Single fixed-time laparoscopic intrauterine insemination as a tool to obtain low-diversity porcine embryos

Abstract

Double fixed-time insemination after ovulation induction is commonly used in pigs to obtain in vivo produced embryos at defined stages of development for downstream biotechnological applications. However, variations in the time of ovulation and fertilisation of the ovulated oocytes by spermatozoa, mainly in one of the inseminations, can cause diversities in embryo development. The aim of the present study was to reduce embryo diversity and to achieve a 'uniform outcome' of porcine embryo stages using single laparoscopic fixedtime insemination (LIUI). Altogether, 48 puberal German Landrace gilts were included in the study. Estrus of gilts was synchronized by 15-day long altrenogest (Regumate ® ) feeding and follicle development was stimulated with 850 IU eCG 24 h after the final altrenogest application. Ovulation was induced with 500 IU hCG 80 h after eCG. LIUI was performed 31 h after hCG treatment. Gilts under general anaesthesia were fixed in a dorsal position, a pneumoperitoneum was produced and three trocar cannulas were inserted into the abdomen for optics and instruments. Each uterine horn was carefully punctured 10-15 cm caudal from the utero-tubal junction with a 2.5 mm trocar. A 2.2 mm catheter was inserted about 3 cm into the uterine lumen and 20 ml of extended fresh boar semen (32.2 × 106 sperm cells/ml) were injected. Embryos were surgically flushed from the genital tract two (Day 2) and three (Day 3) days after insemination. Altogether, 778 oocytes/embryos were recovered (recovery rate 68 ± 17%); 45 of 48 gilts (93.8%) revealed fertilisation and 76.1% of the recovered embryos (n = 592) were at the 2-and 4-cell stage. On Day 2 (n = 22 gilts), a higher percentage of gilts (72.7%, P < 0.05) displayed only 2-cell embryos compared with gilts which had 2-and 4-cell (22.7%), or only 4-cell embryos (4.6%). On Day 3 (n = 23 gilts), the proportion of gilts with 2-cell, 2-and 4-cell, and only 4-cell embryos shifted to 4.3%, 0% and 95.7%, respectively (P < 0.05). The results of the present study demonstrate high rates of fertilisation and homogenously developed embryos after single fixed-time laparoscopic intrauterine insemination in gilts. Additionally, these results were achieved by inseminating a 60% lower number of sperm cells per insemination dose compared to usual doses used for transcervical insemination. In conclusion, LIUI can be recommended for the in vivo production of embryos in a homogeneous developmental stage, and also as an alternative method for low-dose insemination. Keywords: laparoscopic insemination; embryo diversity; pigList of abbreviations BW = body weight, CL = corpus luteum, eCG = equine chorionic gonadotropin, hCG = human chorionic gonadotropin, IU = international unit, LIUI = laparoscopic fixed-time inseminationIn vivo-derived embryos at a defined stage of development are often a necessary requirement for biotechnological applications and research. However, different developmental stages of the embryos (embryo diversity) at the time point of recovery remain an issue pigs (S...

Comparative efficiency of novel laparoscopic and routine vaginal inseminations with cryopreserved semen in rabbits

“…The LAI method is an advanced and well-developed technique in sheep, goat, porcine and bitch (Brüssow et al, 2013;Killen & Caffery, 1982;Martinez et al, 2007;Silva et al, 1995). Pregnancy rates by this technique are reported to be higher even with lower concentrations of sperm cells.…”

Influence of unilateral ovariectomy performed before ovulation on ovarian function, steroid hormone levels and development of porcine fetuses

“…Laparoscopic intrauterine insemination (LIUI): All gilts were inseminated laparoscopically 31 h after GnRH application with 20 ml/uterine horn of extended, fresh boar semen (30 × 106 sperm cells/ml; 65% motility) as described previously [5][6]. Anaesthetized gilts were fixed in a dorsal position.…”

Is the Function of the Porcine Sperm Reservoir Restricted to the Ovulatory Period?

“…Semen was collected from the same proven AI Pietrain boar (AI Station BVN, Malchin, Germany). LIUI was performed as described previously [ 15 , 17 ]. Briefly, general anaesthesia in gilts was induced with ketamine (17.25 mg/kg BW, Ursotamin®, Serumwerk Dessau, Germany) and azaperone (1.2 mg/kg BW, Stresnil®, Elanco Animal Health, Bad Homburg, Germany) and animals were fixed in a dorsal position.…”

“…Since insemination of sows out of estrus bears some difficulties, we used laparoscopic intrauterine insemination (LIUI) to ensure appropriate application of semen at these different time points of the estrus cycle. The LIUI approach has been successfully applied in previous studies [ 12 , 16 , 17 ].…”