9 SINGLE FIXED-TIME LAPAROSCOPIC INTRAUTERINE INSEMINATION IN PIGS TO PRODUCE LOW-DIVERSE EMBRYOS

Abstract

In vivo-derived embryos at a defined stage of development are often a necessary requirement for ongoing biotechnological applications. Because double fixed-time insemination after ovulation induction is commonly used in pigs to produce embryos, variations in the time of ovulation and fertilization of the ovulated oocytes by spermatozoa mainly of 1 of the 2 inseminations can cause, however, diversities in embryo development. To moderate embryo diversity and to realize a uniform outcome of porcine embryo stages, single laparoscopic fixed-time insemination can be used to minimize embryo diversity. The potential of laparoscopic intrauterine insemination (LIUI) has been demonstrated in sperm-mediated gene transfer (Fantinati et al. 2005) and evaluation of sperm migration (Brüssow et al. 2006, 2011). The aim of the present study was to analyze the development and possible diversity of embryos after LIUI. Forty-eight puberal German Landrace gilts were included in the study. Estrus of gilts was synchronized by 15-day Regu-Mate^® (Intervet, Millsboro, DE, USA) feeding and follicle development was stimulated with 850 IU of eCG 24 h after Regu-Mate^® treatment. Ovulation was induced by 500 IU of hCG 80 h after eCG treatment. The LIUI was performed 31 h after hCG treatment. To that, ketamine/azaperone-anaesthetized gilts were fixed in a dorsal position, a pneumoperitoneum was produced and 3 trocar cannulas were inserted into the abdomen for optics and instruments. Laparoscopic handling was observed on a television monitor. Each uterine horn was carefully fixed with an atraumatic forceps 10 to 15 cm caudal from the utero-tubal junction and the uterine wall was punctured with a 2.5-mm diameter trocar. A 2.2-mm catheter connected to a syringe was inserted about 3 cm into the uterine lumen and 20 mL of extended, fresh boar semen (32.2 × 10⁶ sperm cells mL^–1; 65% motility) was deposited in the lumen. Embryos were surgically flushed from the genital tract on Day 2 and 3, respectively. Altogether, 778 oocytes were recovered (recovery rate 68 ± 17%); 45 of 48 gilts (93.8%) revealed fertilization 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 displayed only 2-cell embryos compared with both 2- and 4-cell, and only 4-cell embryos (72.2 v. 22.7 and 4.6%, P < 0.05; chi-square test). On Day 3 (n = 23 gilts), there was a shift regarding the embryo stage. The proportion of gilts with 2-cell, 2- and 4-cell, and only 4-cell embryos was 4.3, 0, and 95.7%, respectively (P < 0.05). Results of the present study demonstrate high rates of fertilization and of non-diverse developed embryos after single fixed-time LIUI in gilts. Additionally, these results were achieved after inseminating a 75% lower number of sperm cells per insemination dose. Laparoscopic intrauterine insemination can be suggested as an alternative for insemination of sex-sorted semen where the number of available sperm cells after the sorting procedure is restricted.