Cysteamine supplementation during in vitro maturation (IVM) of rabbit oocyte improves the developmental capacity after intracytoplasmic sperm injection

The aim of this study was 2-fold: (1) To determine if altrenogest-treated mares will yield higher numbers of quality in vitro-matured (IVM) oocytes than early pregnant mares and cycling/control mares and (2) if the addition of human follicular fluid (HFF) to IVM medium can support IVM and viable pregnancies from in vitro-produced blastocysts. In this study, 18 mares were assigned to 3 equally sized treatment groups and each mare was subjected to follicle aspiration every 10 to 11 days without monitoring follicular growth. The 3 treatment groups were altrenogest-treated mares (0.044 mg kg–1 of PO daily), early pregnant mares (30–110 days) and control/cycling mares. Using transvaginal ultrasound guidance, all visible follicles were aspirated. Altrenogest-treated mares each yielded more follicles (8.75) per aspiration session when compared with the control mare group (5.75) and the pregnant mare group (3.72), but there was no difference in oocyte recovery rates among the groups (Table 1). A limited number of these oocytes were subjected to in vitro maturation. After heated (38.5°C) transport of oocytes to an off-site laboratory, the oocytes were placed in maturation medium containing 10% HFF obtained from preovulatory follicles after ovulation induction, 20% serum substitute supplement and no hormones for 36 h. This approach yielded a maturation rate of 61.8, 68.8 and 82.0% for the altrenogest, pregnant and control treatment groups, respectively (not significant). Mature oocytes (n = 65) were injected with frozen-thawed sperm using a standard intracytoplasmic sperm injection (ICSI) technique. Four expanding blastocysts (Table 1) were selectively transported back to the embryo transfer facility and transcervically transferred into recipient mares on Day 6 post-ICSI. These 4 transfers resulted in 2 viable, normally progressing pregnancies, ongoing beyond 60 days of gestation. Both pregnancies resulted from the altrenogest-treated aspiration group. In this study we concluded that (1) altrenogest-treated mares provide more follicles and may be a better source of viable immature oocytes for the production of ICSI embryos and foals, but their overall advantage is unclear; (2) addition of HFF to IVM media, in the absence of added gonadotropins, can support oocyte maturation, blastocyst production and viable pregnancies; (3) an aspiration schedule of every 10 to 11 days without ultrasonic monitoring can yield viable immature oocytes, capable of producing ICSI blastocysts, resulting in viable pregnancies. Table 1.Altrenogest-treated mares compared to early pregnant mares and control mares

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217 IMPROVEMENT OF INTRACYTOPLASMIC SPERM INJECTION EMBRYO DEVELOPMENT IN BOVINE USING HIGH CYSTEAMINE CONCENTRATION DURING IN VITRO MATURATION AND SPERM CO-CULTURE WITH CUMULUS-OOCYTE COMPLEXES

Reproduction Fertility and Development ◽

10.1071/rdv28n2ab217 ◽

2016 ◽

Vol 28 (2) ◽

pp. 239

Author(s):

N. G. Canel ◽

M. Suvá ◽

R. J. Bevacqua ◽

D. F. Salamone

Keyword(s):

Embryo Development ◽

Intracytoplasmic Sperm Injection ◽

In Vitro Maturation ◽

Critical Role ◽

Standard Condition ◽

Bottom Part ◽

Low Levels ◽

Vitro Development ◽

Sperm Decondensation

In bovine, the intracytoplasmic sperm injection (ICSI) technique remains inefficient probably because of low levels of male sperm decondensation. In species with frequent fertilization failure, high cysteamine (Cys) concentration during in vitro maturation (IVM) has been used to improve IVF. Cysteamine, a precursor of glutathione, plays a critical role on sperm decondensation. The aim of this work was to improve ICSI efficiency in bovine by (1) increasing endogenous glutathione levels from oocytes using high Cys during IVM; and (2) incubating sperm with cumulus-oocyte complexes (COC) before ICSI, to mimic the physiological capacitation process. In experiment 1, we tested the effect of high Cys concentrations during IVM over the development of IVF embryos. In experiment 2, we performed ICSI after IVM with 1 mM Cys, based on IVF results. The COC were collected from slaughtered cow ovaries and IVM for 21 h with 10, 1, and 0.5 mM Cys v. 0.1 mM Cys (standard condition). Then, IVF was performed using 16 × 106 sperm mL–1 for 5 h on BO medium. For ICSI, COC were IVM with 1 mM Cys (ICSI 1 mM groups), and sperm used for injection was previously incubated with COC for 3 h (Inc. groups), as was done for IVF. Sham and diploid parthenogenetic (PA Diplo) controls were also included. Metaphase II oocytes were selected for ICSI, and injected oocytes were activated by a 4-min exposure to 5 μM ionomycin, placed on TCM-199 for 3 h (except for PA Diplo) and treated with 2 mM DMAP for 3 h. For ICSI control groups, COC were matured using 0.1 mM Cys. All embryos were cultured in SOF medium. Cleavage and blastocyst rates were evaluated on Days 2 and 7 post-IVF/ICSI, respectively. The total cell numbers of blastocysts were counted at Day 7, after Hoechst 33342 staining. Results are shown in Table 1. In conclusion, an increase of 5- to 10-fold of Cys concentration during IVM was not detrimental for development to blastocyst after IVF. The use of 1 mM Cys during IVM combined with the use of sperm co-cultured wit IVM COC before sperm injection is a good strategy to improve in vitro development of bovine ICSI embryos. Table 1.Effect of 1 mM cysteamine (Cys) during IVM over the development of IVF bovine embryos (top part) and effect of 1 mM Cys during IVM over embryo development of ICSI embryos, using sperm previously incubated (Inc.) with COC (bottom part)

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