Speed of in vitro embryo development affects the likelihood of foaling and the foal sex ratio

2020 ◽  
Vol 32 (5) ◽  
pp. 468 ◽  
Author(s):  
A. Claes ◽  
J. Cuervo-Arango ◽  
S. Colleoni ◽  
G. Lazzari ◽  
C. Galli ◽  
...  
Keyword(s):  
Sex Ratio ◽  
Embryo Development ◽  
Intracytoplasmic Sperm Injection ◽  
Odds Ratio ◽  
Blastocyst Stage ◽  
Embryo Production ◽  
Time Required

The success of invitro embryo production (IVEP) in horses has increased considerably during recent years, but little is known about the effect of the speed of invitro embryo development. Blastocysts (n=390) were produced by intracytoplasmic sperm injection of IVM oocytes from warmblood mares, cryopreserved, thawed and transferred into recipient mares on Days 3, 4, 5 or 6 after ovulation. The time required for invitro-produced (IVP) embryos to reach the blastocyst stage was recorded (Day 7 vs Day 8). The likelihood of foaling was affected by the speed of invitro embryo development and recipient day after ovulation at transfer. The odds ratio for foaling was ~0.63 for transfer of Day 8 (46%) compared with Day 7 (56%) IVP blastocysts. The highest likelihood of pregnancy (72%) and foaling (60%) was observed when IVP blastocysts were transferred to recipient mares on Day 4 after ovulation. Finally, the sex (colt:filly) ratio was higher after transfer of Day 7 (71%:29%) than Day 8 (54%:46%) IVP blastocysts, suggesting that the speed of embryo development is sex dependent. In conclusion, the speed of invitro embryo development in our IVEP system affects the likelihood of foaling and the sex of the foal.

Use of time-lapse imaging to evaluate morphokinetics of in vitro equine blastocyst development after oocyte holding for two days at 15°C versus room temperature before intracytoplasmic sperm injection

2019 ◽  
Vol 31 (12) ◽  
pp. 1862 ◽  
Author(s):  
N. A. Martino ◽  
G. Marzano ◽  
A. Mastrorocco ◽  
G. M. Lacalandra ◽  
L. Vincenti ◽  
...  
Keyword(s):  
Embryo Development ◽  
Intracytoplasmic Sperm Injection ◽  
Room Temperature ◽  
Time Lapse ◽  
Blastocyst Stage ◽  
Blastocyst Development ◽  
Cell Stage ◽  
Group 13 ◽  
Time Lapse Imaging

Time-lapse imaging was used to establish the morphokinetics of equine embryo development to the blastocyst stage after invitro oocyte maturation (IVM), intracytoplasmic sperm injection (ICSI) and embryo culture, in oocytes held overnight at room temperature (22–27°C; standard conditions) before IVM. Embryos that developed to the blastocyst stage underwent precleavage cytoplasmic extrusion and cleavage to the 2-, 3- and 4-cell stages significantly earlier than did embryos that arrested in development. We then determined the rate of blastocyst formation after ICSI in oocytes held for 2 days at either 15°C or room temperature before IVM (15-2d and RT-2d treatment groups respectively). The blastocyst development rate was significantly higher in the 15-2d than in the RT-2d group (13% vs 0% respectively). The failure of blastocyst development in the RT-2d group precluded comparison of morphokinetics of blastocyst development between treatments. In any condition examined, development to the blastocyst stage was characterised by earlier cytoplasmic extrusion before cleavage, earlier cleavage to 2- and 4-cell stages and reduced duration at the 2-cell stage compared with non-competent embryos. In conclusion, this study presents morphokinetic parameters predictive of embryo development invitro to the blastocyst stage after ICSI in the horse. We conclude that time-lapse imaging allows increased precision for evaluating effects of different treatments on equine embryo development.

197 DOES DURATION OF BOVINE OOCYTE MATURATION IN VITRO AFFECT THE SPEED OF EMBRYO DEVELOPMENT IN VITRO AND SEX RATIO AT THE TWO-CELL OR BLASTOCYST STAGE?

2008 ◽  
Vol 20 (1) ◽  
pp. 177
Author(s):  
P. Bermejo-Álvarez ◽  
A. Gutiérrez-Adán ◽  
P. Lonergan ◽  
D. Rizos
Keyword(s):  
Sex Ratio ◽  
Embryo Development ◽  
Blastocyst Stage ◽  
Early Embryo ◽  
Blastocyst Development ◽  
Maturation In Vitro ◽  
Maturational Stage ◽  
Development In Vitro ◽  
Oviduct Fluid

The faster-developing blastocysts in IVC systems are generally considered more viable and better able to survive following cryopreservation or embryo transfer than those that develop more slowly. However, evidence from several species indicates that embryos that reach the blastocyst stage earliest are more likely to be males than females. The aim of this study was to determine whether the duration of maturation could affect early embryo development and, furthermore, the sex ratio of early- or late-cleaved embryos and blastocysts. Cumulus–oocyte complexes were matured in vitro for 16 h (n = 2198) or 24 h (n = 2204). Following IVF, presumptive zygotes from each group were examined every 4 h between 24 and 48 h postinsemination (hpi) for cleavage, and all embryos were cultured to Day 8 in synthetic oviduct fluid to assess blastocyst development. Two-cell embryos at each time point and blastocysts on Days 6, 7, and 8 from both groups were snap-frozen individually for sexing. Sexing was performed with a single PCR using a specific primer BRY. There was a significantly lower number of cleaved embryos from the 16-h compared with the 24-h maturation group at 28 (10.0 � 1.51 v. 28.8 � 3.57%), 32 (35.3 � 1.48 v. 57.6 � 3.33%), 36 (54.8 � 1.76 v. 67.4 � 2.81%), 40 (63.3 � 1.82 v. 72.0 � 2.54%), and 48 (70.6 � 1.78 v. 77.1 � 2.18%) hpi, respectively (mean � SEM; P d 0.05). However, the blastocyst yields on Day 6 (17.1 � 3.11 v. 16.4 � 2.11%), 7 (30.6 � 4.10 v. 34.6 � 3.51%), or 8 (34.1 � 3.90 v. 39.4 � 4.26%) were similar for both groups (mean � SEM; 16 v. 24 h, respectively). Significantly more 2-cell early cleaved embryos (up to 32 hpi) were male compared with the expected 1:1 ratio from both groups (16 h: 1.24:0.76 v. 24 h: 1.17:0.83, P ≤ 0.05); however, the overall sex ratio among 2-cell embryos was significantly different from the expected 1:1 in favor of males only for the 16-h group (1.18:0.82, P ≤ 0.05). The sex ratio of blastocysts on Day 6, 7, or 8 from both groups was not different from the expected 1:1. However, the total number of male blastocysts obtained after 8 days of culture from the 24-h group was significantly different from the expected 1:1 (1.19:0.81, P ≤ 0.05) and approached significance in the 16-h group. These results show that the maturational stage of the oocyte at the time of fertilization has an effect on the kinetics of early cleavage divisions but not on blastocyst yield. Furthermore, irrespective of the duration of maturation, the sex ratio of early-cleaving 2-cell embryos was weighted in favor of males, and this observation was maintained at the blastocyst stage.

Impact of in vitro fertilization of bovine oocytes with sex-sorted frozen–thawed spermatozoa on developmental kinetics, quality and sex ratio of developing embryos

Zygote ◽  
2010 ◽  
Vol 18 (3) ◽  
pp. 185-194 ◽  
Author(s):  
J. Peippo ◽  
M. Räty ◽  
K. Korhonen ◽  
M. Eronen ◽  
K. Kananen ◽  
...  
Keyword(s):  
In Vitro Fertilization ◽  
Sex Ratio ◽  
Embryo Development ◽  
Bovine Embryos ◽  
Embryo Production ◽  
Male Embryo ◽  
Vitro Fertilization ◽  
Bovine Oocytes

SummaryWe studied whether bovine embryos developing after in vitro fertilization (IVF) with sex-sorted spermatozoa differed in developmental kinetics, quality and sex ratio from embryos produced with unsorted spermatozoa. Abattoir-derived oocytes were fertilized with X-sorted, Y-sorted or unsorted spermatozoa from a single bull. To evaluate economical use of the sex-sorted spermatozoa, washed spermatozoa from a single straw (2 million spermatozoa) were used to fertilize each batch of collected oocytes without any further isolation steps. Concentration of the unsorted spermatozoa was adjusted accordingly. Fertilizations were assessed by staining sperm asters at 10 hpi and pronuclei at 20 hpi. Embryo development and morphological quality were monitored on days 2, 7, 8 and 9 of the development (IVF = day 0). All embryos were sexed using PCR. Following fertilization, penetration and subsequent cleavage rates were compromised in the X-sorted group compared with the Y-sorted and unsorted groups (penetration: 58.0% vs. 89.8% and 90.0%, cleavage: 65.3% vs. 81.5% and 75.0%). The use of the sex-sorted spermatozoa did not, however, reduce the proportion of transferable embryos (sex-sorted 29.6% vs. unsorted 27.7%) or their quality (quality 1: sex-sorted 36.0% vs. unsorted 19.9%). The Y-sorted spermatozoa produced more transferable embryos of better quality than the X-sorted spermatozoa (days 7–8: 31.9% vs. 26.4%, quality 1: 38.9% vs. 30.6%). On average, out of 10 transferable embryos, nine were of the predicted sex in the X- and Y-sorted spermatozoa groups. These results indicate that low numbers of X- and Y-sorted spermatozoa can be used successfully for female and male embryo production in vitro.

249 AN EFFICIENT AND REPEATABLE IN VITRO FERTILIZATION TECHNIQUE IN THE EQUINE FOR PRESERVATION OF ENDANGERED SPECIES

2015 ◽  
Vol 27 (1) ◽  
pp. 214
Author(s):  
C. Douet ◽  
O. Parodi ◽  
F. Reigner ◽  
P. Barrière ◽  
G. Goudet
Keyword(s):  
Embryo Development ◽  
Intracytoplasmic Sperm Injection ◽  
Embryo Production ◽  
Chi Square ◽  
Vitro Fertilization ◽  
In Vitro Development ◽  
Vitro Development

Most wild equids are currently endangered or threatened, as mentioned in the International Union for the Conservation of Nature Red List, and several domestic horse breeds are at risk of extinction. Genome resource banking requires cryoconservation of semen, oocytes, and/or embryos. Embryo production in equids is limited in vivo because routine induction of multiple ovulation is still ineffective. Embryo production in vitro allows the production of several embryos per cycle that could easily be frozen because of their small size. Intracytoplasmic sperm injection has been widely adopted to generate horse embryos in vitro; however, intracytoplasmic sperm injection is time-consuming and requires expensive equipment and expertise in micromanipulation. Several attempts to establish an efficient IVF technique in the equine were performed, but reported IVF rates remain quite low and no repeatable equine IVF technique was available. Our objective was to develop an efficient and repeatable IVF technique in the equine. Immature cumulus-oocyte complexes (COC) were collected either from slaughtered mares in a local slaughterhouse or from our experimental mares by ovum pick up (OPU). The COC were cultured for 26 h in an in vitro maturation (IVM) medium or in preovulatory follicular fluid (FF) collected by OPU, pre-incubated for 30 min in oviducal fluid collected from slaughtered females, co-incubated for 18 h with fresh spermatozoa treated with procain, and cultured in SOF for 30 h. They were fixed and analysed either after 18 h IVF (experiment 1) or after 30 h in vitro development (experiment 2). In experiment 1, COC were collected from slaughtered mares and analysed after 18 h IVF. Zygotes with 2 pronuclei were observed. The IVF rate was similar for oocytes matured in IVM medium (22/33, 67%) or FF (24/42, 57%; chi-square test, P > 0.05). In experiment 2, COC were collected from slaughtered mares and from experimental mares and analysed after 30 h of in vitro development. We observed zygotes with 2 highly decondensed pronuclei, pronuclei decondensation being the first step of embryo development. For oocytes collected from slaughtered mares, the percentage of zygotes was similar for oocytes matured in IVM medium (8/11, 73%) or FF (10/15, 67%). For oocytes collected by ovum pickup, the percentage was similar for IVM medium (3/5, 60%) or FF (6/8, 75%). We also observed some embryonic structures with several nuclei, but the quality of these embryos was poor. In conclusion, we have established an efficient IVM-IVF technique that allows the first step of embryo development. Because we obtained similar results for 4 years, we consider that this efficient technique is repeatable. Further experiments are in progress to improve the quality of the embryos.

182 EFFECTS OF MITOCHONDRIAL ACTIVITY OF INJECTED SPERM ON EARLY DEVELOPMENT IN BOVINE INTRACYTOPLASMIC SPERM INJECTION-DERIVED EMBRYOS

2014 ◽  
Vol 26 (1) ◽  
pp. 205
Author(s):  
Y. Nagao ◽  
H. Yamamoto ◽  
B. Sarentonglaga ◽  
K. Ogata ◽  
M. Yamaguchi ◽  
...  
Keyword(s):  
Embryo Development ◽  
Intracytoplasmic Sperm Injection ◽  
Laser Scanning ◽  
Blastocyst Stage ◽  
Early Embryo ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Mitochondrial Activity ◽  
Early Embryo Development

Intracytoplasmic sperm injection (ICSI) has become the method of choice for bovine ovum pick-up and IVF. However, there are many difficulties with the ICSI technique to obtain viable fetuses. One of the major problems associated with this technique is our lack of knowledge concerning the status of the sperm mitochondria when injected into the oocyte and its effect on embryo development. First, we examined the mitochondrial activity of sperm that had been activated by culturing with methyl-β cyclodextrin (MBCD), in ICSI and in IVF. In vitro-matured oocytes and JC1-labelled sperm were used for the ICSI and IVF. The fluorescence intensity of injected/penetrated sperm mitochondria was measured using confocal laser scanning microscopy. Then, the relative membrane potential of the mitochondria was analysed by a ratiometric method. Second, the reactive oxygen species (ROS) production and capacitation status of the sperm exhibiting normal motility and of the sperm that had been activated by culturing with MBCD were analysed. The ROS levels produced by the sperm were estimated using the luminol assay. The chlortetracycline stain was used to evaluate capacitation status of the sperm. Third, the effect of ROS produced by these sperm types upon embryogenesis following ICSI and IVF was studied. Early developing embryos were examined with a stereomicroscope for cleavage and development to the blastocyst stage after 7 days of culture. Chromosome samples stained with Giemsa solution from the blastocysts were used to analyse the chromosomal integrity. Data were analysed by t-test for Experiments 1 and 2, and ANOVA with Fisher's PLSD test for Experiment 3. The mitochondrial activity immediately after ICSI was higher than at 3 h after insemination (immediately after sperm penetration) in IVF (P < 0.05). The sperm exhibiting activation were capacitated and produced more ROS than the sperm exhibiting normal motility (P < 0.05). The rates of cleaved embryo and blastocyst after ICSI with activated sperm were the same as that in ICSI with normal motility sperm and in IVF (cleaved rate: 66.7, 71.8, and 85.0%, respectively; blastocyst rate: 24.4, 23.3, and 32.0%, respectively). However, chromosomal integrity of blastocysts derived from ICSI with activated sperm was lower than that for ICSI with normal motility sperm or for IVF (23.1, 75.0, and 63.6%, respectively; P < 0.01). In conclusion, capacitated, activated sperm induced chromosomal aberrations during early embryo development following ICSI. Conceivably, the selection of sperm exhibiting progressive motility, which is expected to be activated and to fertilize, would not always be better for early embryo development and fetal growth following ICSI due to the ROS derived from the sperm mitochondria. Injection of sperm exhibiting normal motility, or of mitochondria reduced activated sperm, could improve the quality of ICSI-derived embryos.

scholarly journals Porcine embryos produced after intracytoplasmic sperm injection using xenogeneic pig sperm from neonatal testis tissue grafted in mice

2008 ◽  
Vol 20 (7) ◽  
pp. 802 ◽  
Author(s):  
Ali Honaramooz ◽  
Xiang-Shun Cui ◽  
Nam-Hyung Kim ◽  
Ina Dobrinski
Keyword(s):  
Embryo Development ◽  
Intracytoplasmic Sperm Injection ◽  
Animal Species ◽  
Blastocyst Stage ◽  
Immunodeficient Mice ◽  
Neonatal Pig ◽  
Neonatal Testis ◽  
Porcine Spermatozoa ◽  
Testis Tissue

Embryo development after homologous intracytoplasmic sperm injection (ICSI) with sperm from testis tissue xenografts from pigs or any other farm animal species has not been evaluated critically. Here, we report development of porcine embryos in vitro following ICSI with sperm retrieved from xenografted neonatal pig testis. Small pieces of testis tissue from newborn piglets were grafted under the back skin of castrated immunodeficient mice (n = 4) and the xenografts were collected 8 months after grafting. Spermatozoa were recovered by mincing of the grafted tissue. For comparison, testicular, epididymal and ejaculated spermatozoa were also collected from mature boars. Oocytes injected with xenogeneic spermatozoa were either fixed to determine fertilisation processes (n = 89 in five replicates) or allowed to develop in vitro (n = 143 in four replicates). Xenogeneic porcine spermatozoa were fertilisation competent (24% v. 58%, 68%, 62% or 0% for xenogeneic v. control testicular, epididymal and ejaculated spermatozoa or no spermatozoa, respectively) and embryos developed to the blastocyst stage (8% v. 22%, 27%, 25% or 0%, respectively). These results demonstrate that porcine spermatozoa derived from immature testis tissue xenografted into mice are fertilisation competent, albeit at a lower rate than testicular, epididymal or ejaculated spermatozoa from control boars, and support embryo development after ICSI.

scholarly journals Analysis of Morphokinetic Parameters of Feline Embryos Using a Time-Lapse System

Animals ◽  
2021 ◽  
Vol 11 (3) ◽  
pp. 748
Author(s):  
Joanna Kochan ◽  
Agnieszka Nowak ◽  
Barbara Kij ◽  
Sylwia Prochowska ◽  
Wojciech Niżański
Keyword(s):  
Embryo Development ◽  
Embryo Quality ◽  
Time Lapse ◽  
Blastocyst Stage ◽  
Cavity Formation ◽  
Non Invasive ◽  
Morphokinetic Parameters ◽  
Morphological Defects ◽  
Development In Vitro

The aim of this study was to analyze the morphokinetic parameters of feline embryos using a time lapse system. Oocytes matured in vitro were fertilized (IVF) and in vitro cultured in a time lapse-system (Primo Vision®, Gothenburg, Sweden). The first cell division of embryos occurred between 17 h post insemination (hpi) and 38 hpi, with the highest proportion of embryos (46%) cleaving between 21 and 24 hpi. The timing of the first cleavage significantly affected further embryo development, with the highest development occurring in embryos that cleaved at 21–22 hpi. Embryos that cleaved very early (17–18 hpi) developed poorly to the blastocyst stage (2%) and none of the embryos that cleaved later than 27 hpi were able to reach the blastocyst stage. Morphological defects were observed in 48% of the embryos. There were no statistically significant differences between the timing intervals of the first cleavage division and the frequency of morphological defects in embryos. Multiple (MUL) morphological defects were detected in more than half (56%) of the abnormal embryos. The most frequent single morphological defects were cytoplasmic fragmentation (FR) (8%) and blastomere asymmetry (AS) (6%). Direct cleavage (DC) from 1–3 or 3–5 blastomeres, reverse cleavage (RC) and vacuoles were rarely observed (2–3%). The timing of blastocyst cavity formation is a very good indicator of embryo quality. In our study, blastocyst cavity formation occurred between 127–167 hpi, with the highest frequency of hatching observed in blastocysts that cavitated between 142–150 hpi. Blastocysts in which cavitation began after 161 h did not hatch. In conclusion, the timing of the first and second cleavage divisions, the timing of blastocyst cavity formation and morphological anomalies can all be used as early and non-invasive indicators of cat embryo development in vitro.

scholarly journals 136 EVIDENCE OF A DIRECT EFFECT OF P4 ON IVF-DERIVED BOVINE 8-CELL EMBRYOS

2005 ◽  
Vol 17 (2) ◽  
pp. 219 ◽  
Author(s):  
C.E. Ferguson ◽  
T.R. Davidson ◽  
M.R.B. Mello ◽  
A.S. Lima ◽  
D.J. Kesler ◽  
...  
Keyword(s):  
Embryo Development ◽  
Direct Effect ◽  
Blastocyst Stage ◽  
Control Group ◽  
Bovine Embryo ◽  
Bovine Embryos ◽  
Direct Role ◽  
Treatment Groups ◽  
And Control

There has been much debate over a direct role for progesterone (P4) in early bovine embryo development. While previous attempts to supplement bovine embryos in vitro with P4 produced results that vary and are often contradictory, this may be a response of administering P4 at inappropriate times. Therefore, the objective of these experiments was to determine if P4 could exert a direct effect on developing IVF-derived bovine embryos when administered at an appropriate time of embryo development. In Exp. I, IVF-derived bovine 8-cell embryos were randomly allotted to treatments: (1) control, CR1aa medium (n = 168); (2) vehicle, CR1aa + ETOH (0.01%) (n = 170); and (3) P4, CR1aa + ETOH + P4 (20 ng/mL in 50-μL droplet) (n = 173). In Exp. II, IVF-derived bovine 8-cell embryos were randomly allotted to treatments: (1) control, CR1aa medium (n = 160); (2) vehicle, CR1aa + DMSO (0.01%) (n = 180); and (3) P4, CR1aa + DMSO (0.01%) + P4 (20 ng/mL in 50-μL droplet) (n = 170). All embryos were evaluated on Days 6 to 9 post-insemination and rates calculated from 8-cell embryos. In Exp. I, ETOH tended to have a detrimental effect with significantly fewer (P < 0.05) embryos (53%) developing to the blastocyst stage on Day 7 compared with the control (62%) and P4 (71%) groups. At Day 7, significantly more embryos cultured in P4 (71%) developed to the blastocyst stage compared with the control group (62%). P4 treatment significantly increased the number of Grade 1 blastocysts (25%) on Day 7 compared with vehicle (15%) and control (17%) groups. At the end of culture, there were also significantly more Day 9 hatched blastocysts in the P4 group (33%) compared with vehicle (22%) and control (21%) groups. Supplementing P4 in the culture medium increased the rate of development, resulting in significantly more blastocysts (8%) on Day 6 and hatched blastocysts (21%) on Day 8 compared with vehicle (3% and 12%) and control (0% and 8%) groups, respectively. In Exp. II, there were no significant differences between treatment groups for Day 7 blastocysts (control 54%, DMSO 61%, P4 57%) and Day 9 hatched blastocysts (control 46%, DMSO 51%, P4 46%). However, there were significantly more Grade 1 blastocysts in the P4 group (22% and 36%) on Days 6 and 8 compared with vehicle (11% and 23%) and control (13% and 23%) groups, respectively. The lack of improvement in Day 7 blastocysts and Day 9 hatched blastocysts rates leads to further uncertainty in understanding the P4 vehicle interactions. In conclusion, the results of these two experiments indicate that P4 can exert a direct effect on the developing IVF-derived bovine embryo; however, due to P4 vehicle interactions; other inert vehicles need to be explored to further evaluate the direct effects of P4 on the developing bovine embryo.

Mutations in ZP4 are associated with abnormal zona pellucida and female infertility

2021 ◽  
pp. jclinpath-2020-207170
Author(s):  
Xiaoli Wei ◽  
Youzhu Li ◽  
Qicai Liu ◽  
Wensheng Liu ◽  
Xiaohong Yan ◽  
...  
Keyword(s):  
Embryo Development ◽  
Zona Pellucida ◽  
Intracytoplasmic Sperm Injection ◽  
Treatment Strategies ◽  
Suction Pressure ◽  
Human Female ◽  
Female Reproduction ◽  
Efficient Treatment ◽  
Whole Exome

BackgroundThe zona pellucida (ZP) of human oocytes plays essential protective roles in sperm–egg interactions during fertilisation and embryo development. ZP4-null female rabbits exhibit a thin and irregular ZP, which severely impairs embryo development and fertility. However, the effects of ZP4 defect on human female reproduction remain unknown.Methods and resultsWe performed whole-exome sequencing in 26 female patients with abnormal (thin and irregular) ZP and identified heterozygous variants in ZP4 (OMIM: 613514) from 3 patients (approximately 11%). No ZP4 variant was found in the 30 control women with proven fertility. We constructed ZP4-mutated plasmids and found that the variants reduced the secretion of ZP4 in vitro. Lower suction pressure facilitated egg retrieval, and intracytoplasmic sperm injection (ICSI) was a desirable treatment for ZP4-mutated patients with abnormal ZP.ConclusionsWe identified ZP4 as a novel gene for human abnormal ZP and found that lower suction pressure and ICSI are efficient treatment strategies.

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