The quality after culture in vitro or in vivo of porcine oocytes matured and fertilized in vitro and their ability to develop to term

Abstract

The quality of porcine blastocysts produced in vitro is poor in comparison with those that develop in vivo. We examined the quality of in vitro-matured and fertilized (IVM/IVF) oocytes, their abilities to develop to blastocysts under in vivo and in vitro conditions, and the potential of the embryos to develop to term after transfer. IVM/IVF oocytes were either transferred and the embryos recovered on Days 5 and 6 (100% and 87.5%, respectively) ('ET-vivo' embryos), or cultured in vitro for 5 or 6 days ('IVC' embryos). The proportion of blastocysts differed significantly between the two groups on Day 5 (20.6% and 8.0%, respectively), but not on Day 6 (23.8% and 21.2%, respectively). The mean number of cells in ET-vivo blastocysts on Days 5 or 6 was significantly higher (72.8 and 78.7, respectively) than that in IVC blastocysts (22.1 and 39.7, respectively). When IVM/IVF oocytes and IVC blastocysts on Day 6 were transferred, all (three and three, respectively) developed to piglets (16 and 16, respectively), without any difference in the rates of development to term (2.1% and 2.6%, respectively). These data suggest that, although blastocyst production differs between the two culture conditions, IVM/IVF oocytes possess the same ability to develop to term.

Utilization of porcine in vitro‐produced parthenogenetic embryos for co‐transfer with vitrified and warmed embryos

“…; Nakamura et al . ). Collected COCs were cultured in modified NCSU‐37 solution (Petters & Wells ) containing 10% porcine follicular fluid, 0.6 mmol/L cysteine, 1 mmol/L dibutyryl cyclic adenosine monophosphate (cAMP), 10 IU/mL equine chorionic gonadotropin (eCG, Peamex; Sankyo Lifetech Co., Ltd., Tokyo, Japan), 10 IU/mL human chorionic gonadotropin (hCG, Puberogen 500 U; Novartis Animal Health, Tokyo, Japan) and antibiotics for 20 h. They were subsequently cultured in modified NCSU‐37 solution without dibutyryl cAMP and hormones for 24 h. Culture was carried out at 39°C under O 2, CO 2 and N 2 adjusted to 5%, 5% and 90%, respectively.…”

“…; Nakamura et al . ). The frozen spermatozoa were thawed and preincubated for 15 min at 38°C in Medium 199 (with Earle's salts, Gibco) adjusted to pH 7.8 (Nagai et al .…”

“…We consider that co-transfer of in vitro-produced embryos may be a promising avenue for achieving this goal, and two sources of embryos produced in vitro for cotransfer could be potentially utilized: (i) PA embryos and (ii) in vitro fertilization (IVF) embryos. Utilization of PA embryos has already been reported (Kawarasaki et al 2009), and we have shown that in vitro-matured (IVM)/IVF oocytes have the ability to develop to term even after in vitro culture (IVC) to the blastocyst stage (Nakamura et al 2017). In the present study, we compared the abilities of both PA and IVM/IVF oocytes to develop to the blastocyst stage, and their quality after in vitro culture.…”

“…Porcine ovaries were obtained from prepubertal cross-bred (Landrace, Large White and Duroc breeds) gilts at a local slaughterhouse and transported to the laboratory in Dulbecco's phosphate-buffered saline (PBS) (Nissui Pharmaceutical Co., Ltd., Tokyo, Japan) at 37°C within 2 h. Cumulus-oocyte complexes (COCs) were collected by scraping 3-6 mm follicles in a collection medium, Medium 199 (with Hanks' salts) supplemented with 10% (v/v) fetal bovine serum (FBS; Gibco, Thermo Fisher Scientific Inc., Waltham, MA, USA), 20 mmol/L HEPES (Dojindo Laboratories, Kumamoto, Japan), antibiotics (100 units/mL penicillin G potassium; Meiji Seika Kaisha Ltd., Tokyo, Japan) and 0.1 mg/mL streptomycin sulfate (Meiji) (Kikuchi et al 1993). IVM of oocytes was carried out as previously reported (Kikuchi et al 2002;Men et al 2016;Nakamura et al 2017). Collected COCs were cultured in modified NCSU-37 solution (Petters & Wells 1993) containing 10% porcine follicular fluid, 0.6 mmol/L cysteine, 1 mmol/L dibutyryl cyclic adenosine monophosphate (cAMP), 10 IU/mL equine chorionic gonadotropin (eCG, Peamex; Sankyo Lifetech Co., Ltd., Tokyo, Japan), 10 IU/mL human chorionic gonadotropin (hCG, Puberogen 500 U; Novartis Animal Health, Tokyo, Japan) and antibiotics for 20 h. They were subsequently cultured in modified NCSU-37 solution without dibutyryl cAMP and hormones for 24 h. Culture was carried out at 39°C under O 2, CO 2 and N 2 adjusted to 5%, 5% and 90%, respectively.…”

“…), and we have shown that in vitro ‐matured (IVM)/IVF oocytes have the ability to develop to term even after in vitro culture (IVC) to the blastocyst stage (Nakamura et al . ). In the present study, we compared the abilities of both PA and IVM/IVF oocytes to develop to the blastocyst stage, and their quality after in vitro culture.…”