Embryo development capacity of oocytes fertilized by immature sperm and sperm treated with motility stimulants

1994 ◽  
Vol 6 (1) ◽  
pp. 113 ◽  
Author(s):  
O Lacham-Kaplan ◽  
AO Trounson
Keyword(s):  
Embryonic Development ◽  
Embryo Development ◽  
Sperm Motility ◽  
Cumulus Cells ◽  
Progressive Motility ◽  
Development Capacity ◽  
Fertilizing Ability ◽  
Embryonic Arrest ◽  
Human And Mouse

The fertilizing ability of mouse spermatozoa develops during maturation and coincides with the acquisition of motility. The lack of progressive motility of spermatozoa from the testis and precaudal segments of the epididymis interferes with their ability to fertilize oocytes after insemination in vitro. The removal of cumulus cells for insemination in vitro and the use of subzonal injection of a single spermatozoon resulted in a higher number of oocytes fertilized by immature caput and corpus spermatozoa. High rates of embryonic arrest and retarded development were observed in oocytes fertilized by caput and corpus spermatozoa when compared with oocytes fertilized by cauda spermatozoa. However, when the oocytes were enclosed in their cumulus cells or microinjected with a single spermatozoon, these effects were reduced. A block in embryonic development was also observed after human and mouse oocytes were exposed to the sperm motility stimulants pentoxifylline (PTF) and 2-deoxyadenosine (DOA). These observations suggest that exposure of oocytes to PTF and DOA should be avoided.

scholarly journals Early embryonic development of bovine oocytes challenged with LPS in vitro or in vivo

Reproduction ◽  
2019 ◽  
Vol 158 (5) ◽  
pp. 453-463
Author(s):  
Joao Alveiro Alvarado Rincón ◽  
Patricia Carvalho Gindri ◽  
Bruna Mion ◽  
Ferronato Giuliana de Ávila ◽  
Antônio Amaral Barbosa ◽  
...  
Keyword(s):  
Embryonic Development ◽  
Embryo Development ◽  
Cumulus Cells ◽  
Early Embryo ◽  
Cleavage Rate ◽  
Early Embryo Development ◽  
Lps Challenge ◽  
Bovine Oocytes

The aim of this study was to evaluate the effect of exposing bovine oocytes to lipopolysaccharides (LPS) in vivo and in vitro on early embryo development. In experiment 1, cumulus oocyte complexes (COCs, n = 700/group) were challenged with 0, 0.1, 1.0 or 5.0 μg/mL of LPS during in vitro maturation (IVM). Later, in vitro fertilization (IVF) and in vitro culture (IVC) were performed. In experiment 2, COCs (n = 200/group) matured and in vitro fertilized without LPS were subjected to IVC with the same doses of LPS from experiment 1. In experiment 3, heifers received two injections of saline solution (n = 8) or 0.5 μg/kg of LPS (n = 8) 24 h apart, and 3 days later, COCs were recovered and submitted to IVM, IVF, and IVC. In experiments 1 and 3, the expression of TLR4, TNF, AREG and EREG genes in cumulus cells was evaluated. Exposure to 1 and 5 μg/mL of LPS during IVM decreased nuclear maturation (39.4 and 39.6%, respectively) compared with control (63.6%, P < 0.05). Despite that, no effect on cleavage and blastocyst rates were observed. Exposure to LPS during IVC did not affect embryonic development. In vivo exposure to LPS decreased the in vitro cleavage rate (54.3 vs 70.2%, P = 0.032), but cleaved embryos developed normally. Number of cells per embryo and gene expression were not affected by the LPS challenge in any experiment. In conclusion, although in vitro exposure to LPS did not affect early embryo development, in vivo LPS exposure reduced cleavage rate.

scholarly journals Mouse sperm delivery via cauda epididymis without transport medium

2016 ◽  
Author(s):  
Man-Xi Jiang ◽  
Mei-Shan Wang ◽  
Xiang-Hong Ou ◽  
Xue-Jin Chen ◽  
Yan Zhu
Keyword(s):  
Embryo Development ◽  
Sperm Motility ◽  
Room Temperature ◽  
Sperm Quality ◽  
Mouse Sperm ◽  
Spermatozoa Motility ◽  
Transport Medium ◽  
Fertilization Rates ◽  
Progressive Motility

This study was aimed to investigate the effects of room temperature (RT, 20-25 oC) and absence of medium during cauda epididymis transport on spermatozoa quality, fertility and embryo development. In the first experiment, fresh sperm from one side of cauda epididymis was used for in vitro fertilization, and another side was delivered at RT or 4-8 oC either with or without M2. In the second experiment, each side of cauda epididymis obtained from the same mouse was individually delivered at RT or 4 oC with or without M2. Finally, sperm motility, progressive motility scores and fertility of fresh spermatozoa or those from transported cauda epididymis, and IVF embryo development were evaluated. Progressive motility scores and fertilization rates were higher in fresh spermatozoa than transported sperm; sperm motility of transported cauda epididymis at 4-8 oC was comparable to fresh spermatozoa, but spermatozoa motility of transported cauda epididymis at RT was inferior to fresh spermatozoa. Spermatozoa motillty of transported cauda epididymis at 4-8 oC with transport medium was much higher than that without transport medium; absence of transport medium did not affect sperm motility of transported cauda epididymis at 4-8 oC but affected sperm motility of transported cauda epididymis at RT. Sperm quality from transported cauda epididymis can be efficiently kept at 4-8 oC, and cauda epididymis transport at 4-8 oC without M2 is more beneficial on keeping their fertility. Moreover cauda epididymis transport at RT without medium could sufficiently produce embryos for obtainning live offsprings.

scholarly journals Mouse sperm delivery via cauda epididymis without transport medium

2016 ◽  
Author(s):  
Man-Xi Jiang ◽  
Mei-Shan Wang ◽  
Xiang-Hong Ou ◽  
Xue-Jin Chen ◽  
Yan Zhu
Keyword(s):  
Embryo Development ◽  
Sperm Motility ◽  
Room Temperature ◽  
Sperm Quality ◽  
Mouse Sperm ◽  
Spermatozoa Motility ◽  
Transport Medium ◽  
Fertilization Rates ◽  
Progressive Motility

This study was aimed to investigate the effects of room temperature (RT, 20-25 oC) and absence of medium during cauda epididymis transport on spermatozoa quality, fertility and embryo development. In the first experiment, fresh sperm from one side of cauda epididymis was used for in vitro fertilization, and another side was delivered at RT or 4-8 oC either with or without M2. In the second experiment, each side of cauda epididymis obtained from the same mouse was individually delivered at RT or 4 oC with or without M2. Finally, sperm motility, progressive motility scores and fertility of fresh spermatozoa or those from transported cauda epididymis, and IVF embryo development were evaluated. Progressive motility scores and fertilization rates were higher in fresh spermatozoa than transported sperm; sperm motility of transported cauda epididymis at 4-8 oC was comparable to fresh spermatozoa, but spermatozoa motility of transported cauda epididymis at RT was inferior to fresh spermatozoa. Spermatozoa motillty of transported cauda epididymis at 4-8 oC with transport medium was much higher than that without transport medium; absence of transport medium did not affect sperm motility of transported cauda epididymis at 4-8 oC but affected sperm motility of transported cauda epididymis at RT. Sperm quality from transported cauda epididymis can be efficiently kept at 4-8 oC, and cauda epididymis transport at 4-8 oC without M2 is more beneficial on keeping their fertility. Moreover cauda epididymis transport at RT without medium could sufficiently produce embryos for obtainning live offsprings.

PSV-5 Relationship between sire conception rate, sperm motility, sperm SERPINA5 relative concentration, and in vitro produced embryos in dairy bulls

2021 ◽  
Vol 99 (Supplement_3) ◽  
pp. 310-310
Author(s):  
Saulo Menegatti Zoca ◽  
Julie Walker ◽  
Taylor Andrews ◽  
Adalaide C Kline ◽  
Jerica J Rich ◽  
...  
Keyword(s):  
Embryo Development ◽  
Sperm Motility ◽  
Relative Concentration ◽  
Early Embryo ◽  
Conception Rate ◽  
Cleavage Rate ◽  
Blastocyst Rate ◽  
Positive Correlations ◽  
Sire Conception Rate

Abstract Sire conception rate (SCR) is a field measure of fertility among bulls, but it can be influenced by several factors (Sperm transport, sperm-egg binding, early embryo development, etc). The objective of this study was to evaluate the relationship between SCR, sperm motility, SERPINA5 concentrations, and in vitro embryo development. Measurements were performed in 19 bulls with SCR values ranging from -7.7 to 4.45. For each bull, an aliquot of frozen-thawed semen was used for analyses of total (TMOT) and progressive (PROG) motility. Remaining semen was fixed with 2% formaldehyde, and concentration of SERPINA5 was determined by immunolocalization (antibody SERPINA5/Dylight405; PA5-79976-Invitrogen / ab201798-Abcam). Mean fluorescence intensity was determined in ~200 sperm heads/bull. Approximately 149 oocytes/bull were fertilized in vitro for embryo development analysis (cleavage and blastocyst rates). Statistical procedures were performed in SAS (9.4) using the procedures CORR for correlations (SCR, TMOT, PROG, SERPINA5, cleavage and blastocyst) and GLIMMIX for comparison of “field-fertility” (SCR divided in HIGH or LOW) and “field-embryo-fertility” (LOW-SCR sires were divided based on blastocyst rate (HIGH or LOW) resulting in two classifications; LOW-HIGH≥31% and LOW-LOW≤26%, respectively). There were positive correlations (P &lt; 0.05) between cleavage-blastocyst (r=0.50), SERPINA5-cleavage (r=0.48), and TMOT-PROG (r=0.76). Sire SCR was not associated with SERPINA5, TMOT, PROG, cleavage and blastocyst rate (P &gt; 0.52). Among LOW-SCR sires, LOW-LOW sires (-4.83±0.60) tended to have a better SCR score than LOW-HIGH (-6.18±0.42) sires (P = 0.08), but there were no differences (P &gt; 0.43) between LOW-HIGH, LOW-LOW, and HIGH sires for SERPINA5, TMOT, PROG, and cleavage. In conclusion, some LOW SCR sires have good embryo development indicating a different mechanism for their low SCR; however, these differences in SCR could not be explained by TMOT, PROG, SERPINA5, cleavage and blastocyst. There were, however, positive correlations between cleavage-blastocyst rate, and SERPINA5-cleavage rate.

Effect of the number of inseminated spermatozoa on subsequent human and mouse embryonic development in vitro

1992 ◽  
Vol 7 (7) ◽  
pp. 1010-1013 ◽  
Author(s):  
John C.M. Dumoulin ◽  
Marijke Bras ◽  
Jolande A. Land ◽  
Math H.E.C. Pieters ◽  
M.Engin Enginsu ◽  
...  
Keyword(s):  
Embryonic Development ◽  
Development In Vitro ◽  
Human And Mouse

374 THE EFFECTS OF POLYVINYLPYRROLIDONE ON BOVINE SPERM FUNCTION AND EMBRYO DEVELOPMENT

2007 ◽  
Vol 19 (1) ◽  
pp. 302 ◽  
Author(s):  
Y. Kato ◽  
M. Fukushima ◽  
A. Kenmotsu ◽  
K. Chikazawa ◽  
Y. Nagao
Keyword(s):  
Embryonic Development ◽  
Embryo Development ◽  
Staining Method ◽  
Blastocyst Stage ◽  
Sperm Function ◽  
Bovine Sperm ◽  
Developmental Capacity ◽  
Chromatin Integrity ◽  
Triple Staining

In assisted reproduction by ICSI, PVP has been successfully used to replicate the viscosity of sperm solution, thus facilitating the handling and immobilization of spermatozoa. Sperm is suspended in medium containing polyvinylpyrrolidone (PVP), then injected into the oocytes together with a small amount of the medium in ICSI. However the effects of PVP on sperm function and embryo development have not been investigated in detail. In the present study, we investigated the effects of PVP solution on sperm function and embryonic development. Frozen–thawed spermatozoa from a Japanese Black bull and immature COCs from slaughterhouse bovine ovaries were used for all experiments. In experiment 1, bovine sperm was cultured in SOF or SOF containing 10% PVP. For detection of sperm acrosomal and chromatin integrity, sperm cultured in each medium were stained by the triple staining method and acridine orange after 0, 15, 30, and 60 min of culture. In experiment 2, zygotes were injected with PVP solution and cultured in vitro; subsequent cleavage and development to blastocysts were examined. In experiment 3, zygote injected with PVP solution was fixed by 4% paraformaldehyde after 1–3 h of PVP injection. The location of PVP solution in zygote was observed. In experiment 4, two-cell embryos were microinjected with a solution of dextran conjugated with fluorescein (FITC-dextran) and cultured in vitro. The location of FITC-dextran in the embryo was examined. In experiment 1, acrosome reactions of the sperm were enhanced after 15 min of incubation in PVP solution (P &lt; 0.05), but chromatin integrity of the sperm was not influenced (P &gt; 0.05). In experiment 2, PVP suppressed the development of the zygote to 2-cell, morula and blastocyst (75.0%, 35.1%, and 26.3% vs. 61.3%, 20.2%, and 12.9% for control and PVP group, respectively, P &lt; 0.05). In experiment 3, the locations of PVP solution in the zygote were observed 1–3 h after injection. In experiment 4, FITC-dextran was observed in ICM at the blastocyst stage. These findings suggest that PVP affects the acrosome but not the chromatin of sperm in ICSI. PVP solution exists locally in embryos injected and affects the developmental capacity of the embryos.

224 LOCALIZATION OF NITRIC OXIDE SYNTHASE ACTIVITY IN BUFFALO (BUBALUS BUBALIS) OOCYTES AND EMBRYOS

2011 ◽  
Vol 23 (1) ◽  
pp. 211
Author(s):  
K. R. Babu ◽  
R. Sharma ◽  
K. P. Singh ◽  
A. George ◽  
M. S. Chauhan ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Embryo Development ◽  
In Vitro Maturation ◽  
Cumulus Cells ◽  
Epifluorescence Microscopy ◽  
Rt Pcr ◽  
Cell Stage ◽  
Vitro Fertilization ◽  
Nos Isoforms

Ovarian nitric oxide (NO) and that produced within the oocytes and embryos have been reported to play important roles in oocyte meiotic maturation and embryo development. Production of NO is catalyzed by NO synthase (NOS), which exists in 3 isoforms, the constitutive endothelial (eNOS) and neuronal (nNOS) isoforms and the inducible (iNOS) isoform. We have previously shown that low concentrations of NO stimulate and high concentrations inhibit embryo development, and that endogenous NO produced by iNOS is necessary for optimal embryo development in the buffalo. The present study was aimed at localizing different isoforms of NOS and examining their relative mRNA abundance in buffalo oocytes and embryos. Oocytes from slaughterhouse ovaries were subjected to in vitro maturation in 100-μL droplets (10 to 15 oocytes/droplet) of in vitro maturation medium (TCM-199 + 10% FBS + 5 μg mL–1 of pFSH + 1 μg mL–1 of oestradiol-17β + 0.81 mM sodium pyruvate + 10% buffalo follicular fluid + 50 μg mL–1 of gentamicin) for 24 h in a CO2 incubator (5% CO2 in air) at 38.5°C. In vitro fertilization was carried out by incubating in vitro-matured oocytes with 2 to 4 million spermatozoa mL–1 for 18 h. The presumed zygotes were cultured on original beds of cumulus cells in in vitro culture medium (mCR2aa + 0.6% BSA + 10% FBS) for up to 8 days post-insemination. Immature and in vitro-matured oocytes and embryos at the 2-cell, 4-cell, 8- to 16-cell, morula, and blastocyst stages were examined for the presence of NOS isoforms by indirect immunofluorescence staining using epifluorescence microscopy and RT-PCR. Each experiment was repeated in triplicate, and data were analysed using one-way ANOVA, after arcsine transformation of percentage values. Expression of all 3 NOS isoforms was detected inside the cytoplasm, in all the stages of oocytes and embryos examined, by both immunofluorescence and RT-PCR. Abundance of the iNOS transcript was significantly higher (P ≤ 0.01) in the morula and blastocyst stages compared with that in immature and in vitro-matured oocytes and in embryos at the 2-cell, 4-cell, and 8- to 16-cell stages, indicating that its expression was up-regulated at the 8- to 16-cell stage. The expression of eNOS was significantly higher (P ≤ 0.05) in the immature and mature oocytes and in 8- to 16-cell stage embryos, morulae, and blastocysts than in the early-cleavage embryos at the 2- and 4-cell stages, indicating that it was down-regulated after fertilization and was up-regulated again at the 8- to 16-cell stage. Abundance of the nNOS transcript was not significantly different among all the stages of oocytes and embryos examined. These results demonstrate that different NOS isoforms are expressed in a dynamic manner during embryonic development in the buffalo. The role of an increase in expression of iNOS and eNOS at the 8- to 16-cell stage, at which a developmental block occurs in this species, needs to be examined.

256 EFFECT OF SPERM SELECTION ON THE RATE OF IN VITRO FERTILIZATION IN ALPACA (VICUGNA PACOS)

2015 ◽  
Vol 27 (1) ◽  
pp. 217
Author(s):  
E. Mellisho ◽  
V. Rivas ◽  
J. Ruiz ◽  
G. Mamani
Keyword(s):  
Extended Period ◽  
Cumulus Cells ◽  
Sperm Selection ◽  
Cleavage Rate ◽  
Slow Cooling ◽  
Vitro Fertilization ◽  
Progressive Motility ◽  
Frozen Semen ◽  
Washing Method

In alpacas, improvement of reproductive efficiency of male camelids is limited by the small size of the testes, extended period of ejaculation, and low quality of semen. This study was designed to determine the effect of 2 sperm preparation treatments before IVF on the cleavage rate. The sperm was obtained by slicing the head of the epididymis of slaughtered male alpacas (n = 8), diluting in Tris-yolk-glycerol, and freezing with the slow-cooling method. Frozen semen straws per each male were thawed in a water bath at 37°C for 15 s and evaluated for percentage of progressive motility (32 ± 8.6%) and concentration (66.5 ± 24 × 106 sperm mL–1) post-thawing. Sperm selection by the swim-up method was performed by centrifugation at 1077 × g for 5 min with washing sperm medium eliminating the supernatant; sperm were settled in inclined tube with fertilization medium (without capacitating agent) for 60 min, after which 100 μL from the surface was recovered for use in IVF. The washing method consisted in repeated washing (twice) of sperm in washing sperm medium and fertilization medium by centrifugation at 1077 × g for 5 and 3 min, respectively, and recovery of 50 μL from the bottom of the tube for use in IVF. Sperm selected by swim-up or washing methods had similar characteristics of progressive motility (18 and 23%); however, the concentration was higher for the washing v. swim-up method (52 v. 14 × 106 sperm mL–1, respectively). Cumulus-oocyte complexes (COC) were recovered from 278 ovaries of alpacas killed at abattoirs and classified (Grade 1 and 2) for in vitro maturation (38.5°C at 5% CO2 in air for 27 h in 50 μL of 10 COC per drop). A total of 839 oocytes cultured for 27 h in maturation medium were partially stripped out of cumulus cells by gentle aspiration with a pipette. Sperm suspensions in Fert TALP medium (5 μL) from each treatment group were added to each fertilization drop with 10 oocytes per drop of 45 μL obtaining a final concentration of 10 × 106 sperm mL–1 and cultivated for 72 h until their evaluation. The data for the 13 repetitions of the rate of cleavage (2 to 8 cells) were converted to angular values (angle = arcsin √%) with the object of normalizing the distribution of the data; the analysis of variance was performed (complete randomised design with sub-sampling, P < 0.05) using SAS® version 8.0 for Windows. The rate of cleavage (cell division) did not show statistical differences (P = 0.67) for the swim-up method (37%; 155/421) v. washing method (35%; 147/418). The methods of sperm selection (swim-up and washing) did not affect the rate of IVF.

271 THE EFFECT OF TEMPERATURE ON HUMAN TESTICULAR TISSUE TO OPTIMIZE THE IN VITRO MATURATION OF PRE-FREEZE MOTILITY

2009 ◽  
Vol 21 (1) ◽  
pp. 233
Author(s):  
M. C. Schiewe ◽  
A. Spitz ◽  
R. E. Anderson
Keyword(s):  
Sperm Motility ◽  
Seminiferous Tubules ◽  
Type I ◽  
Obstructive Azoospermia ◽  
Percentage Increase ◽  
Testicular Sperm ◽  
Incubation Condition ◽  
Progressive Motility ◽  
Over Time

The development of intracytoplasmic sperm injection has made the use of testicular sperm a viable option for infertile men with obstructive and nonobstructive azoospermia. Over the past decade, testicular biopsies have been handled and processed using a variety of different methods. Whole biopsy pieces can be effectively cryopreserved in a 10% glycerol diluent (Schiewe et al. 1997 67, S115 abst); however, the ability to find viable, motile sperm post-thaw is improved when prefreeze motility exists. The purpose of this study was to comparatively document in vitro sperm motility enhancement over time at different temperatures, and to prove that an intermediate temperature (28 to 30°C) would optimize sperm longevity for up to 1 week. In this study, 10 men with obstructive azoospermia underwent a surgical, open testicular biopsy procedure. Each biopsy was placed in HEPES buffered-human tubal fluid (mHTF) medium supplemented with 5% human serum albumin (HSA; Irvine Sci., Santa Ana, CA, USA) and transported to the laboratory at room temperature. Each testis biopsy (TBx) was dissected into 8 equal pieces (approximately 2 × 2 × 1 mm). Five intact pieces of TBx were cryopreserved in separate cryovials for future use. The remaining TBx tissue was subdivided into 1 of 3 temperature treatment groups (24, 30, or 37°C) for extended IVM. The 30°C incubation condition was achieved by placing the dish(es) in a Styrofoam box placed on a 37°C warming plate. Each TBx was placed into a separate 100 × 35 mm Falcon dish in 150-μL droplets of mHTF under oil and shredded by needle dissection to disperse the contents of the seminiferous tubules. Reminant tissue was placed into another droplet for additional dissection, as needed. Sperm were analyzed for motility (graded as Type I = twitching, II = undulating, III = slow progression, and IV = rapid progression) at 0, 24, 96, and 144 h without replacing the IVM medium. The percentage increase in motility, compared with 0 h, was statistically contrasted across temperature treatments by chi-squared analysis. Testicular sperm motility ranged from 5 to 25% at 0 h and increased in all groups at 24 h. There was no difference in total motility at 24 h, but progressive motility was higher (P < 0.05) at 37°C compared with 24°C. Significant differences (P < 0.05) were observed in total percentage of motility/percentage of progressive motility at 96 h with treatment differences (*) being 30°C (66%*/44%*) > 24°C (42%*/14%) > 37°C (18%*/10%). This statistical trend continued at 144 h with 30°C (42%*/23%*) > 24°C (24%*/8%) > 37°C (9%*/5%). During this study, a viable pregnancy was achieved using a 30°C sample 8 days post biopsy, exceeding 2 previous healthy triplet pregnancies, which were successful using 4-day-old TBx specimens in 1996 and 2004. This study confirms that an intermediate IVM temperature of 30°C is optimal to enhance TBx cryopreservation, which in our laboratory is generally performed at 24 to 72 h of IVM. Our routine pregnancy success in applying ICSI with IVM, thawed TBx sperm discounts the concern some individuals express regarding DNA fragmentation of sperm over time.

168 INHIBITION OF BOVINE OOCYTE CUMULUS CELL PROGESTERONE SYNTHESIS DURING IN VITRO MATURATION AFFECTS CHROMOSOME ALIGNMENT AND SPINDLE INTEGRITY IN FERTILIZED EGGS AND EARLY EMBRYOS

2014 ◽  
Vol 26 (1) ◽  
pp. 198
Author(s):  
E. Daly ◽  
A. G. Fahey ◽  
M. M. Herlihy ◽  
T. Fair
Keyword(s):  
Embryo Development ◽  
Cumulus Cell ◽  
Cumulus Cells ◽  
Early Embryo ◽  
Developmental Competence ◽  
Bovine Oocyte ◽  
Spindle Formation ◽  
Vitro Fertilization ◽  
Time Treatment

We have previously demonstrated the importance of progesterone (P4) synthesis by cumulus cells during oocyte maturation in vitro (IVM) for bovine oocyte acquisition of developmental competence and subsequent embryo development (Aparicio et al. 2011 Biol. Reprod. 84). The aim of this study was to identify key processes that may be deregulated by the inhibition of P4 signalling in the cumulus–oocyte complex (COC) during IVM. To this end, good quality immature COC were placed in IVM medium [TCM-199 supplemented with 10% (vol/vol) FCS and 10 ng mL–1 epidermal growth factor] and cultured at 39°C for 22 h in a humidified atmosphere containing 5% CO2, in the presence or absence of 10 μM trilostane (which blocks P4 synthesis by inhibiting 3 β-hydroxysteroid dehydrogenase; Stegram Pharmaceuticals Ltd., Surrey, UK). Matured COC were washed and placed in 250 μL of fertilization medium (25 mM bicarbonate, 22 mM Na-lactate, 1 mM Na-pyruvate, 6 mg mL–1 fatty acid-free BSA, and 10 mg mL–1 heparin). In vitro fertilization (IVF) was performed with 250 μL of frozen–thawed semen at a final concentration of 1 × 106 spermatozoa mL–1 at 39°C under 5% CO2 during 20 h. Presumptive zygotes were denuded, washed, and transferred to 25-μL culture droplets (SOF + 5% FCS) at 39°C under 5% CO2, 90% of N2, and 5% O2 atmosphere with maximum humidity. Subsets of presumptive fertilized eggs and developing embryos were recovered at 6, 72, 120, and 192 h postinsemination (hpi) and processed for confocal whole-mount immunocytochemistry. The meiotic and mitotic spindles and chromosomes were visualised by immunofluorescent labelling of α-tubulin and 4′,6-diamindino-2-phenylindole (DAPI), respectively, and classified as normal if the chromosomes were correctly aligned or appropriately segregated, or abnormal if lagging chromosomes or abnormal chromosome segregation were observed. Samples were collected from 5 replicates (n = 50 zygotes/embryos per treatment, per timepoint) and a total of 157 spindles were observed. Logistic regression analysis was conducted to determine the probability of abnormal spindle formation. The incidence of spindle abnormality was regressed on time, treatment, and treatment by time. For all time points, there was significant reduction in the odds of abnormal spindle formation in control samples versus trilostane-treated samples (P < 0.001). In conclusion, our data imply a role for P4 signalling in maintaining spindle integrity during oocyte meiotic maturation and progression through the initial mitotic divisions of early embryo development in cattle.

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