33 PRODUCTION OF A CLONED FOAL USING MITOCHONDRIAL DNA-IDENTICAL OOCYTES

2014 ◽  
Vol 26 (1) ◽  
pp. 131
Author(s):  
Y. H. Choi ◽  
J. Ritthaler ◽  
K. Hinrichs
Keyword(s):  
Mitochondrial Dna ◽  
Vitamin C ◽  
In Vitro Maturation ◽  
Transvaginal Ultrasound ◽  
Maternal Line ◽  
Exact Test ◽  
Sperm Extract ◽  
C 50 ◽  
The Individual

Recently we reported the birth of a viable foal produced by nuclear transfer (NT) using oocytes recovered from immature follicles of live mares by transvaginal ultrasound-guided aspiration (TVA; 2013 Theriogenology 79, 791–796). This procedure opens the door for production of mitochondrial DNA-identical cloned foals; typically, use of heteroplastic oocytes results in cloned offspring that have different mitochondrial DNA from that of the donor. We selected 2 mares (BL and SM) from the maternal line of the donor, a 23-year old stallion. Genetic analysis confirmed that the mares’ mitochondrial genotype was identical to that of the donor. Oocytes were obtained from the mares by TVA of all follicles ≥5 mm diameter, and were matured in vitro for 20 to 26 h. Donor fibroblasts were treated with 15 μM roscovitine for 24 h, then were directly injected into enucleated oocytes using a Piezo drill. Reconstructed oocytes were activated with 5 μM ionomycin for 4 min followed by injection with sperm extract, then incubation in 2 mM 6-dimethylaminopurine for 4 h. Oocytes from mare SM were assigned to treatment with either Scriptaid (500 nM) or Scriptaid plus vitamin C (50 μg mL–1) for 14 to 16 h, starting at the onset of 6-dimethylaminopurine exposure; mare BL did not provide sufficient oocytes for treatment grouping. Presumptive zygotes were cultured in vitro for 7 to 11 days and blastocysts were shipped for transfer to recipient mares, 1 embryo per mare. In mare BL, 10 aspiration sessions were conducted, 78 follicles were aspirated and 45 oocytes were collected, of which 4 were degenerating. After in vitro maturation, 12/40 (30%) oocytes were mature. Five of 12 oocytes lysed during manipulation; the remaining 7 were cultured and 1 blastocyst (14%) was obtained, which did not yield a pregnancy. In mare SM, 3 aspiration sessions were conducted and 53 oocytes were recovered from 81 follicles. After in vitro maturation, 31/53 (58%) were mature. Four oocytes were lysed during manipulation, 27 were cultured, and 4 blastocysts (15%) were produced, 2 from scriptaid treatment and 2 from scriptaid plus vitamin C. Transfer of these blastocysts yielded one pregnancy (scriptaid treatment); the mare delivered a healthy foal at 328 days of gestation. These results indicate that NT can be successful using low numbers of immature oocytes from selected mares. However, the individual mare may greatly affect the outcome in terms of oocyte number and quality; in this case, mare BL not only yielded fewer oocytes per aspiration session (4.5 v. 17.7 for mare SM; P < 0.001, t-test), but also fewer reconstructed oocytes per oocyte recovered (7/45 v. 27/53, respectively; P < 0.001, Fisher's exact test). Efficiency (14 to 15% blastocysts per reconstructed oocyte cultured; 1 foal from 5 embryos transferred) was similar to that achieved previously in our laboratory using heteroplastic oocytes. This work was supported by the Link Equine Research Endowment Fund, Texas A&M University, by Kit Knotts, and by Jack Waggoner.

Effect of resveratrol administration on ovarian morphology, determined by transvaginal ultrasound for the women with PCOS

2021 ◽  
pp. 1-18
Author(s):  
Amir Pejman Hashemi Taheri ◽  
Behnaz Moradi ◽  
Amir Reza Radmard ◽  
Milad Sanginabadi ◽  
Mostafa Qorbani ◽  
...  
Keyword(s):  
Repeated Measures ◽  
Polycystic Ovary ◽  
Transvaginal Ultrasound ◽  
Antral Follicle ◽  
Antral Follicle Count ◽  
Ovarian Volume ◽  
Exact Test ◽  
Ovarian Morphology ◽  
The Mean

Abstract Background: Intake of resveratrol has been associated with improved ovarian morphology under in vitro and in the animal models; however, this finding has not been confirmed in trials. The aim of our study was, therefore, to use a placebo-controlled approach with the detailed assessment of the ovarian morphology by applying transvaginal ultrasound to examine the effectiveness of this therapeutic approach in this group of women. Methods: Forty-one women with polycystic ovary syndrome (PCOS) were randomly assigned (1:1) to 3 months of daily 1000 mg resveratrol or placebo. Random assignment was done by blocked randomisation. Our primary endpoints were the change in the ovarian volume, stromal area and antral follicle count per ovary (FNPO) from the baseline to 3 months. Secondary endpoints were improvement in the distribution of follicles and ovarian echogenicity. Differences between the resveratrol and control groups were evaluated by Chi-square, fisher’s exact test and repeated-measures of ANOVA. Results: The mean age of all participants was 28.61 ± 4.99 years, with the mean BMI of 28.26 ± 5.62 kg/m2. Resveratrol therapy, as compared with placebo, was associated with a significantly higher rate of improvement in the ovarian morphology (p= 0.02). Women who received resveratrol had a more dominant follicle than those getting placebo, with a significant reduction in the ovarian volume (p<0.05). However, the number of FNPO, stromal area, ovarian echogenicity and distribution of follicles were not significantly altered (P>0.05). Conclusions: Treatment with resveratrol significantly reduced the ovarian volume and PCOM, thus suggesting a disease-modifying effect in PCOS. Trial registration: IRCT, IRCT2017061917139N2. Registered 7 July 2017, http://irct.ir/trial/15836.

51 Effect of Cryoprotectant Exposure Time on Development of Vitrified-Warmed Immature Equine Oocytes

2018 ◽  
Vol 30 (1) ◽  
pp. 164
Author(s):  
H. S. Canesin ◽  
J. G. Brom-de-Luna ◽  
Y.-H. Choi ◽  
A. M. Pereira ◽  
G. G. Macedo ◽  
...  
Keyword(s):  
Ethylene Glycol ◽  
Propylene Glycol ◽  
Exposure Time ◽  
In Vitro Maturation ◽  
Initial Exposure ◽  
Exact Test ◽  
Fetal Bovine ◽  
Follicle Aspiration ◽  
Meiotic Competence

Effective methods for cryopreservation of equine oocytes have not yet been established. Vitrification involves use of high cryoprotectant (CPA) concentrations, which can be cytotoxic. Thus, it is critical to determine a CPA concentration and exposure time able to protect the cell during cooling but with a minimal toxicity. Using a rapid non-equilibrating system, we fixed the time in the first, lower CPA concentration solution (V1) at 40 s, based on the time to maximal shrinkage. We then evaluated different exposure times in the final vitrification solution (V2). Cumulus-oocyte complexes (COC) were collected from slaughterhouse-derived ovaries and held overnight in commercial embryo holding medium. Fetal bovine serum was used as the base medium (BM). In experiment 1, COC were held in BM, incubated in V1 (2% propylene glycol + 2% ethylene glycol) for 40 s followed by incubation in V2 (17.5% propylene glycol + 17.5% ethylene glycol + 0.3 M trehalose) for 0, 45, 75, or 110 s, and then loaded in groups of 6 to 10 oocytes on a 75-µm steel mesh and plunged into liquid nitrogen. Warming was performed in decreasing trehalose concentrations in BM: 0.4 M (60-70 s), 0.2 M (5 min), 0.1 M (5 min), 0.05 M (5 min), and 0 M. After warming, oocytes were cultured for in vitro maturation (IVM) and evaluated after staining with Hoechst 33258. Differences between treatments were analysed by Fisher’s exact test. The maturation (metaphase II, MII) rate of the Control (non-vitrified oocytes; 38.8%, 31/80) was similar to that of the 75-s treatment (34.8%, 16/46; P = 0.71), and higher (P < 0.05) than those of the 0, 45, and 110 s treatments (0.0%, 0/10; 11.4%, 4/35; and 3.6%, 1/28; respectively). In experiment 2, timings in V2 focusing around 75 s were evaluated. The COC were collected and vitrified as for experiment 1, except that time in V2 was 50, 60, 70, 80, 90, or 100 s. The vitrified COC were then shipped to the intracytoplasmic sperm injection (ICSI) laboratory. After warming and IVM, oocytes were subjected to ICSI and embryo culture. Control oocytes were recovered by transvaginal follicle aspiration. The MII rate of the Control (60%, 33/55) was similar (P > 0.05) to that of the 60- and 70-s treatments (38.9%, 7/18, and 35.3%, 6/17, respectively), and higher (P < 0.05) than those of the 50-, 80-, 90-, and 100-s treatments (5.6 to 31.6%). The cleavage rates were 94% (31/33) for the Control and 71 to 100% for vitrified oocytes (P > 0.05). No blastocyst was produced from vitrified oocytes compared with 8/33 (24.2%) for Control. This work demonstrates that a rapid, non-equilibrating vitrification technique using a 40-s initial exposure and 70- to 80-s final exposure to CPA is associated with maintenance of meiotic competence of immature equine oocytes; however, further work is required to optimize embryonic development with this method. Research supported by the Clinical Equine ICSI Program and the Link Equine Research Fund, Texas A&M University.

188 HUMAN FOLLICULAR FLUID IN EQUINE IN VITRO MATURATION MEDIUM SUPPORTS IN VITRO MATURATION AND VIABLE INTRACYTOPLASMIC SPERM INJECTION-BLASTOCYST PREGNANCIES

2012 ◽  
Vol 24 (1) ◽  
pp. 206
Author(s):  
C. Makloski ◽  
R. Gotti ◽  
K. Harris ◽  
J. Bottger ◽  
M. Meintjes
Keyword(s):  
Follicular Fluid ◽  
Intracytoplasmic Sperm Injection ◽  
In Vitro Maturation ◽  
Transvaginal Ultrasound ◽  
Human Follicular Fluid ◽  
Immature Oocytes ◽  
Treatment Groups ◽  
Maturation Medium ◽  
And Control

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

344 EFFECTS OF BMP4 AND ITS INHIBITOR, NOGGIN, ON OOCYTE MATURATION AND DEVELOPMENT OF BOVINE PREIMPLANTING EMBRYOS

2010 ◽  
Vol 22 (1) ◽  
pp. 328
Author(s):  
I. La Rosa ◽  
R. Fernandez y Martín ◽  
D. A. Paz ◽  
D. F. Salamone
Keyword(s):  
Embryo Development ◽  
Oocyte Maturation ◽  
In Vitro Maturation ◽  
Cell Number ◽  
Bmp Signaling ◽  
Control Group ◽  
Cleavage Rate ◽  
Exact Test ◽  
Student’S T

BMP4 regulates different events during development in all vertebrates and Noggin is one of its powerful inhibitors that blocks BMP4 interaction with its receptors (Groppe et al. 2002). In this work, the effect of these factors on bovine oocyte maturation and subsequent embryo development has been investigated. COCs were aspirated from abattoir ovaries and in vitro-matured for 22 h or 24 h in a 5% CO2 humidified atmosphere at 39°C in TCM containing 0.6% BSA, 2 mM FSH, 10 mM cysteamine, 1% antibiotic and 1% pyruvate, control group (C), plus 100 ng mL-1 of BMP4 (B), or 100 ngmL-1 of Noggin (NOG). Oocytes were stained with Hoechst 33342 and classified by their nuclear stage. Effects on embryo development were investigated for embryos produced by parthenogenic activation (PA) and IVF For PA, denuded oocytes were chemically activated in 5 μM ionomycine for 4 min, and immediately incubated in 1.9 mM of 6-dimethilaminopurine for 3 h. For IVF, frozen-thawed semen was centrifuged and resuspended in Bracket and Oliphant (BO) solution and incubated with 22 h matured COCs for 5 h. Embryos were cultured in CR2 medium free of serum and co-culture. Cleavage and blastocyst formation were registered at Day 2 and 9 respectively. Fischer’s exact test was used and P ≤ 0.05 was considered significant. Nuclear progression was not affected by maturation treatments [% of MII: 79.4(C, n = 102), 72.4 (B, n = 98), 80.9 (NOG, n = 89)]. For PA, both factors significantly increased cleavage rates [%: 51.7 (C, n = 284), 65 (B, n = 186), 62.1 (NOG, n = 198)] while blastocyst rates were not affected [%: 8.8 (C), 7.5 (B), and 8.6 (NOG)]. On the other hand, for IVF, cleavage rate was statistically lower for Noggin group [%: 70.7 (C, n = 140), 71.3 (B, n = 157), 64 (NOG, n = 159)] while blastocyst rates were similar between groups [%: 15.7 (C), 13.4 (B), 14.5 (NOG)]. Any of the added factors affected cell number of the embryos at Day 2. Blastocysts did not differ in the number of cells at Day 9 (Student’s t-test was used) neither for PA [mean ± SD: 100 ± 33 (C, n = 9), 88 ± 14 (B, n = 3) and 68 ± 8,(NOG, n = 3)] nor for IVF [mean ± SD: 90 ± 24 (C, n = 9), 132 ± 18 (B, n =4) and 99 ± 8 (NOG, n = 3)]. It is noticeable that addition of these factors during in vitro maturation showed different effects on subsequent embryo development depending on whether the embryos were PA or IVF. Probably, these responses represent differences in the BMP signaling system between these embryos which could be associated with different imprinting pattern. Further experiments are needed to elucidate clearly the mechanisms implicated. To our knowledge, this is the first work to study BMP4 inhibition during bovine in vitro maturation. To “Merlo” and “Nueva Escocia” Slaughterhouses

202 CHARACTERIZATION OF BOVINE OOCYTE CYTOPLASMIC MATURATION WITH COMMON IN VITRO MATURATION PROTOCOLS

2016 ◽  
Vol 28 (2) ◽  
pp. 232
Author(s):  
B. A. Foster ◽  
F. A. Diaz ◽  
P. T. Hardin ◽  
E. J. Gutierrez ◽  
K. R. Bondioli
Keyword(s):  
Gap Junctions ◽  
Gap Junction ◽  
In Vitro Maturation ◽  
Transvaginal Ultrasound ◽  
Bovine Oocyte ◽  
Hoechst 33342 ◽  
Nuclear Maturation ◽  
Mitochondrial Distribution ◽  
Cytoplasmic Maturation

Modulators of 3′-5′-cyclic adenosine monophosphate have been extensively researched to delay nuclear maturation in in vitro maturation (IVM) systems to improve synchronization of nuclear and cytoplasmic maturation. However while normal maturation for many organelles has been characterised, there is a lack of information on how modulators affect cytoplasmic maturation. The goal of this study was to identify the effect of different components of bovine oocyte maturation systems on 3 aspects of cytoplasmic maturation. Bovine oocytes were collected from mixed breed beef cattle using transvaginal ultrasound guided oocyte aspiration. Oocytes were assigned to 1 of 4 treatments; staining immediately after collection (n = 249) or after 24 h of IVM (n = 270), 2 h of pre-IVM in Forskolin and 3-isobutyl-1-methylxanthine (IBMX; n = 254), or 2 h of pre-IVM followed by IVM (n = 259). Following treatment, half of the recovered oocytes were stained with Hoechst 33342 to determine nuclear maturation status, and Calcein AM for gap junction status. The other half were stained with Hoechst 33342, Mitotracker deep red to identify mitochondria distribution patterns and Alexa Fluor 488 conjugated phalloidin for F actin microfilament distribution. Organelle patterns were coded and statistically analysed using linear models to determine if treatment had an effect on the indicators of cytoplasmic maturation or their agreement with nuclear maturation. Results indicated that there was a high degree of variability in both cytoplasmic and nuclear maturation of oocytes irrespective of treatment group, with many oocytes exhibiting aberrant patterns in both mitochondrial and microfilament distribution. Gap junctions were classified as open (immature), partially open or closed (mature), based on the strength of Calcein fluorescence within the ooplasm. Both treatment and nuclear maturation had a significant effect on gap junction status (P < 0.001) with gap junctions tending to close as oocytes matured, while treatment in pre-IVM maintained open gap junctions, even as meiosis progressed. Mitochondria were classified as peripheral (immature), diffuse, central (mature) or too sparse to accurately classify. There was an unexpectedly high proportion of oocytes with few mitochondria (17%), suggesting an incomplete growth phase before collection. There was no correlation between meiotic stage and mitochondrial distribution (P = 0.73), with the majority of oocytes having diffuse mitochondrial distribution. As normal maturation proceeds, microfilaments aggregate and migrate peripherally. However, neither microfilament aggregation nor redistribution were correlated with nuclear maturation (P = 0.6 and P = 0.11 respectively) or mitochondrial distribution (P = 0.33 and P = 0.06 respectively). Overall, results show that while pre-IVM maintains open gap junctions, the system studied here is not sufficient for improving correlation between cytoplasmic and nuclear maturation. Many deviations from normal cytoplasmic maturation are seen with IVM and these irregularities are maintained with prematuration in Forskolin and IBMX.

scholarly journals RESPON PENGGUNAAN BERBAGAI BAHAN AKTIVATOR PADA AKTIVASI PARTENOGENESIS OOSIT KAMBING HASIL IVM

SAINSTIS ◽  
2012 ◽  
Author(s):  
Kholifah Holil, Eva Ari Wahyuni, Hari Soepriandono Gatot Ciptadi
Keyword(s):  
In Vitro Maturation ◽  
Fetal Bovine Serum ◽  
Reproductive Technologies ◽  
Cleavage Rate ◽  
Parthenogenetic Activation ◽  
Sperm Extract ◽  
Fetal Bovine ◽  
Humidified Air

Parthenogenetic activation is one method that can be used to determine the quality of IVM oocytes results before further use to other reproductive technologies (IVF and transfer core). In parthenogenetic activation can be used various activators such as ethanol, Ca Ionophore, and Crude Sperm Extract (CSE). Therefore, the aim of this experiment is to know the response use a variety of materials activator of parthenogenetic activation of goat oocytes IVM.<br />The sample used in this experiment was oocytes aspirated from goat ovarian follicles taken from RPH Sukun of Malang. Oocytes were matured for 24 hr in TCM-199 supplemented with fetal bovine serum (FBS), follicle-stimulating hormone (FSH) and lutheinizing hormone (LH) at a temperature of 38,5oC and 5% CO2 in humidified air. After another 30 hours of in vitro maturation, they were then activated by various treatments. The treatment of experiment are treatment 1, activation using ethanol 7% for 7 minute, treament 2, activation using Ca Ionophore 20 µM for 7 minute. Treatment 3, activation using CSE 2,5 µg/ml for 2 hr.<br />Based on the result of research, it is showed that activation by using 7% ethanol for 7 minutes is able to produce cleavage rate of 70.40%. Activation by Ca Ionophore 20 μM for 7 minutes is able to produce cleavage rate of 52.75%. While the use of CSE activation with 2.5 ug / ml for 2 hours produces cleavage rate of 36.33%. Thus it can be concluded that the goat oocyte IVM able to respond to a variety of materials activators on parthenogenetic activation performed. The highest response given by the successive results goat IVM oocytes activated using 7% ethanol for 7 minutes, 20 μM Ca Ionophore for 7 minutes, and the CSE 2.5 microg / ml for 2 hours.<br /><br />Keywords: Parthenogenetic activation, goat oocytes IVM, etanol, calsium ionophore, Crude Sperm Extract (CSE). <br /><br />

270 HIGH GASEOUS PRESSURE PRETREATMENT IN IN VITRO MATURATION OF CANINE OOCYTES

2013 ◽  
Vol 25 (1) ◽  
pp. 282
Author(s):  
B. A. Rodrigues ◽  
C. A. Rodrigues ◽  
M. B. Salviano ◽  
B. R. Willhelm ◽  
F. J. F. Collares ◽  
...  
Keyword(s):  
In Vitro Maturation ◽  
P Value ◽  
Sample Collection ◽  
In Vitro Production ◽  
Exact Test ◽  
Treatment Groups ◽  
Nuclear Maturation ◽  
Grand Island ◽  
Laboratory Protocol

Stress processes, such as hydrostatic pressure treatment of oocytes in different species, have been reported to increased embryo rate following in vitro maturation (IVM). However, studies on high gaseous pressure (HGP) pretreatment in IVM of oocytes from domestic animals are lacking in the literature. This experiment aimed to test HGP pretreatment of canine oocytes to increase meiosis achievement (metaphase II) after IVM. A total of 502 canine oocytes (6 replicates) were used in this study. Ovaries from 15 bitches were obtained from local shelters or rescue organizations after ovariohysterectomy. Sample collection was blind as to reproductive stage and dog age. The ovaries were transported to the laboratory in 0.9% NaCl and were processed within 3 h of collection. The ovarian cortex was sliced and washed in PBS with 1% FCS to release cumulus–oocyte complexes. Grade 1 and 2 cumulus–oocyte complexes were selected for IVM and randomly distributed into 3 treatment groups: HGP (oocytes placed in PBS and subjected to pressure chamber; 206 oocytes), ambient control (oocytes maintained in TCM-HEPES at room temperature for 60 min; 130 oocytes), and laboratory protocol (oocytes IVM after morphologic selection; 166 oocytes). The average pressure, initial and final temperature, and duration of oocytes in the HGP pretreatment were 76.19 atm (±0.92), 32.20°C (±5.17) and 27.71°C (±3.17), and 60 min, respectively. In vitro maturation was carried out for 72 h at 37°C in a high-glucose medium, consisting of TCM-199 with 2.2 mg mL–1 of sodium bicarbonate (11150, Gibco, Grand Island, NY, USA), and supplemented with 0.1% polyvinyl alcohol (P-8136, Sigma, St. Louis, MO, USA), 0.991 mg mL–1 of glucose (108337, Merck, Darmstadt, Germany), 50 µg mL–1 of gentamicin, 22 µg mL–1 of pyruvic acid, 20 µg mL–1 of oestradiol (E-8875, Sigma), 0.5 µg mL–1 of FSH (Folltropin-V, Vetepharm Inca), 0.03 IU mL–1 of hCG (Chorulon®, Intervet, Kenilworth, NJ, USA), under 20% oxygen tension. The number of oocytes at each stage (prophase to metaphase II) was recorded according to the morphology of nuclear content after staining with Hoechst 33342. For comparison purposes of nuclear maturation in oocytes, data were analysed by Fisher’s exact test. Differences at a P-value ≤0.05 were considered significant. Oocytes from the HGP, ambient control, and laboratory protocol groups had similar meiotic progression to the metaphase II stage (metaphase I–anaphase I–telophase I–metaphase II), and were 35.4% (73/206), 30.8% (40/130), and 34.9% (58/166), respectively (P ≥ 0.05). The proportion of oocytes without chromatin or having an irregular organisation was not different among groups. In conclusion, results indicate that HGP pretreatment as used in this experiment did not improve meiosis rates in IVM canine oocytes. Further investigations to understand the significance of HGP pretreatment in IVM and in vitro production of canine embryos are ongoing in our laboratory.

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