126 Interference of mastitis with ovulation and oocyte and granulosa cell quality in dairy cows

2019 ◽  
Vol 31 (1) ◽  
pp. 188
Author(s):  
G. Santos ◽  
M. P. Bottino ◽  
A. P. C. Santos ◽  
R. E. Orlandi ◽  
L. M. S. Simões ◽  
...  
Keyword(s):  
Dairy Cows ◽  
Follicular Fluid ◽  
Rate Control ◽  
Cumulus Cell ◽  
Transcript Abundance ◽  
Cumulus Cells ◽  
Subclinical Mastitis ◽  
Oocyte Quality ◽  
Ovulation Rate ◽  
Intravaginal Device

The objective of this study was to evaluate the effect of mastitis diagnosed by somatic cell count (SCC) on follicular growth, ovulation, oocytes and cumulus cells quality and the concentration and size of exosomes in follicular fluid of dairy cows. In the study, crossbred cows (Bos taurus-Holstein×Bos indicus-Gir) were classified for analysis as control (SCC <200.000 cells mL−1) and mastitis (SCC >400.000 cells mL−1) groups. In Experiment 1 (follicular dynamics), cows (n=57: control=31; mastitis=26) received a progesterone intravaginal device (Sincrogest®, Ourofino Saude Animal, Cravinhos, Brazil) and 2mg of oestradiol benzoate (Sincrodiol®, Ourofino Saude Animal) injected IM. Eight days later (D8), the progesterone device was removed and cows received IM 500mg of cloprostenol (Sincrocio®, Ourofino Saude Animal), 1mg of oestradiol cypionate (SincroCP®, Ourofino Saude Animal) and 300IU of eCG (SicroeCG®, Ourofino Saude Animal). Ultrasound exams (Mindray 4900, probe linear de 5MHz, Shenzhen, China) were performed every 24h from removal of the progesterone-releasing intravaginal device (D8) until 48h later. Thereafter, evaluations were performed every 12h, until ovulation or up to 96h after removal of the progesterone-releasing intravaginal device. In Experiment 2 (oocyte, cumulus complexes, and follicular fluid evaluation), cows (n=26: control=13; mastitis=13) were submitted to follicular aspiration (ovum pickup) for oocyte quality and cumulus cells transcript evaluation. Transcript abundance of apoptosis markers (BCL2, BAX, PI3K, PTEN, FOXO3) was determined by real-time RT-PCR. Moreover, 7 days after the ovum pickup session, the dominant follicle was aspirated and follicular fluid samples were obtained. Exosomes were isolated from the follicular fluid by serial centrifugations, which were also performed for evaluation of particle size and concentration. Statistical analyses were performed using the SAS (SAS Institute Inc., Cary, NC, USA), and the GLIMMIX procedure was used to determine significant differences between groups. Gene expression and exosome data were submitted to the Student’s t-test. Ovulation rate [control 77.4% (24/31) and mastitis 57.7% (15/26); P=0.09] and viable oocytes rate [control 59.1% (130/220) and mastitis 41.9% (125/298); P=0.01] were higher in control animals. Additionally, there was a greater number of degenerate oocytes (control 6.7±1.2 and mastitis 13.3±5.5; P=0.001) in subclinical mastitis cows. There was greater abundance (P=0.003) of BAX cumulus cell transcripts and exosome mean (P=0.03) was smaller in subclinical mastitis cows. However, BCL2, PI3K, PTEN, nd FOXO3 cumulus cell transcripts was similar between treatments. In conclusion, ovulation rate, oocyte quality, and exosome diameter were smaller in cows with SCC >400.000 cells mL−1, demonstrating that subclinical mastitis can influence the fertility of dairy cows.

117 Subclinical Mastitis Reduces Ovulation and Oocyte Quality in Milk-Producing Cows

2018 ◽  
Vol 30 (1) ◽  
pp. 198
Author(s):  
G. Santos ◽  
M. P. Bottino ◽  
M. B. D. Ferreira ◽  
J. C. Silveira ◽  
A. C. F. C. M. Avila ◽  
...  
Keyword(s):  
Follicular Fluid ◽  
Target Genes ◽  
Bos Taurus ◽  
Bos Indicus ◽  
Cumulus Cell ◽  
Cumulus Cells ◽  
Subclinical Mastitis ◽  
Oocyte Quality ◽  
Ovulation Rate ◽  
Follicular Dynamics

The aim was to evaluate the effect of subclinical mastitis by somatic cell count (SCC) on follicular dynamics, ovulation, oocyte and cumulus cell quality, exosome size and concentration in milk-producing cows. Crossbred cows (Bos taurus × Bos indicus; that is, Holstein × Gyr) were randomly allocated to control (SCC <200,000 cells mL−1] and mastitis (SCC >400,000 cells mL−1) groups. In experiment 1 (follicular dynamics), cows (n = 57) were submitted to ultrasonographic evaluations every 24 h, after removal of an intravaginal progesterone device (Day 8) up to Day 10. From Day 10, ultrasound evaluations were performed every 12 h, until ovulation or until 96 h after progesterone device withdrawal, in order to follow final dominant follicle growth and ovulation. In experiment 2 (oocyte, cumulus cells, and follicular fluid evaluation), cows (n = 23) were submitted to follicular aspirations, preceded by synchronization of the emergence of the follicular wave. The levels of target genes in cumulus cells (BCL2, BAX, PI3K, PTEN, FOXO3) were evaluated by RT-qPCR. In the follicular fluid, the exosomes were isolated for evaluation of particle size. Data were analysed by the Glimmix procedure of SAS (SAS Institute Inc., Cary, NC, USA). Ovulation rate (P = 0.09) was higher in control cows [control 77.42% (24/31) and mastitis 57.69% (15/26)]. Viable oocyte rate (P = 0.01) was also higher in control cows [control 59.1% (130/220) and mastitis 41.9% (125/298)]. The dynamics of follicular growth did not differ between groups. The number of degenerate oocytes (P = 0.001) was higher in cows of the mastitis group. In the evaluation of cumulus cell gene expression, there was a higher abundance of BAX transcripts (P = 0.003) in cells of mastitis cows. Additionally, the mean and mode of exosome diameter in mastitis cows were smaller (P = 0.03 and P = 0.02, respectively). In conclusion, ovulation rate, oocyte quality, and follicular fluid exosome diameter were lower in cows with subclinical mastitis, demonstrating a link between mammary gland sanitary status and reproduction.

scholarly journals Predictive value of bovine follicular components as markers of oocyte developmental potential

2014 ◽  
Vol 26 (2) ◽  
pp. 337 ◽  
Author(s):  
Satoko Matoba ◽  
Katrin Bender ◽  
Alan G. Fahey ◽  
Solomon Mamo ◽  
Lorraine Brennan ◽  
...  
Keyword(s):  
Follicular Fluid ◽  
Cumulus Cell ◽  
Principal Component ◽  
Transcript Abundance ◽  
Cumulus Cells ◽  
Blastocyst Stage ◽  
Developmental Potential ◽  
Thecal Cells ◽  
Total Fatty Acids

The follicle is a unique micro-environment within which the oocyte can develop and mature to a fertilisable gamete. The aim of this study was to investigate the ability of a panel of follicular parameters, including intrafollicular steroid and metabolomic profiles and theca, granulosa and cumulus cell candidate gene mRNA abundance, to predict the potential of bovine oocytes to develop to the blastocyst stage in vitro. Individual follicles were dissected from abattoir ovaries, carefully ruptured under a stereomicroscope and the oocyte was recovered and individually processed through in vitro maturation, fertilisation and culture. The mean (± s.e.m.) follicular concentrations of testosterone (62.8 ± 4.8 ng mL–1), progesterone (616.8 ± 31.9 ng mL–1) and oestradiol (14.4 ± 2.4 ng mL–1) were not different (P > 0.05) between oocytes that formed (competent) or failed to form (incompetent) blastocysts. Principal-component analysis of the quantified aqueous metabolites in follicular fluid showed differences between oocytes that formed blastocysts and oocytes that degenerated; l-alanine, glycine and l-glutamate were positively correlated and urea was negatively correlated with blastocyst formation. Follicular fluid associated with competent oocytes was significantly lower in palmitic acid (P = 0.023) and total fatty acids (P = 0.031) and significantly higher in linolenic acid (P = 0.036) than follicular fluid from incompetent oocytes. Significantly higher (P < 0.05) transcript abundance of LHCGR in granulosa cells, ESR1 and VCAN in thecal cells and TNFAIP6 in cumulus cells was associated with competent compared with incompetent oocytes.

The follicular microenvironment in low (++) and high (I+B+) ovulation rate ewes

Reproduction ◽  
2020 ◽  
Vol 159 (5) ◽  
pp. 585-599
Author(s):  
Zaramasina L Clark ◽  
Derek A Heath ◽  
Anne R O’Connell ◽  
Jennifer L Juengel ◽  
Kenneth P McNatty ◽  
...  
Keyword(s):  
Follicular Fluid ◽  
Cumulus Cell ◽  
Single Copy ◽  
Oocyte Quality ◽  
Ovulation Rate ◽  
Fluid Composition ◽  
Embryo Survival ◽  
Expression Levels ◽  
Follicular Maturation ◽  
Metabolic Substrates

Ewes with single copy mutations in GDF9, BMP15 or BMPR1B have smaller preovulatory follicles containing fewer granulosa cells (GC), while developmental competency of the oocyte appears to be maintained. We hypothesised that similarities and/or differences in follicular maturation events between WT (++) ewes and mutant ewes with single copy mutations in BMP15 and BMPR1B (I+B+) are key to the attainment of oocyte developmental competency and for increasing ovulation rate (OR) without compromising oocyte quality. Developmental competency of oocytes from I+B+ animals was confirmed following embryo transfer to recipient ewes. The microenvironment of both growing and presumptive preovulatory (PPOV) follicles from ++ and I+B+ ewes was investigated. When grouped according to gonadotropin-responsiveness, PPOV follicles from I+B+ ewes had smaller mean diameters with fewer GC than equivalent follicles in ++ ewes (OR = 4.4 ± 0.7 and 1.7 ± 0.2, respectively; P < 0.001). Functional differences between these genotypes included differential gonadotropin-responsiveness of GC, follicular fluid composition and expression levels of cumulus cell-derived VCAN, PGR, EREG and BMPR2 genes. A unique microenvironment was characterised in I+B+ follicles as they underwent maturation. Our evidence suggests that GC were less metabolically active, resulting in increased follicular fluid concentrations of amino acids and metabolic substrates, potentially protecting the oocyte from ROS. Normal expression levels of key genes linked to oocyte quality and embryo survival in I+B+ follicles support the successful lambing percentage of transferred I+B+ oocytes. In conclusion, these I+B+ oocytes develop normally, despite radical changes in follicular size and GC number induced by these combined heterozygous mutations.

P–219 mtDNA content in bovine cumulus cells does not predict oocytés developmental competence

2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
Á Martíne. Moro ◽  
I Lamas-Toranzo ◽  
L González-Brusi ◽  
A Pérez-Gómez ◽  
P Bermejo-Álvarez
Keyword(s):  
Cumulus Cell ◽  
Cumulus Cells ◽  
Oocyte Quality ◽  
Blastocyst Stage ◽  
Early Embryo ◽  
Developmental Competence ◽  
Success Rates ◽  
Developmental Potential ◽  
Mtdna Sequence

Abstract Study question Does cumulus cell mtDNA content correlate with oocyte developmental potential in the bovine model? Summary answer The relative amount of mtDNA content did not vary significantly in oocytes showing different developmental outcomes following IVF What is known already Cumulus cells are closely connected to the oocyte through transzonal projections, serving essential metabolic functions during folliculogenesis. These oocyte-supporting cells are removed and discarded prior to ICSI, thereby constituting an interesting biological material on which to perform molecular analysis aimed to predict oocyte developmental competence. Previous studies have positively associated oocytés mtDNA content with developmental potential in both animal models and women. However, it remains debatable whether mtDNA content in cumulus cells could be used as a proxy to infer oocyte developmental potential. Study design, size, duration Bovine cumulus cells were allocated into three groups according to the developmental potential of the oocyte: 1) oocytes developing to blastocysts following IVF (Bl+Cl+), 2) oocytes cleaving following IVF but arresting their development prior to the blastocyst stage (Bl-Cl+), and 3) oocytes not cleaving following IVF (Bl-Cl-). Relative mtDNA content was analysed in 40 samples/group, each composed by the cumulus cells from one cumulus-oocyte complex (COC). Participants/materials, setting, methods Bovine cumulus-oocyte complexes were obtained from slaughtered cattle and individually matured in vitro (IVM). Following IVM, cumulus cells were removed by hyaluronidase treatment, pelleted, snap frozen in liquid nitrogen and stored at –80 ºC until analysis. Cumulus-free oocytes were fertilized and cultured in vitro individually and development was recorded for each oocyte. Relative mtDNA abundance was determined by qPCR, amplifying a mtDNA sequence (COX1) and a chromosomal sequence (PPIA). Statistical differences were tested by ANOVA. Main results and the role of chance Relative mtDNA abundance did not differ significantly (ANOVA p &gt; 0.05) between the three groups exhibiting different developmental potential (1±0.06 vs. 1.19±0.05 vs. 1.11±0.05, for Bl+Cl+ vs. Bl-Cl+ vs. Bl-Cl-, mean±s.e.m.). Limitations, reasons for caution Experiments were conducted in the bovine model. Although bovine folliculogenesis, monoovulatory ovulation and early embryo development exhibit considerable similarities with that of humans, caution should be taken when extrapolating these data to humans. Wider implications of the findings: The use of molecular markers for oocyte developmental potential in cumulus cells could be used to enhance success rates following single-embryo transfer. Unfortunately, mtDNA in cumulus cells was not found to be a good proxy for oocyte quality. Trial registration number Not applicable

205 CUMULUS CELL MORPHOLOGY BEFORE AND AFTER IN VITRO MATURATION AS AN INDICATOR OF PORCINE OOCYTE DEVELOPMENTAL COMPETENCE

2010 ◽  
Vol 22 (1) ◽  
pp. 260
Author(s):  
M. Bertoldo ◽  
P. K. Holyoake ◽  
G. Evans ◽  
C. G. Grupen
Keyword(s):  
Cell Morphology ◽  
In Vitro Maturation ◽  
Cumulus Cell ◽  
Cumulus Cells ◽  
Oocyte Quality ◽  
Developmental Competence ◽  
Oocyte Developmental Competence ◽  
Before And After ◽  
Follicle Size

Effective in vitro maturation (IVM) is essential for successful in vitro embryo production. The morphology of the cumulus investment before and after IVM may be a useful noninvasive indicator of oocyte quality. In pigs, oocyte developmental competence is reduced during the summer months. The aim of this study was to determine whether the morphology of cumulus-oocyte complexes (COC) before and after IVM are associated with oocyte quality, using COC collected from small and large follicles in summer and winter as models of poor and good oocyte quality. Ovaries were collected from sows slaughtered 4 days after weaning. The COC recovered from small (3-4 mm) and large (5-8 mm) antral follicles were morphologically graded and parthenogenetically activated following IVM during winter (n = 1419; 10 replicates) and summer (n = 2803; 10 replicates). Grade 1 and 2 COC had >2 layers of compact cumulus cells and a homogenous cytoplasm. Grade 3 COC were either partially or fully denuded, had a heterogeneous cytoplasm, or were vacuolated or dark in color. Grade 4 COC had expanded cumulus cells. Cumulus expansion was also assessed subsequent to IVM. The COC recorded as having a cumulus expansion index (CEI) of 1 had the poorest expansion with no detectable response to IVM, whereas those with a CEI of 4 had the greatest amount of expansion, including that of the corona radiata. Data were analyzed using a generalized linear mixed model in GenStat® (release 10, VSN International, Hemel Hempstead, UK). There was an effect of follicle size for Grade 1 COC, with COC from large follicles in both seasons yielding better quality COC (P < 0.05). The proportion of COC in Grade 2 was higher in small follicles during winter compared with large follicles, but there were no differences between follicle sizes during summer (P < 0.05). The proportion of COC with CEI 1 was highest in COC from small follicles during summer (P < 0.05). The proportion of COC from large follicles with CEI 2 was higher during summer compared with winter (P < 0.05). There were no seasonal or follicle size effects on COC with CEI 3 or 4 (P > 0.05). The proportion of oocytes that developed to blastocysts was greater in winter than in summer (39.06% ± 5.67 v. 22.27% ± 4.01; P < 0.05). Oocytes derived from large follicles had a greater ability to form blastocysts compared with those from small follicles (37.13% ± 5.65 v. 23.32% ± 4.56; P < 0.06). Morphological assessment of cumulus cells before and after IVM may be a useful tool to evaluate the effects of follicle size on oocyte developmental competence. However, the results of the present study indicate that cumulus cell morphology is not a good indicator of the effect of season on oocyte developmental competence.

38 Developmental competence of bovine cumulus–oocyte complexes collected from cows fed rumen-protected methionine and lysine

2021 ◽  
Vol 33 (2) ◽  
pp. 126
Author(s):  
M. Ritz ◽  
A. Gonzalez ◽  
A.-S. Fries ◽  
T. Scheu ◽  
N. Blad-Stahl ◽  
...  
Keyword(s):  
Dairy Cows ◽  
Follicular Fluid ◽  
Oocyte Quality ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
Isopropyl Ester ◽  
Lactating Dairy Cows ◽  
Endogenous Lipid ◽  
Developmental Rates ◽  
Methionine Supplementation

Supplementation of rumen-protected amino acids (RPAA) has proven to be an effective tool to supply limiting AA in dairy diets. Methionine and lysine are the two most limiting AA for lactating dairy cows. Recently, it has been shown that methionine supplementation seems to affect pre-implantation embryos collected from superovulated cows enhancing their developmental competence because there is strong evidence that endogenous lipid reserves serve as an energy substrate (Acosta et al. 2016 Theriogenology 85, 1669–1679). Moreover, higher concentrations of methionine were determined in the follicular fluid of the first dominant follicle postpartum in cows supplemented with rumen-protected methionine and rumen-protected choline from 21 days before calving to 30 days postpartum and it was assumed that higher methionine concentrations in the follicular fluid could affect oocyte quality (Acosta et al. 2017 Theriogenology 96, 1–9). There is no information available so far regarding the effect of a combined methionine and lysine supplementation (each rumen-protected) on oocyte quality. Therefore, the objective of this study was to evaluate the effect of a combined methionine and lysine supplementation during early to mid-lactation on the developmental competence of oocytes collected from lactating dairy cows (days 0 to 100 p.p.). Thirty pregnant multiparous German Holstein dairy cows were grouped 3 weeks before their expected calving date, receiving identical diets. After calving, they were randomly allocated to 2 groups fed a total mixed ration supplemented with (N=14 cows; RPAA) or without (N=16 cows; CON) LysiGEMTM (encapsulated lysine; Kemin Industries) and Metasmart DryTM (isopropyl ester of the hydroxylated analogue of methionine adsorbed onto a silicon dioxide carrier; Adisseo). Starting from 45 days p.p., animals from both groups were submitted to an ovum pickup (OPU) session once a week for at least 8 weeks. Collected cumulus–oocyte complexes (COC) were subjected to a standard invitro production (IVP) protocol (Stinshoff et al. 2014 Reprod. Fertil. Dev. 26, 502–10) including IVM, IVF, and invitro culture (IVC). Cleavage and developmental rates up to the morula/blastocyst stage were recorded on Days 3, 7, and 8. In total, 1211 follicles have been aspirated from RPAA animals compared with 1413 from CON animals, from which 742 and 885 COC were collected, respectively. The calculated recovery rate based on the number of aspirated follicles and collected COC was similar for both groups (61.3±29.4% vs. 62.6±33.5%). Cleavage and developmental rates based on 240 (RPAA group) and 299 (CON group) COC also showed similar results [RPAA: 84.1±5.9% (202/240), 18.3±4.4% (44/240), 18.8±4.7% (45/240); CON: 81.9±8.6% (245/299), 15.4±8.9% (46/299), 16.7±8.4% (50/299)]. In conclusion, supplementation of RPAA (methionine and lysine) had no beneficial effect on the developmental competence of COC obtained from these animals compared with those collected from cows fed the diet without RPAA supplementation.

scholarly journals Sequential exposure of porcine cumulus cells to FSH and/or LH is critical for appropriate expression of steroidogenic and ovulation-related genes that impact oocyte maturation in vivo and in vitro

Reproduction ◽  
2008 ◽  
Vol 136 (1) ◽  
pp. 9-21 ◽  
Author(s):  
Ikkou Kawashima ◽  
Tetsuji Okazaki ◽  
Noritaka Noma ◽  
Masahide Nishibori ◽  
Yasuhisa Yamashita ◽  
...  
Keyword(s):  
Oocyte Maturation ◽  
Chorionic Gonadotropin ◽  
Follicular Fluid ◽  
Culture System ◽  
Expression Patterns ◽  
Cumulus Cell ◽  
Cumulus Cells ◽  
Developmental Competence ◽  
Antral Follicles

In this study, we collected follicular fluid, granulosa cells, and cumulus cells from antral follicles at specific time intervals following equine chorionic gonadotropin (eCG) and human chorionic gonadotropin (hCG) treatment of gilts. The treatment with eCG increased the production of estrogen coordinately with up-regulated proliferation of granulosa and cumulus cells. eCG also induced the expression ofLHCGRandPGRin cumulus cells and progesterone accumulation was detected in follicular fluid prior to the LH/hCG surge. Moreover, progesterone and progesterone receptor (PGR) were critical for FSH-inducedLHCGRexpression in cumulus cells in culture. The expression ofLHCGRmRNA in cumulus cells was associated with the ability of LH to induce prostaglandin production, release of epidermal growth factor (EGF)-like factors, and a disintegrin and metalloprotease with thrombospondin-like repeats 1 expression, promoting cumulus cell oocyte complexes (COCs) expansion and oocyte maturation. Based on the unique expression and regulation ofPGRandLHCGRin cumulus cells, we designed a novel porcine COCs culture system in which hormones were added sequentially to mimic changes observedin vivo. Specifically, COCs from small antral follicles were pre-cultured with FSH and estradiol for 10 h at which time progesterone was added for another 10 h. After 20 h, COCs were moved to fresh medium containing LH, EGF, and progesterone. The oocytes matured in this revised COC culture system exhibited greater developmental competence to blastocyst stage. From these results, we conclude that to achieve optimal COC expansion and oocyte maturation in culture the unique gene expression patterns in cumulus cells of each species need to be characterized and used to increase the effectiveness of hormone stimulation.

Mitochondrial SIRT5 is present in follicular cells and is altered by reduced ovarian reserve and advanced maternal age

2014 ◽  
Vol 26 (8) ◽  
pp. 1072 ◽  
Author(s):  
Leanne Pacella-Ince ◽  
Deirdre L. Zander-Fox ◽  
Michelle Lane
Keyword(s):  
Follicular Fluid ◽  
Ovarian Reserve ◽  
Protein Function ◽  
Maternal Age ◽  
Cumulus Cells ◽  
Advanced Maternal Age ◽  
Oocyte Quality ◽  
Carbamoyl Phosphate ◽  
Protein Activity

Women with reduced ovarian reserve or advanced maternal age have an altered metabolic follicular microenvironment. As sirtuin 5 (SIRT5) senses cellular metabolic state and post-translationally alters protein function, its activity may directly impact on oocyte viability and pregnancy outcome. Therefore, we investigated the role of SIRT5 in relation to ovarian reserve and maternal age. Women (n = 47) undergoing routine IVF treatment were recruited and allocated to one of three cohorts based on ovarian reserve and maternal age. Surplus follicular fluid, granulosa and cumulus cells were collected. SIRT5 mRNA, protein and protein activity was confirmed in granulosa and cumulus cells via qPCR, immunohistochemistry, western blotting and desuccinylation activity. The presence of carbamoyl phosphate synthase I (CPS1), a target of SIRT5, was investigated by immunohistochemistry and follicular-fluid ammonium concentrations determined via microfluorometry. Women with reduced ovarian reserve or advanced maternal age had decreased SIRT5 mRNA, protein and desuccinylation activity in granulosa and cumulus cells resulting in an accumulation of follicular-fluid ammonium, presumably via alterations in activity of a SIRT5 target, CPS1, which was present in granulosa and cumulus cells. This suggests a role for SIRT5 in influencing oocyte quality and IVF outcomes.

217 TRANSCRIPT ABUNDANCE OF CATHEPSIN GENES IN CUMULUS CELLS AS A MARKER OF CATTLE OOCYTE QUALITY

2013 ◽  
Vol 25 (1) ◽  
pp. 257
Author(s):  
E. Warzych ◽  
A. Wolc ◽  
A. Cieslak ◽  
D. Lechniak-Cieslak
Keyword(s):  
Real Time Pcr ◽  
Copy Number ◽  
Transcript Abundance ◽  
Cumulus Cells ◽  
Oocyte Quality ◽  
Mtdna Copy Number ◽  
Cdna Synthesis ◽  
Fa Composition ◽  
Grade 3

Dynamics of follicular growth and atresia is closely connected with apoptosis. Cathepsins (CTS) are involved in diverse biological functions, whereas one member of this family, cathepsin B, plays a major regulatory role in the process of apoptosis. Oocyte quality is a complex trait shaped by the follicular components (e.g. cumulus cells, CC; follicular fluid, FF). A negative relationship between relative transcript abundance (RA) of CTSB, CTSS, and CTSZ genes in CC with the quality of corresponding oocytes was reported in cattle (Bettegowda et al. 2008). Fatty acid (FA) composition of the FF and mtDNA copy number in the oocyte are other markers of oocyte quality. Therefore, in this study, we analysed relations between selected traits of the 3 follicular components (FF, CC, oocyte) within the individual follicle with the focus on oocyte quality. The experiment was based on cumulus–oocyte-complexes (COC) and FF obtained from individual follicles of slaughterhouse ovaries. Each follicle was measured and assigned into 1 of 3 classes (small <6 mm; medium 6 to 8 mm; large >8 mm). The COC morphology (grades 1 to 4) was evaluated according to Stojkovic et al. (2001). The following analyses were performed: CC, mRNA abundance of CTSB, CTSS, CTSZ, and CTSK genes (real-time PCR, 100 replicates, ACTB as a reference gene); the oocyte, mtDNA copy number (real-time PCR, 93 replicates, COX1 gene); and FF and FA composition (gas chromatography). The following procedures were employed: total RNA isolation, mirVana Paris Kit (Ambion); total DNA isolation, High Pure PCR Template Preparation Kit (Roche, Indianapolis, IN, USA); cDNA synthesis, Transcriptor High Fidelity cDNA Synthesis Kit (Roche); and the standard curve method, to analyse the qPCR data. For statistical analysis, the Kruskal-Wallis test as well as Spearman rank correlation were applied. The highest RA of CTSB gene was noted in CC from the grade 3 COC (P < 0.05), whereas that of CTSK and CTSZ genes in CC from the grade 4 COC (P < 0.01). Because grade 3 and 4 COC are not suitable for IVM, we assumed that high RA of CTS gene in CC may indicate reduced quality of the corresponding oocyte. Surprisingly, the highest RA for CTSB gene was observed in CC from the medium follicles (P < 0.05). Significant (P < 0.05) correlations were estimated between the following: RA of CTSB gene in CC and mtDNA copy number in the oocyte (r = 0.27), RA of CTSB gene in CC and C18.3 n-3 concentration in FF (r = 0.32), RA of CTSZ gene in CC and C18.3 n-3 concentration in FF (r = 0.37), as well as RA of CTSZ gene in CC and n-3 concentration in FF (r = 0.34). Although an increase in RA of the CTS genes in CC was accompanied by the inferior oocyte morphology, it was also correlated with higher mtDNA copy number in the oocyte and FA content in FF. The last 2 features have been previously attributed to oocytes of better quality, what contrasts with the high RA of the CTS genes. Thus, higher RA of CTS genes within CC may not mark the bovine oocyte of reduced quality. Funding–National Science Center, grant no. N N302 604438.

132 Influence of oocyte retrieval methods and maturation media on invitro development of polar body extrusion in pig oocytes

2021 ◽  
Vol 33 (2) ◽  
pp. 174
Author(s):  
K. M. Honneysett ◽  
M. L. Mphaphathi ◽  
A. M. Maqhashu ◽  
E. C. Webb
Keyword(s):  
Follicular Fluid ◽  
Cell Layer ◽  
Polar Body ◽  
Cumulus Cell ◽  
Oocyte Retrieval ◽  
Cumulus Cells ◽  
Reproductive Technology ◽  
Maturation Stage ◽  
Significant Difference ◽  
Polar Body Extrusion

Oocyte recovery is a reproductive technology that can be done by using two techniques, aspiration and slicing. Invitro maturation (IVM) is an additional reproductive technology used to advance an oocyte to a maturation stage; thereafter, it may be used during IVF. The objectives of the present study were (1) to compare two different oocyte retrieval methods (aspiration and slicing) from pig ovaries on oocyte quality and quantity, and (2) to compare three different IVM media [NCSU 37, TCM-199, and modified porcine follicular fluid (mpFF=porcine follicular fluid+FSH+LH] on oocytes’ polar body extrusion. During aspiration, an 18G needle was attached to a 10-mL syringe and all visible follicles were aspirated. During slicing, a surgical blade was used to slice the ovaries held in mDPBS. Follicular fluid collected from both methods was assessed for the presence of oocytes with the aid of a microscope. The collected oocytes were then categorized as Grade A, B, or C: Grade A=oocytes with compacted, multilayered cumulus cells and a homogeneous ooplasm; Grade B=oocytes with a compact cumulus cell layer with homogeneous ooplasm; Grade C=oocytes with a less compact cumulus cell layer with irregular ooplasm containing dark granules. The IVM media were placed in a four-well multidish; thereafter Grades A and B oocytes were allocated per treatment groups and matured for 44h. The treatment means were compared using the Fisher’s protected t-test least significant difference. The results showed significant differences between the grades of oocytes (P&lt;0.05) with Grade A and B oocytes accounting for 50.8% of total oocytes (193.8) for aspiration and 58.7% of total oocytes (488.6) for slicing. The oocytes polar body extrusion was recorded as 25.3, 84.2, and 73.8% for NCSU 37 (P&lt;0.05) and TCM-199 and mpFF respectively (P&gt;0.05). In conclusion, the slicing method proved to be better than aspiration with regards to the retrieval of Grades A and B oocytes as well as the total number of oocytes retrieved. The TCM-199 and mpFF media had a higher percentage of oocytes with polar body extrusion than NCSU 37.

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