Effect of the Presence of Corpus Luteum on Oocyte Recovery and Subsequent in vitro Maturation and Fertilization in Buffaloes

The laparoscopic method of recovering oocytes in goats and sheep is one of the minimally invasive methods used in the biotechnology of animal reproduction. It allows for good quality oocytes that are suitable for in vitro maturation and fertilization to be recovered. The limitation of using the laparoscopic ovum pick-up (L-OPU) method in goat and sheep is its changing effectiveness and the lack of repeatability of results, as well as the varying effectiveness of different variants of the method. Therefore, it is necessary to develop effective non-invasive techniques allowing for multiple good quality oocyte recovery that would be suitable for in vitro maturation and fertilization. In this study, four different L-OPU variants were described in goats and sheep. Various techniques of recovering oocytes were discussed, including the techniques of conducting the operation, various tools for recovering oocytes, and different plans of hormonal stimulation. Recovery rates were 35% (Variant I), 57% (Variant II), 72% (Variant III), and 67% (Variant IV). After evaluation, 94% (both Variant I and II), 93% (Variant III), and 84% (Variant IV) of the oocytes were qualified for in vitro maturation. The results of the study show that the proposed technique of laparoscopic recovery of oocytes allows a sufficient number of ovarian cells suitable for in vitro culture to be obtained and as a consequence it makes them useful in in vitro maturation/in vitro fertilization (IVM/IVF) programs or cloning. The method allows for a fast and effective conduct of the operation in a living donor with minimal invasiveness while preserving the excellent condition of animals.

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243 EFFECT OF OOCYTE RECOVERY TECHNIQUES ON IN VITRO MATURATION OF SWINE OOCYTES

Reproduction Fertility and Development ◽

10.1071/rdv21n1ab243 ◽

2009 ◽

Vol 21 (1) ◽

pp. 219

Author(s):

F. R. O. de Barros ◽

M. G. Marques ◽

M. D. Goissis ◽

M. A. Peres ◽

M. P. Milazzotto ◽

...

Keyword(s):

Follicular Fluid ◽

Recovery Rate ◽

In Vitro Maturation ◽

Metabolic Stress ◽

Water Bath ◽

Epifluorescence Microscopy ◽

Nuclear Maturation ◽

Oocyte Recovery ◽

Centrifuge Tube

The aim of this study was to compare 2 different techniques to obtain swine oocytes from abattoir ovaries. Ovaries were washed in saline at 35°C and submitted to slashing or aspiration, simultaneously. For the slashing group, ovaries were held with a hemostat inside a beaker containing 35 mL of HEPES-buffered Tyrode’s media (HbT) and follicles (2–6 mm) were incised with a scalpel. For every 5 slashed ovaries, HbT-containing follicular fluid was transferred to 50-mL centrifuge tubes. For the aspiration group, follicles (2–6 mm) were aspirated using an 18-gauge needle and a 5-mL syringe. The follicular fluid of each ovary was transferred to a 50-mL centrifuge tube. Tubes from both techniques were placed in a water bath at 35°C for 15 min to allow settling of the cumulus–oocyte complexes (COC). The supernatant was removed and the sediment was resuspended in HbT and placed in water bath at 35°C for an additional 15 min. The sediment was resuspended in 15 mL of HbT and COC were recovered under stereomicroscopy. Oocytes were in vitro matured for 44 h in TCM-199 added with 10% porcine follicular fluid (PFF) and hormones (LH and FSH) at 38.5°C, 5% CO2 and high humidity. The oocyte recovery rate of each technique was determined by the ratio between the number of COC and ovaries used. To verify nuclear maturation by epifluorescence microscopy (Zeiss), oocytes were fixed, permeabilized, and incubated in 10 μg mL–1 of RNAse for 30 min and in 10 μg mL–1 of propidium iodide for 10 min. Heat shock protein 70 (HSP70) content was assessed as described in Kawarsky and King (2001 Zygote 9(3), 39–50) to verify the metabolic stress. Data were analyzed by ANOVA and Tukey’s test using the software Statistica for Windows. A level of 5% was considered significant in all assessments. The oocyte recovery rate (COC/ovary) was higher for the slashing group (2.665 ± 0.38) compared with the aspiration group (1.762 ± 0.15). The percentage of oocytes that reached the germinative vesicle (GV) stage (h 0 of maturation) did not differ between groups (100 ± 0 and 86.66 ± 13.36, slashing and aspiration group, respectively). The same was observed for the percentage of oocytes that reached the metaphase II stage (MII, after 44 of maturation; 79.99 ± 9.74 and 96.00 ± 4.00, slashing and aspiration group, respectively). Moreover, no difference at pixel quantification of HSP70 was observed between groups (256.50 ± 42.42 and 238.61 ± 71.18, slashing and aspiration group, respectively). In conclusion, the slashing procedure provided a better oocyte recovery rate compared with the aspiration of ovaries. This technique does not affect nuclear maturation, because no differences were observed regarding the percentage of oocytes that reached the GV and MII stages. In addition, it does not affect HSP70 content, suggesting that the slashing of ovaries does not increase the basal stress of oocytes in an in vitro-maturation system.

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Effect of time during transport of excised mare ovaries on oocyte recovery rate and quality after in vitro maturation

Theriogenology ◽

10.1016/s0093-691x(99)00169-7 ◽

1999 ◽

Vol 52 (5) ◽

pp. 757-766 ◽

Cited By ~ 22

Author(s):

F. Guignot ◽

J. Bezard ◽

E. Palmer

Keyword(s):

Recovery Rate ◽

In Vitro Maturation ◽

Oocyte Recovery

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Effect of administering a crude equine gonadotrophin preparation to mares on follicular development, oocyte recovery rate and oocyte maturation in vivo

Reproduction ◽

10.1530/reprod/118.2.351 ◽

2000 ◽

pp. 351-360 ◽

Cited By ~ 15

Author(s):

I Bruck ◽

J Bezard ◽

M Baltsen ◽

B Synnestvedt ◽

I Couty ◽

...

Keyword(s):

Oocyte Maturation ◽

Recovery Rate ◽

In Vitro Maturation ◽

Follicular Development ◽

Vitro Fertilization ◽

Nuclear Maturation ◽

Oocyte Yield ◽

Oocyte Recovery

In mares, the shortage of oocytes and the variability in nuclear maturation at a certain time of the oestrous cycle hinders the optimization of methods for in vitro maturation and in vitro fertilization. Increasing the number of small-to-medium-sized follicles available for aspiration in vivo may increase the overall oocyte yield. The aims of the present study were to investigate whether administration of crude equine gonadotrophins affects follicular development, oocyte recovery rate, in vivo oocyte maturation and follicular concentrations of meiosis-activating sterols. During oestrus, all follicles >/= 4 mm were aspirated from 19 pony mares (first aspiration: A1). Over the next 8 days, the mares were treated daily with either 25 mg crude equine gonadotrophins (n = 10) or physiological saline (n = 9). Between day 1 and day 8, follicular growth was monitored by ultrasonography. On day 8, all follicles >/= 4 mm were evacuated (second aspiration: A2) and nuclear maturation of the recovered oocytes was assessed after orcein staining. Follicular growth between A1 and A2, as well as the number and size of follicles at A2 were similar for control mares and mares treated with crude equine gonadotrophins. The oocyte recovery rates at A1 and A2 were similar. At A2, the oocyte recovery rate and oocyte maturation in vivo were not affected by treatment with crude equine gonadotrophins. The number of expanded cumulus oophorus complexes recovered from follicles </= 29 mm was significantly higher at A1 than at A2. The number of oocytes at the germinal vesicle stage was significantly higher at A2 (41.5%) than at A1 (17.8%). Meiosis-activating sterols (FF-MAS and T-MAS) were identified in follicular fluid recovered at A2. Follicular concentrations of FF-MAS and T-MAS were unaffected by treatment with crude equine gonadotrophins. The present study demonstrates that follicular aspiration during oestrus allowed a new follicular population to develop and resulted in a higher degree of synchronization of oocyte development with respect to cumulus expansion and nuclear maturation. The availability of a more homogeneous population of oocytes might facilitate a better optimization of in vitro maturation and in vitro fertilization techniques in mares. Administration of crude equine gonadotrophins during early dioestrus did not affect the growth of small follicles, the oocyte yield after aspiration or oocyte maturation in vivo.

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Comparative seasonal assessment on the quality of Black Bengal goat oocytes in view of in vitro embryo production

Asian-Australasian Journal of Bioscience and Biotechnology ◽

10.3329/aajbb.v5i3.53870 ◽

2020 ◽

Vol 5 (3) ◽

pp. 78-87

Author(s):

Shikha Sarkar ◽

Md Saiful Islam ◽

Md Enayet Kabir ◽

Falguni Dadok ◽

Md Abdullah Al Zaber ◽

...

Keyword(s):

Corpus Luteum ◽

Recovery Rate ◽

In Vitro Maturation ◽

Winter Season ◽

Excellent Opportunity ◽

Different Seasons ◽

In Vitro Embryo Production ◽

Black Bengal Goat

In vitro maturation (IVM) of oocytes provide an excellent opportunity for cheap and abundant embryos for carrying out animal improvement. With the aim for studying the quality of oocyte in different seasons of Black Bengal goat, both right and left ovaries were collected from the slaughter houses. For each of the specimens, gross parameters such as right, left, corpus luteum (CL)-present and corpus luteum (CL)-absent group were evaluated on the basis of weight (g), length (cm), width (cm), follicles aspirated and number and state of cumulus-oocyte-complexes (COCs), normal COCs and abnormal COCs, the effect of season on cumulus oocyte complexes (COCs) of goat oocytes. Our study revealed that the average number of follicles and oocytes recovery rate were higher in summer than in winter season and recovery rate of grade A and B oocytes were higher in summer than that of in winter and recovery rate of grade C and D were higher in winter than that of in summer. In the present study a significantly greater number of oocytes per ovary were recovered from ovaries without a corpus luteum than from ovaries with a corpus luteum. So the summer was the best season for recovery of COCs which have a worthy competence to be matured in vitro. Asian Australas. J. Biosci. Biotechnol. 2020, 5 (3), 78-87

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Collection grading and evaluation of cumulus-oocyte-complexes for in vitro maturation in sheep

Bangladesh Veterinarian ◽

10.3329/bvet.v28i1.8811 ◽

1970 ◽

Vol 28 (1) ◽

pp. 31-38

Author(s):

MNS Talukder ◽

A Iqbal ◽

MAMY Khandoker ◽

MZ Alam

Keyword(s):

Corpus Luteum ◽

Culture Condition ◽

In Vitro Maturation ◽

Type I ◽

Type Ii ◽

In Vitro Production ◽

Length Width ◽

Left And Right ◽

Vitro Production

An alternative to superovulation is in-vitro production (IVP) of embryos where the efficient collection and grading of oocytes is important. Ovaries from an abattoir were collected and categorized as type I with no corpus luteum (CL), and type II with CL. The length, width and weight of type I and type II ovary were 1.4 ± 0.03 and 1.5 ± 0.08 cm; 0.8 ± 0.04 and 1.0 ± 0.07 cm; 0.6 ± 0.07 and 0.7 ± 0.04 gm, respectively, each significantly (P<0.05) higher in type II ovaries. A total of 80 and 78 follicles were observed and 60 and 61 follicles aspirated from left and right ovaries, respectively, from each of 25 ovaries. Out of 133 follicles 100 were aspirated from 40 type-I ovaries, and 21 aspirated from 10 type-II ovaries. The differences in the number of normal, abnormal and total cumulus-oocytecomplex (COCs) per ovary between left and right ovaries were not significant (P>0.05). The number of normal (1.9 ± 0.11) and total (2.5 ± 0.14) COCs per ovary were significantly (P<0.05) higher in ovaries without than in those with CL (1.2 ± 0.36 and 2.0 ± 0.30, respectively). But the number (0.80 ± 0.13) of abnormal COCs per ovary was significantly (P<0.05) higher in ovaries with CL than in those without (0.7 ± 0.09). Significantly (P<0.05) higher percentage of COCs expansion was grade A (6.9 ± 2.05) than grade B (53.1 ± 1.27) COCs. It is suggested that type I (without CL) ovaries and follicles of 2-6 mm diameter are suitable to collect good quality COCs for in-vitro maturation (IVM) of oocytes and the culture condition for IVM of sheep COCs are reported. DOI: http://dx.doi.org/10.3329/bvet.v28i1.8811 Bangl. vet. 2011. Vol. 28, No. 1, 31-38

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Follicular Flushing Avoids Multiple Vaginal Punctures and May Aid in Oocyte Recovery in In Vitro Maturation (IVM)

Fertility and Sterility ◽

10.1016/j.fertnstert.2009.01.005 ◽

2009 ◽

Vol 91 (3) ◽

pp. S5 ◽

Cited By ~ 4

Author(s):

P.S. Uzelac ◽

G.L. Christensen ◽

S.T. Nakajima

Keyword(s):

In Vitro Maturation ◽

Oocyte Recovery

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Oocyte Quality and Subsequent In Vitro Maturation of Sheep Oocyte-Cumulus Complex from Ovary with Presence and Absence of Corpus Luteum

KnE Life Sciences ◽

10.18502/kls.v3i6.1125 ◽

2017 ◽

Vol 3 (6) ◽

pp. 166 ◽

Cited By ~ 1

Author(s):

Rini Widyastuti ◽

Mas Rizky A.A. Syamsunarno ◽

Takdir Saili ◽

Arief Boediono

Keyword(s):

Corpus Luteum ◽

In Vitro Maturation ◽

Polar Body ◽

Cumulus Cells ◽

Oocyte Quality ◽

Maturation Stage ◽

Cumulus Oocyte Complex ◽

First Polar Body ◽

Maturation Rate

In vitro maturation is the crucial step for in vitro embryo production. It needs a large number of oocytes as source gamet cells recovered. The present study is aimed to assess the influence of corpus luteum on the average number oocytes harvested, COCs quality and subsequent maturation of immature oocytes recovered from sheep ovaries. Sheep ovaries were collected from local slaughterhouse and COCs were collected by using slicing method. Collected COCs were graded into three categories dependent upon cumulus cells surrounding them and the homogenous of cytoplasm. COCs were maturated in maturation media at 5% CO2 for 24 hours. Maturation of oocytes evaluated base on the expansion of cumulus cells and extrusion of the first polar body. There was significantly higher on average of COCs harvested from ovaries with corpus luteum compared without corpus luteum. The presence of Corpus luteum did not affect the COCs quality and ability to reach the maturation stage. However, there was a dramatic effect of cultured COCs quality on maturation rate both groups. Collectively, these results indicate that COCs quality is the main factor affecting the subsequent of oocytes matured in vitro. Keywords: Corpus luteum; cumulus oocyte complex; in vitro maturation; maturation rate; ovaries

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Influence of different protein supplements on the recovery and in vitro maturation of bovine oocytes

Revista Colombiana de Ciencias Pecuarias ◽

10.17533/udea.rccp.v33n3a01 ◽

2019 ◽

Vol 33 (3) ◽

pp. 172-181

Author(s):

Pedro H Fernandes-França ◽

Maria V Oliveira-Santos ◽

Gabriela P Oliveira-Lira ◽

Alana Azevedo-Borges ◽

Alexsandra Fernandes-Pereira

Keyword(s):

Bovine Serum ◽

In Vitro Maturation ◽

Cumulus Cells ◽

Oocyte Quality ◽

Protein Supplementation ◽

Protein Supplements ◽

Oocyte Recovery ◽

Stage 1 ◽

Bovine Oocytes

Background: Oocyte quality and maturation are influenced by protein supplementation. Objective: To evaluate the influence of fetal bovine serum (FBS) and bovine serum albumin (BSA) concentrations on the recovery and in vitro maturation (IVM) of bovine oocytes. Methods: The study was divided into Stage 1 (oocyte recovery), and Stage 2 (IVM). In the first stage, three experiments were conducted according to the recovery (R) medium used: (R1) 10 vs. 20% FBS; (R2) 5 vs. 10% BSA; and (R3) the best results from R1, R2, and the combination of FBS+BSA (5+5%). Within the second stage, the maturation medium was supplemented according to three experiments: (M1) 5 vs. 10% FBS; (M2) 0.4 vs. 0.8% BSA; and (M3) better results of M1, M2, and the combination of FBS+BSA (5+0.8%). Results: In Stage 1 (R1 and R2), the media with 10% FBS and 10% BSA showed better oocyte quality results and were defined for experiment R3. In R3, the 10% FBS and the combination of FBS+BSA (5+5%) allowed recovery of better-quality oocytes. In Stage 2 (M1 and M2), media with both levels of FBS (5 and 10%) and 0.8% BSA were defined as better according to the maturation and viability rates of cumulus cells, so they were defined for experiment M3. In M3, no difference was noted among the supplements. Conclusions: For oocyte recovery, 10% FBS and the combination of FBS+BSA (5+5%) can be used to obtain immature oocytes. For the in vitro maturation, FBS (both levels, 5 and 10%) and BSA (0.8%) can be used alone or in combination.

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