Impact of oxidative stress on oocyte competence for in vitro embryo production programs

The benefit of superstimulation with exogenous FSH before ovum pickup for in vitro embryo production has been the subject of significant controversy. In addition, there is limited information on different dose regimens. Thus, the objective of the present study was to evaluate the effect of dose of porcine (p)-FSH during superstimulation before ovum pickup (OPU) on in vitro embryo production in pregnant heifers. Pregnant Holstein heifers (n=36) were assigned to a complete 3×3 crossover design. Three treatment groups were evaluated as follows: p-FSH 0mg (FSH0), p-FSH 160mg (FSH160) and p-FSH 300mg (FSH300). Three sessions of OPU were performed on each animal at 48, 62 and 76 days of gestation, with a washout interval between sessions of 14 days. Follicular wave emergence was synchronized by dominant follicle removal. Heifers in the FSH0 group received no further treatment, whereas the remaining groups received a total of 4 injections 12h apart as follows: FSH160 (48.0, 42.7, 37.3 and 32.0mg) or FSH300 (90.0, 80.0, 70.0 and 60.0mg), beginning 36h after dominant follicle removal. Ovum pickup was performed in all heifers 40h after the last p-FSH injection. Heifers were subjected to OPU for oocyte recovery, and number of follicles was determined. Recovered oocytes were processed and in vitro embryo production performed. Differences between treatment groups were evaluated by generalized linear mixed models. Data are presented (Table 1) as mean±standard error of the mean. There was no effect of days in gestation for any of the outcomes evaluated (P>0.05). Follicle numbers at the time of oocyte recovery were different (P<0.01) between groups. Heifers in the FSH300 group had a greater (P<0.05) number of medium, large and total follicles than heifers in the FSH0 group, whereas heifers in the FSH160 were intermediate. Total number of recovered, viable and cleaved oocytes were greater (P<0.01) in FSH300- than in FSH160- and FSH0-treated heifers. Cleavage rate and blastocyst development rate were not different (P>0.10) between groups. The number of grade 1 and 2 blastocysts was greater in FSH300- than in FSH160- and FSH0-treated heifers (P<0.03). In summary, the use of 300mg of p-FSH before OPU in pregnant heifers increases the number of follicles, oocytes and blastocysts produced per heifer with no detrimental effect on oocyte competence. Table 1.Ovum pickup and in vitro embryo production in pregnant heifers treated with different doses of porcine FSH

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State-of-the-art production, conservation and transfer of in-vitro-produced embryos in small ruminants

Reproduction Fertility and Development ◽

10.1071/rd04029 ◽

2004 ◽

Vol 16 (4) ◽

pp. 437 ◽

Cited By ~ 57

Author(s):

Yves Cognié ◽

Nati Poulin ◽

Yann Locatelli ◽

Pascal Mermillod

Keyword(s):

Oocyte Maturation ◽

Small Ruminants ◽

Reproductive Technologies ◽

Defined Medium ◽

Blastocyst Stage ◽

Embryo Production ◽

Oocyte Competence ◽

Epidermal Growth ◽

In Vitro Embryo Production

Today, although not efficient enough to replace multiple ovulation and embryo transfer, in vitro embryo production for small ruminants is a platform for new reproductive technologies, such as embryo sexing, transgenesis and cloning. The in vitro embryo-production system developed for sheep and goats is more efficient now than 15 years ago, but could still be improved. Laparoscopic collection of oocytes in live animals treated with gonadotrophin indicates a promising future for the application of this technology to genetic improvement programmes. Oocyte maturation in defined medium with epidermal growth factor and cysteamine appears as efficient as oocyte maturation in follicular fluid-supplemented medium and allows future study of the effect of other factors involved in the cytoplasmic maturation of oocytes from these species. Further efforts have to be made to standardise the semen-capacitating process and to improve the quality and freezability of in-vitro-produced (IVP) embryos. The optimisation of IVP procedures for deer species has required the study of the seasonal variation of oocyte competence and the development of a specific methodology to allow the culture of embryos up to the blastocyst stage.

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159 IN VITRO EMBRYO PRODUCTION FROM HOLSTEIN CALF OOCYTES RECOVERED BY LAPAROSCOPIC OVUM PICKUP

Reproduction Fertility and Development ◽

10.1071/rdv28n2ab159 ◽

2016 ◽

Vol 28 (2) ◽

pp. 209

Author(s):

H. Baldassarre ◽

L. Currin ◽

L. Michalovic ◽

W. Glanzner ◽

K. Gutierrez ◽

...

Keyword(s):

Reproductive Organs ◽

Blastocyst Stage ◽

Developmental Competence ◽

Bovine Embryo ◽

Embryo Production ◽

Oocyte Competence ◽

Veterinary Practitioner ◽

Collection Tube ◽

In Vitro Embryo Production

Oocyte competence and reproductive biology in prepubertal heifer calves are not fully understood. Multiple publications have reported high oocyte yields recovered from calves aged 2–6 months old but low embryo development rates following in vitro embryo production. The objective of this study was to characterise the developmental competence of oocytes from young calves. We report herein the oocyte/embryo yields obtained from 6 Holstein heifer calves that were subjected to gonadotropin stimulation and laparoscopic ovum pick-up (LOPU) every 2 weeks, starting at 2 months of age and ending at 5 months of age. The LOPU was conducted under general anaesthesia with the animal lying in dorsal recumbency on a table with a 45-degree angle to facilitate the visualisation of reproductive organs. Briefly, looking through the laparoscope, the ovarian surface was exposed by pulling from the fimbria with an atraumatic grasping forceps. The follicle contents were aspirated using a 20 G needle mounted on a pipette, which was connected to a collection tube and a vacuum pump. Media and procedures for aspiration, in vitro maturation (IVM), IVF, and in vitro culture (IVC) were standard in use for commercial adult bovine embryo production. Because of the small number of animals and the multifactorial variables in play (age, number of previous treatments and aspirations, etc.), in this preliminary study we focused on the overall oocyte/embryo yield and the potential effects of LOPU on ovarian integrity. A total of 766 follicles were aspirated (avg. 17/calf per session) resulting in 625 cumulus-oocyte complexes (COC) recovered (avg. 14/calf per session; 82% recovery rate). A total of 457 (73%) COC were graded eligible for IVM, of which 353 cleaved (77%) and 109 (24%) reached a viable blastocyst stage at the end of IVC, of which 42 (38.5%) were graded as freezable. In balance, ~2 viable blastocysts/calf per session were produced. No adhesions or sequels were observed in the animals up to the last LOPU session, as well as 2 weeks after the last LOPU when the animals were evaluated by rectal palpation by an experienced OPU veterinary practitioner. Further studies will look into other aspects of oocyte developmental competence to better understand this biological process.

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297 ADDITION OF GLUTATHIONE TO THAWING MEDIUM FOR BULL SPERMATOZOA IMPROVES THE IN VITRO EMBRYO PRODUCTION

Reproduction Fertility and Development ◽

10.1071/rdv19n1ab297 ◽

2007 ◽

Vol 19 (1) ◽

pp. 264

Author(s):

L. Grullón ◽

J. C. Gardó ◽

F. García-Vázquez ◽

J. Gadea

Keyword(s):

Oxidative Stress ◽

Defense Mechanism ◽

Developmental Stages ◽

Ros Generation ◽

Cleavage Rate ◽

Embryo Production ◽

Short Period ◽

In Vitro Embryo Production ◽

Bull Sperm

The cold shock of spermatozoa is associated with oxidative stress induced by reactive oxygen species (ROS) generation (Chatterjee et al. 2001 Mol. Reprod. Dev. 60, 498–506). Glutathione (l-�-glutamyl-l-cysteinylglycine; GSH) plays an important role as an intracellular defense mechanism against oxidative stress. The process of freezing is associated with a significant reduction in GSH content in novine spermatozoa (Bilodeau et al. 2000 Mol. Reprod. Dev. 55, 282–288). The main objective of this study was to evaluate the effect of GSH supplementation of the thawing extender on bull sperm used for in vitro embryo production. To examine the effect of GSH supplementation during the thawing process, spermatozoa from 6 different bulls were incubated without addition of GSH (control), and with addition of 1 mM or 5 mM GSH to the sperm-TALP medium, and maintained 30 min at 37�C in these media before assay. IVM and IVF were performed as described Coy et al. (2005 Reproduction 129, 19–26, 747–755). Oocytes were fertilized with frozen–thawed semen (106 total sperm/mL). After 18 h co-incubation, they were cultured in KSOM medium for 7 days. Embryos were examined for developmental stages at Day 2 (2–4 cells), Day 3 (8–16 cells), and Day 7 (morula–blastocysts). Finally, the total number of nuclei of blastocysts was determined by staining with Hoechst 33342 (10 mg mL-1; 20 min). The percentage of cleavage in the different embryo stages was higher in the 5 mM GSH group [n = 233, Day 2 (64.63%), Day 3 (56.92%), and Day 7 (31.28%)] than in control [n = 229, Day 2 (47.64%), Day 3 (42.16%), and Day 7 (22.32%)], and the 1 mM group maintained an intermediate position with [n = 246, Day 2 (54.88%), Day 3 (49.56%), and Day 7 (28.98%)]. The bull sperm affected significantly the cleavage parameters studied. The number of nuclei within the blastocysts ranged from 39.93 to 47.71, and no differences were observed between experimental groups. It is known that addition of GSH produces a reduction in ROS generation (Gadea et al. 2005 J. Androl. 26, 749–756). The results showed that in the treatment group there is a reduction in ROS generation in spermatozoa and an improvement in the cleavage rate and blastocyst formation when GSH was used. This study demonstrates that only a short period of incubation of the spermatozoa in an antioxidant could improve the spermatozoa functionality of producing more and better embryos. This work was supported by AGL2003-03144, BIOCARM 10BIO2005/01-6463 and Seneca 03018/PI/05.

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Beneficial effects of melatonin on in vitro embryo production from juvenile goat oocytes

Reproduction Fertility and Development ◽

10.1071/rd17170 ◽

2018 ◽

Vol 30 (2) ◽

pp. 253 ◽

Cited By ~ 20

Author(s):

Sandra Soto-Heras ◽

Montserrat Roura ◽

Maria G. Catalá ◽

Irene Menéndez-Blanco ◽

Dolors Izquierdo ◽

...

Keyword(s):

Embryo Quality ◽

Cell Number ◽

Blastocyst Development ◽

Embryo Production ◽

Melatonin Concentration ◽

Oocyte Competence ◽

Beneficial Effects ◽

In Vitro Embryo Production ◽

Ovarian Follicular Fluid

Melatonin is a universal antioxidant that improves in vitro embryo production in several species. The aims of this study were to determine the melatonin concentration in the ovarian follicular fluid (FF) of juvenile goats and the effect of melatonin during in vitro maturation (IVM) on embryo development. The FF melatonin concentration was 0.57–1.07 × 10−9 M, increasing with follicular diameter. Oocytes were matured, fertilised and cultured under conventional conditions. Blastocyst development, embryo quality and levels of reactive oxygen species (ROS) and reduced glutathione were assessed. In Experiment 1 different melatonin concentrations (10−3, 10−7, 10−9, 10−11 M) were added to the IVM medium, which contained cysteamine as antioxidant, and no differences were observed. In Experiment 2, melatonin (10−7 M) was tested in the presence or absence of cysteamine (experimental groups: melatonin, cysteamine, melatonin + cysteamine, non-antioxidant). The melatonin group presented a higher blastocyst rate than the non-antioxidant group (28.9 vs 11.7%; P < 0.01) and a higher total cell number than the cysteamine group (225.1 vs 129.0; P < 0.05). Oocytes from the melatonin and cysteamine groups had lower ROS levels than those from the non-antioxidant group. This study shows that melatonin is an interesting tool for improving oocyte competence in juvenile goats as it increases embryo production and quality.

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Laparoscopic Ovum Pick-Up Followed by In Vitro Embryo Production and Transfer in Assisted Breeding Programs for Ruminants

Animals ◽

10.3390/ani11010216 ◽

2021 ◽

Vol 11 (1) ◽

pp. 216

Author(s):

Hernan Baldassarre

Keyword(s):

Pregnancy Rate ◽

Commercial Application ◽

Embryo Production ◽

Breeding Programs ◽

Sheep And Goats ◽

Embryo Recovery ◽

Marker Selection ◽

Holstein Calves ◽

In Vitro Embryo Production

The potential of laparoscopic ovum pick-up (LOPU) followed by in vitro embryo production (IVEP) as a tool for accelerated genetic programs in ruminants is reviewed in this article. In sheep and goats, the LOPU-IVEP platform offers the possibility of producing more offspring from elite females, as the procedure is minimally invasive and can be repeated more times and more frequently in the same animals compared with conventional surgical embryo recovery. On average, ~10 and ~14 viable oocytes are recovered by LOPU from sheep and goats, respectively, which results in 3–5 transferable embryos and >50% pregnancy rate after transfer. LOPU-IVEP has also been applied to prepubertal ruminants of 2–6 months of age, including bovine and buffalo calves. In dairy cattle, the technology has gained momentum in the past few years stemming from the development of genetic marker selection that has allowed predicting the production phenotype of dairy females from shortly after birth. In Holstein calves, we obtained an average of ~22 viable oocytes and ~20% transferable blastocyst rate, followed by >50% pregnancy rate after transfer, declaring the platform ready for commercial application. The present and future of this technology are discussed with a focus on improvements and research needed.

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