equine oocytes (n = 321). Extender CMLE resulted in a lower cleavage rate (35%) than the other treatment groups (MFR5: 74%, CMMFR5: 62%, LE: 68%; P < 0.05). Extender MFR5 yielded a higher blastocyst rate per injected oocyte (21/82 [26%]) than the Groups LE (8/77 [10%]), CMLE (4/80 [5%]) or CMMFR5 (4/82 [5%]; P < 0.05). Extender MFR5 also yielded a higher blastocyst rate per cleaved oocyte (34%) than Groups LE, CMLE or CMMFR5 (15%, 14%, 8%; respectively P < 0.05). In Experiment 2, ejaculated (EJ) and epididymal (EPD) sperm from a fertile stallion which was initially cryopreserved in the CMLE extender, was thawed and re-cryopreserved in MFR5 extender for use in ICSI. Sperm from both groups (EJ vs. EPD) were used for ICSI on in vitro matured oocytes (n = 127). Differences were not detected for cleavage rate (EJ: 36/63 [57%] vs. EPD: 49/64 [77%]), blastocyst rate per injected oocyte (EJ: 11/63 [17%] vs. EPD: 11/64 [17%]), or blastocyst rate per cleaved oocyte (EJ: 31% vs. EPD: 22%) between treatment groups (P > 0.05). In Experiment 3, morphologically normal sperm (N), or sperm with proximal droplets (PD) or bent tails (BT), were obtained from a single fertile stallion and were used for ICSI on in vitro matured oocytes (n = 75). No differences were detected among treatment groups for cleavage rate (N: 19/25 [77%] vs. PD: 20/25 [88%] vs. BT: 18/25 [72%]), blastocyst rate per injected oocyte (N: 6/25 [24%] vs. PD: 5/25 [20%] vs. BT: 2/25 [8%]), and blastocyst rate per cleaved oocyte (N: 32% vs. PD: 23% vs. BT: 11%; P > 0.05). In conclusion, the current study indicates that freezing extender composition used for stallion sperm cryopreservation has an impact on the developmental competence of in vitro-matured equine oocytes after ICSI and in vitro culture. Furthermore, we were unable to detect differences on cleavage and blastocyst rates when performing ICSI when using: 1) ejaculated or epididymal sperm; or 2) sperm with different morphologic features. The results from the current study provide additional insight regarding stallion-related factors that should be considered when performing ICSI in horses.
胞浆内精子注射（ICSI）使用冷冻/解冻精子是从肥沃或半肥沃的母马和种马中获得胚胎的常见程序。与母马相关的影响 ICSI 效率的因素研究得还不如与种马相关的因素多。进行了三项实验：实验 1：冷冻扩展剂成分和冷冻保护剂的影响；实验 2：精子在冷冻前暴露于精液对受精的影响（射出的与附睾的精子；每个冷冻/解冻循环两次）；实验 3：用于受精的精子形态特征的影响（正常的与细胞质液滴的与弯曲尾巴的）；对 ICSI 后的囊胚率的影响。在实验 1 中，使用市售的含有以下成分的扩展剂冷冻保存种马精子：a）2%蛋黄+牛奶+4%甘油（MFR5）；b）2%蛋黄+牛奶+2%甘油+3%甲酰胺（CMMFR5）；c）20%蛋黄+4.75%甘油（LE）；或 d）20%蛋黄+2%甘油+3%甲酰胺（CMLE）。来自每个处理组的精子用于在体外成熟的马卵母细胞上进行 Piezo 驱动的 ICSI（n=321）。与其他处理组（MFR5：74%，CMMFR5：62%，LE：68%；P<0.05）相比，扩展剂 CMLE 导致更低的卵裂率（35%）。扩展剂 MFR5 比 LE（21/82 [26%]）、CMLE（4/80 [5%]）或 CMMFR5（4/82 [5%]）组产生更高的囊胚率每注射卵母细胞（P<0.05）。与 LE、CMLE 或 CMMFR5 组（15%、14%、8%；分别为 P<0.05）相比，MFR5 扩展剂还产生了更高的每卵裂卵母细胞的囊胚率（34%）。在实验 2 中，从最初在 CMLE 扩展剂中冷冻保存的可育种马中解冻并重新冷冻保存的射出（EJ）和附睾（EPD）精子，用于在体外成熟的卵母细胞上进行 ICSI（n=127）。两组（EJ 与 EPD）的精子用于 ICSI。卵裂率（EJ：36/63 [57%] 与 EPD：49/64 [77%]）、每注射卵母细胞的囊胚率（EJ：11/63 [17%] 与 EPD：11/64 [17%]）或每卵裂卵母细胞的囊胚率（EJ：31% 与 EPD：22%）在处理组之间没有差异（P>0.05）。在实验 3 中，从单个可育种马中获得形态正常的精子（N）或带有近端液滴的精子（PD）或弯曲尾巴的精子（BT），并用于在体外成熟的卵母细胞上进行 ICSI（n=75）。处理组之间在卵裂率（N：19/25 [77%] 与 PD：20/25 [88%] 与 BT：18/25 [72%]）、每注射卵母细胞的囊胚率（N：6/25 [24%] 与 PD：5/25 [20%] 与 BT：2/25 [8%]）或每卵裂卵母细胞的囊胚率（N：32% 与 PD：23% 与 BT：11%）方面没有差异（P>0.05）。总之，本研究表明，用于种马精子冷冻保存的冷冻扩展剂成分对 ICSI 后体外成熟的马卵母细胞的发育能力以及体外培养有影响。此外，当进行 ICSI 时，我们无法检测到使用：1）射出的或附睾的精子；或 2）具有不同形态特征的精子时，在卵裂和囊胚率方面的差异。本研究的结果提供了关于在马中进行 ICSI 时应考虑的与种马相关的因素的更多见解。
