136 IMPACT OF SELECTION SYSTEM BY KINETICS ON THE EARLY EMBRYONIC DEVELOPMENT IN BOVINE OVUM PICKUP-IVF EMBRYOS

2017 ◽  
Vol 29 (1) ◽  
pp. 176
Author(s):  
M. Takayama ◽  
M. Moriyoshi ◽  
O. Dochi ◽  
K. Imai
Keyword(s):  
Embryo Transfer ◽  
Calf Serum ◽  
Cumulus Cells ◽  
Conception Rate ◽  
Culture Dish ◽  
Fresh Embryo Transfer ◽  
Chi Square ◽  
Predicting Factors

Recently, in vitro-produced (IVP) embryos have been increasingly produced using ovum pickup (OPU) and IVF in cows worldwide. However, the conception rate of IVP embryos is lower than that of in vivo-derived embryos. This study was conducted to determine the proportion of embryos that led to a high conception rate when the embryos were selected according to the 4 predicting factors. A total of 30 Holstein and 20 Japanese Black cows were used, and 81 OPU-IVF sessions were performed from October 2014 to May 2016. The collected cumulus-oocyte complexes (COC) were cultured for 22 h. Capacitated sperm (at a final concentration of 5 × 106 spermatozoa/mL) were incubated with COC for 6 h. After insemination, presumptive zygotes were separated from cumulus cells and sperm by pipetting. Then, the presumptive zygotes were cultured for 9 days in CR1aa supplemented with 5% calf serum by using a micro-well culture dish (Dai Nippon Printing, Tokyo, Japan). The kinetics of embryo development was observed at 27, 31, and 55 h post-insemination (hpi). The 4 factors used to select embryos were as follows: (1) time at which first cleavage occurred (less than 27 hpi, or less than 31 hpi, in case any of the zygotes did not cleave at 27 hpi in each culture dish); (2) 2 blastomeres after first cleavage at 31 hpi; (3) absence of fragments after first cleavage at 31 hpi; and (4) 8 or more blastomeres at 55 hpi. The number of blastocysts was analysed at 7, 8, and 9 days post IVF. Additionally, the number of produced embryos that could be used for embryo transfer (ET) was determined. The data were analysed using the chi-square test. The total numbers of blastocysts and produced embryos were 615 and 503, respectively. The numbers of blastocysts and produced embryos selected using the combination of factors 1 to 4 were 200 (32.5%) and 169 (27.5%), respectively. The numbers of blastocysts and produced embryos selected using factor 1 were 397 (64.6%) and 340 (67.6%), using factor 2 were 445 (71.3%) and 378 (75.1%), using factor 3 were 364 (81.8%) and 307 (81.2%), and using factor 4 were 374 (60.8%) and 308 (61.2%), respectively. The numbers of blastocysts and produced embryos that were rejected using a combination of factors 1 to 4 were 123 (27.5%) and 90 (17.9%), respectively. The conception rate of fresh embryo transfer was 46.6% (n = 73). We found that the conception rate of the embryos selected using factors 1 to 4 was significantly (P < 0.05) higher than that of embryos without one factor or more [60.0% (n = 35) v. 29.4% (n = 34)]. These results show the applicability and efficiency of the 4 factors for producing embryos with a high competence for conception.

164 EFFECTS OF KINETICS AND MORPHOLOGY ON EARLY EMBRYONIC DEVELOPMENT IN BOVINE OPU-IVF EMBRYOS

2016 ◽  
Vol 28 (2) ◽  
pp. 212
Author(s):  
M. Takayama ◽  
O. Dochi ◽  
K. Imai
Keyword(s):  
Embryo Transfer ◽  
Calf Serum ◽  
Conception Rate ◽  
Culture Dish ◽  
Fresh Embryo Transfer ◽  
Cell Stage ◽  
Vitro Fertilization ◽  
Observation Method

In recent years, the use of ovum pick up (OPU) and IVF for embryo production has increased worldwide; however, the conception rate of embryo transfer is lower for OPU-IVF embryos than for in vivo-derived embryos. This study aimed to determine the efficacy of embryo selection by a 3-step observation method by using a micro-well culture dish (Dai Nippon Printing, Tokyo, Japan). In this study, 9 Holstein and 15 Japanese Black cows were used, and the OPU-IVF was conducted from October 2014 to May 2015. The collected cumulus-oocyte complexes (COC) were cultured for 22 h in 25 mM HEPES-buffered TCM-199 supplemented with 5% calf serum (CS) and 0.02 AU mL–1 of FSH. Sperm (at a final concentration of 5 × 106 spermatozoa mL–1) were incubated with COC for 6 h. After insemination, presumptive zygotes were separated from cumulus cells and sperm by pipetting. Then, the presumptive zygotes were cultured for 9 days in CR1aa supplemented with 5% CS by using a micro-well culture dish. Kinetics and morphology were observed at 27, 31, and 55 h post-insemination (hpi). The presumptive zygotes were divided to 3 groups (more than 2 cells, 2 cells, and no cleavage) at 27 and 31 hpi. Then, embryos at the 2-cell stage at 31 hpi were divided into 2 groups: 2-cell with normal cleavage and 2-cell embryos with abnormal cleavage (unequal cleavage, 2-cell with fragments, and 2-cell with protrusion). Subsequently, embryos were classified as 8-cell and more than 8 cell, or less than 8 cell at 55 hpi. The blastocyst rate (BL%) was analysed at 7, 8, and 9 days post IVF. Embryos selected by the 3-step observation method were used for fresh embryo transfer. The data were analysed by chi-squared test. In total, 856 oocytes were collected by OPU and 633 oocytes were cultured, of which 39.7% (263/663) developed to the blastocyst stage. The BL% of 2-cell embryos (72.5%, 116/160) was significantly higher (P < 0.01) than that of no cleavage (47.0%, 117/249) at 27 hpi. The BL% of 2-cell (65.4%, 206/315) and more than 2-cell (53.0%, 35/66) was significantly higher (P < 0.01 and P < 0.05) than that of embryos divided as no cleavage (25.9%, 22/85) at 31 hpi. The BL% was not significantly different between 2-cell with normal cleavage (68.5%, 172/251) and abnormal cleavage (53.1%, 34/64). The BL% of 8-cell and more than 8-cell stage (72.8%, 182/250) was significantly higher (P < 0.01) than that of embryos with less than 8 cells (38.8%, 81/209) at 55 hpi. Overall, 2-cell embryos at 27 hpi, 2-cell embryos with normal cleavage at 31 hpi, and 8-cell and more than 8 cell at 55 hpi showed the highest BL% (82.1%, 78/91). The conception rate was higher for following the selected fresh embryo transfer that was 70.6% (12/17) than average of in vitro fertilization embryos transfer that was 40.0%. These results demonstrate that the 3-step observation method used in this study can be effectively applied for the selection of IVF embryos that have a strong ability to develop into blastocysts and high competence for conception.

44 IN VITRO AND IN VIVO SURVIVABILITY ON VITRIFIED EMBRYOS OBTAINED BY BOVINE SOMATIC CELL NUCLEAR TRANSFER

2006 ◽  
Vol 18 (2) ◽  
pp. 131
Author(s):  
K. Kaneyama ◽  
S. Kobayashi ◽  
S. Matoba ◽  
Y. Hashiyada ◽  
K. Imai ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Survival Rates ◽  
Calf Serum ◽  
Cumulus Cells ◽  
Nuclear Transplantation ◽  
Embryo Cryopreservation

Although many studies have been conducted on somatic cell nuclear transfer, there are only a few reports on cryopreservation of reconstructed embryos after nuclear transplantation. The objective of this study was to examine in vitro or in vivo development of vitrified blastocysts obtained by nuclear transfer. Nuclear transfer was carried out according to the procedure of Goto et al. (1999 Anim. Sci. J. 70, 243–245), and conducted using abattoir-derived oocytes and cumulus cells derived by ovum pickup from Holstein and Japanese Black cows. Embryos were vitrified as described by Saito et al. (1998 Cryobiol. Cryotech. 43, 34–39). The vitrification solution (GESX solution) was based on Dulbecco's PBS containing 20% glycerol (GL), 20% ethylene glycol (EG), 0.3 M sucrose (Suc), 0.3 M xylose (Xyl), and 3% polyethylene glycol (PEG). The blastocysts were equilibrated in three steps, with 10% GL, 0.1 M Suc, 0.1 M Xyl, and 1% PEG for 5 min (1); with 10% GL, 10% EG, 0.2 M Suc, 0.2 M Xyl, and 2% PEG for 5 min (2) and GESX solution (3). After transfer to GESX, equilibrated embryos were loaded to 0.25-mL straws and plunged into liquid nitrogen for 1 min. The vitrified blastocysts were warmed in water (20°C) and diluted in 0.5 M and 0.25 M sucrose for 5 min each. Equilibration and dilution procedures were conducted at room temperature (25–26°C). After dilution, the vitrified blastocysts were cultured in TCM-199 supplemented with 20% fetal calf serum and 0.1 mM β-mercaptoethanol at 38.5°C under gas phase of 5% CO2 in air. In Experiment 1, survival rates after vitrification were compared between the nuclear transfer and the IVF blastocysts. Survival rates of vitrified nuclear transfer blastocysts (n = 60, Day 8) at 24 and 48 h were 70.0% and 56.7%, respectively, and those of vitrified IVF blastocysts (n = 41) were 82.9% and 82.9%, respectively. There were no significant differences in survival rates at 24 and 48 h between the two groups. In Experiment 2, one (VIT-single) or two (VIT-double) vitrified and one (nonVIT-single) or two (nonVIT-double) nonvitrified reconstructed blastocysts per animal were transferred into Holstein dry cows. The result of Experiment 2 is shown in Table 1. This experiment demonstrated that the vitrification method in this study can be used for cloned embryo cryopreservation but the production rate should be improved. Table 1. Comparison of survival rates of vitrified or nonvitrified cloned embryos after transfer

188 IMPROVEMENT OF IN VITRO CANINE OOCYTE MATURATION BY OVIDUCTAL SECRETOME

2017 ◽  
Vol 29 (1) ◽  
pp. 202 ◽  
Author(s):  
A. Lange-Consiglio ◽  
C. Perrini ◽  
P. Esposti ◽  
F. Cremonesi
Keyword(s):  
Oocyte Maturation ◽  
In Vitro Maturation ◽  
Cumulus Cell ◽  
Follicular Development ◽  
Cumulus Cells ◽  
Chi Square ◽  
Cell Layers ◽  
Hoechst Staining

The in vitro maturation of canine oocyte is problematic because it is difficult to reproduce the oviducal microenvironment where the in vivo maturation occurs. Because cells are able to communicate with each other by paracrine action, oviducal cells could be in vitro cultivated to obtain the conditioned medium (CM) consisting of soluble factors and microvesicles (MV), which represent a carrier for nonsoluble molecules including microRNA. The aim of the present work was to investigate the effect of the addition of CM or MV, secreted by oviducal cells, to the canine in vitro maturation medium. To generate CM, cells from oviducts of 3 animals in late oestrus were cultured for 5 days at 38.5°C in a humidified atmosphere of 5% CO2. Supernatants were collected, pooled, centrifuged at 2500 × g, and stored at −80°C. Microvesicles were obtained by ultracentrifugation of CM at 100,000 × g for 1 h at 4°C and measured for concentration and size by a Nanosight instrument. Ovaries were obtained from 50 healthy domestic bitches (1–4 years old) of different breeds that underwent ovariectomy regardless of the oestrous cycle. Cumulus-oocyte complexes were released by slicing the ovarian cortex with a scalpel blade, and only Grade 1 cumulus-oocyte complexes (darkly granulated cytoplasm and surrounded by 3 or more compact cumulus cell layers) 110 to 120 µm in diameter were selected for culture. Maturation was performed at 38.5°C in a humidified atmosphere of 5% CO2 and 5% of O2 in bi-phasic systems: 24 h in SOF with 5.0 μg mL−1 of LH followed by 48 h in SOF supplemented with 10% of oestrous bitch serum and 10% CM or 50, 75, 100, or 150 × 106 MV mL−1 labelled with PKH-26. Control was the same medium without CM or MV. Oocytes were observed under a fluorescent microscope to detect metaphase II (MII), by Hoechst staining, and the incorporation of MV. Statistical analysis was performed by chi-square test. Results show that canine oviducal cells secreted MV of 234 ± 23 nm in size, underling that these MV fall within the shedding vesicles category. The incorporation of labelled MV occurred at first in cumulus cells, at 48 h of maturation, and then, at 72 h, in oocyte cytoplasm. These MV had a positive effect on maturation rate (MII) at the concentration of 75 and 100 × 106 MV mL−1 compared with CM and control (20.34 and 21.82 v. 9.09 and 3.95%, respectively). The concentration of 150 × 106 MV mL−1 provided only 9.26% of MII. To understand the role of MV, we assessed the expression of 3 microRNA (miRNA-30b, miR-375, and miR-503) that are involved in some key pathways (WNT, MAPK, ERbB, and TGFβ) regulating follicular development and meiotic resumption. The lower rate of MII with the higher concentration of MV is possibly due to the high level of miR-375, which recent literature shows to suppress the TGFβ pathway, leading to impaired oocyte maturation. In conclusion, the oviducal MV, or specific microRNA, are involved in cellular trafficking during oocyte maturation, and their possible use in vitro could facilitate the exploitation of canine reproductive biotechnologies.

188 EFFECT OF TRANSFER OF FROZEN-THAWED IVF EMBRYOS ON PREGNANCY IN REPEAT-BREEDER INSEMINATED OR NON-INSEMINATED HOLSTEIN CATTLE

2006 ◽  
Vol 18 (2) ◽  
pp. 202 ◽  
Author(s):  
O. Dochi ◽  
M. Tanisawa ◽  
S. Goda ◽  
H. Koyama
Keyword(s):  
Pregnancy Rate ◽  
Embryo Transfer ◽  
Calf Serum ◽  
Pregnancy Rates ◽  
Holstein Cattle ◽  
Chi Square ◽  
Vitro Fertilization ◽  
Chi Square Test ◽  
Repeat Breeder

Repeat-breeding is one of the important factors that affect dairy management. The objective of this study was to investigate the effect of transfer of frozen–thawed IVF embryos on pregnancy in repeat-breeder Holstein cattle. Cumulus–oocyte complexes (COCs) were collected by aspiration of 2–1-mm follicles from ovaries obtained at a local abattoir. COCs were matured for 20 h in TCM-199 supplemented with 5% calf serum (CS) and 0.02 mg/mL of FSH at 38.5°C under a 5% CO2 atmosphere in air. Matured oocytes were inseminated with spermatozoa of 5 × 106/mL in BO solution (Brackett and Oliphant 1975 Biol. Reprod. 12, 260–274) containing 10 mM hypotaurine and 4 units/mL heparin. After 18 h of gamete co-culture, presumptive zygotes were cultured in CR1aa (Rosenkrans et al. 1991 Theriogenology 35, 266) supplemented with 5% CS for 8 days at 38.5°C under 5% CO2, 5% O2, 90% N2 atmosphere in air. After in vitro fertilization, Day 7 and Day 8 blastocysts were frozen in 1.5 M ethylene glycol (EG) in Dulbecco's PBS (DPBS) supplemented with 0.1 M sucrose and 20% CS. Embryos were transferred into a freezing medium, loaded into 0.25-mL straws, and allowed to stand for 15–20 min for equilibration. The straws were then plunged into a −7°C methanol bath of a programmable freezer for 1 min, seeded at −7°C, maintained at −7°C for 15 min, cooled to −30°C at the rate of −0.3°C/min, and then plunged into liquid nitrogen. Recipient animals (43 heifers, 131 cows) included those that did not conceive after being artificially inseminated (AI) 3 to 15 times. The frozen–thawed IVF embryos were directly transferred to the recipient animals 7 days after estrus or AI. Pregnancy rates were analyzed by chi-square test. The results are presented in Table 1. There were no significant differences in the pregnancy rates between treatments. However, a slightly higher pregnancy rate was achieved by embryo transfer after AI. These results suggest that embryo transfer may increase the pregnancy rate in repeat-breeder Holstein cattle. Table 1. Pregnancy rates after transfer of IVF frozen–thawed embryos in repeat-breeder Holstein cattle

153 EFFECT OF DIFFERENT HOLDING AND TRANSPORT MEDIA ON CONCEPTION RATES FOLLOWING TRANSFER OF IN VIVO AND IN VITRO FERTILIZATION-DERIVED BOVINE EMBRYOS

2011 ◽  
Vol 23 (1) ◽  
pp. 180
Author(s):  
C. A. Zanenga ◽  
C. M. Martins ◽  
N. C. Rodovalho ◽  
F. Aidar ◽  
J. F. Hasler ◽  
...  
Keyword(s):  
Animal Health ◽  
Conception Rate ◽  
Bovine Embryos ◽  
Chi Square ◽  
Mato Grosso ◽  
Conception Rates ◽  
Chi Square Test ◽  
Donor Cows

Two experiments were conducted to compare conception rates following embryo transfer (ET) of bovine embryos held and transported in Syngro® holding medium (Bioniche, Belleville, Ontario, Canada) with other 2 holding media: Emcare® (ICPbio, Auckland, New Zealand) for in vivo-derived embryos and HEPES-buffered synthetic oviduct fluid (H-SOF) for IVF-derived embryos. The first trial was performed in the period from October through December 2006 at the Curitiba farm in Poços de Caldas, Minas Gerais, Brazil. A total of 140 in vivo-derived embryos were produced from 20 Nelore donor cows and transferred fresh at the same farm. After each donor recovery, embryos were equally separated per stage (morula or blastocyst) and classification (grades 1, 2, and 3) into 2 Petri dishes, each containing either Syngro or Emcare. The embryos were held for an average of 3 h after recovery, loaded into 0.25-mL straws, and transferred fresh into recipients heifers, which were all previously synchronized with the same hormonal protocol treatment and presented a corpus luteum on the day of transference. Conception rate was checked at approximately 60 days of conception by rectal palpation. The chi-square test was used for statistical analysis. The conception rate of embryos maintained in Syngro was significantly higher than those in Emcare: 64.2% (43/67) v. 47.9% (35/73; P < 0.05). A second experiment was performed between September and December 2008 at Embriza Biotechnology Laboratory, Campo Grande, Mato Grosso do Sul, Brazil. A total of 1689 IVF-derived embryos (stage = 7, quality = 1), produced from Nelore donor cows, were randomly assigned to be held and transported in either Syngro (769) or H-SOF transport medium (920). Transportation time ranged from 1 to 9 h, and the recipient farms ranged from 100 to 1200 km in distance from the Embriza Laboratory. Crossbred recipient heifers (Bos taurus × Bos indicus) were synchronized with prostaglandin or vaginal progesterone device protocols. Pregnancy diagnosis was performed by ultrasonography approximately 60 days after ET. Statistical comparisons were performed using the chi-square test. Conception rates resulting from embryos transported in Syngro (45.1%, 347/769) and in H-SOF (42.0%, 386/920) were not different (P = 0.19). Financial support from Embriza Biotecnology, Tecnopec LTDA, and Bioniche Animal Health

137 EFFECTS OF X-SORTED SPERM IN QUALITY OF BOVINE BLASTOCYST DERIVED FROM IN VIVO-MATURED OOCYTES

2014 ◽  
Vol 26 (1) ◽  
pp. 182
Author(s):  
K. Imai ◽  
M. Ohtaku ◽  
Y. Aikawa ◽  
H. Matsuda ◽  
S. Kobayashi ◽  
...  
Keyword(s):  
Prognostic Factors ◽  
In Vitro Culture ◽  
Calf Serum ◽  
Cumulus Cell ◽  
Early Embryo ◽  
Lag Phase ◽  
Culture Dish

Recently, we reported on a promising system for selecting healthy IVF embryos in cattle using kinetics of early embryo development and oxygen consumption of blastocyst [Sugimura et al. 2012 PLoS ONE 7, e36627]. The present study was conducted to examine the differences in embryo quality of bovine blastocysts obtained after IVF of in vivo-matured oocytes with X-sorted and unsorted sperm. Holstein dry cows (n = 8) were reared under the same feeding and environmental conditions. Two ovum pickup (OPU) sessions were conducted in each cow to fertilize with or without X-sorted sperm. In vivo-matured oocytes were collected by OPU just before ovulation after superstimulation treatment. The oocytes were inseminated with 5 × 106 sperm mL–1 of each sperm, and presumptive zygotes were cultured in CR1aa supplemented with 5% newborn calf serum and 0.25 mg mL–1 of linolenic acid albumin at 38.5 C in 5% CO2, 5% O2, and 90% N2 for 168 h. Embryo kinetics were observed individually using a microwell culture dish (Dai-Nippon Print) and time-lapse cinematography (CCM-1.4MZS; Astec, Fukuoka, Japan; Sugimura et al. 2010 Biol. Reprod. 83, 970–978). Photographs of each embryo were taken every 15 min during the in vitro culture period and images were analysed by CCM-1.4 software (Astec). By assessing the quality of blastocysts, a combination of identified prognostic factors were used: (1) timing of the first cleavage (less than 27 h post-insemination); (2) two blastomeres at the end of the first cleavage; (3) absence of fragments at the end of the first cleavage; and (4) six or more blastomeres at the onset of the lag-phase. Data were analysed by ANOVA. In total, 34.1 ± 18.4 oocytes per session per donor were collected by OPU, and 23.7 ± 13.4 oocytes had an expanded cumulus cell. Oocyte recovery rates were recorded at 77.1 ± 15.1%. After IVF and in vitro culture, 10.6 ± 7.7 blastocysts per session per donor were produced in this study. There was no significantly difference in cleavage rates and blastocyst formation rates between X-sorted sperm and unsorted sperm (87.1 ± 10.8 and 82.6 ± 12.1% and 38.4 ± 23.6 and 57.1 ± 23.4%, respectively). However, blastocysts derived from X-sorted sperm showed significantly (P < 0.05) lower quality in the prognostic factor (1) and combined (1) to (4) than that in unsorted sperm (35.3 v. 54.0 and 14.7 v. 42.9%, respectively). Pregnancy rates were higher for the blastocysts that had a high score in the prognostic factors (1) to (4) compared to those that had a low score (75.0%, n = 8 v. 36.4%, n = 22). These results suggest that quality of blastocysts, based on the prognostic factors studied, derived from X-sorted sperm is lower than that from unsorted sperm. Supported by the Research and Development projects for application in promoting new policy of agriculture, forestry and fisheries (22016).

363 FACTORS INFLUENCING PREGNANCY RATES FOLLOWING TRANSFER OF BOVINE IN VIVO EMBRYOS BIOPSIED FOR SEX DETERMINATION

2007 ◽  
Vol 19 (1) ◽  
pp. 297
Author(s):  
S. Li ◽  
W. Yu ◽  
J. Fu ◽  
Y. Bai ◽  
F. Jin ◽  
...  
Keyword(s):  
Pregnancy Rate ◽  
Embryo Transfer ◽  
Pregnancy Rates ◽  
Chi Square ◽  
Embryo Survival ◽  
Chi Square Test ◽  
First Time ◽  
Fresh Embryos

Data collected from commercial embryo transfer programs in 63 farms in China during June 2002 to December 2005 was analyzed to examine the effects of various factors (biopsy, freezing, sample size, embryo development and quality, in vitro culture, and recipient quality) on pregnancy rates of in vivo-biopsied embryos. Embryos were flushed from superovulated dairy cattle and subjected to a biopsy for sexing determination using protocols and sexing kits supplied by AB Technology Ltd. Fresh embryos were implanted on the same day or frozen with AG freeze medium (AB Technology Ltd., Pullman, WA, USA) for later transfer. Recipients were synchronized with CIDA + PG protocols. Embryos were cultured in 6-well dishes containing 1.3 mL of holding medium (AB Technology Ltd.) in each well at room temperature (20–25�C) for examination of embryo survival in vitro. The chi-square test was used in statistic analysis. The implantation of fresh embryos after biopsy did not affect pregnancy rates (49.6%, 257/518) compared to that of non-biopsied fresh and frozen–thawed embryo groups (52.9%, 47/140 and 46.6%, 177/380, respectively). However, for biopsied embryos subjected to frozen and thawed procedures before implantation, particularly for those subjected to the removal of a larger biopsy, a reduced pregnancy rate was observed (41.8%, 297/710; P &lt; 0.01). Pregnancy rates among biopsied embryos at 3 different development stages (morula-early blastocyst, blastocyst, and expanded blastocyst) were not different. Similar results were found between embryo groups of grade 1 and 2. A significant decrease in pregnancy rate (0/10) was observed with embryos held in vitro for a longer period of time (&gt;5 h), suggesting detrimental effects of in vitro conditions on embryo survival. The highest pregnancy rate (68.0%) was observed in recipients synchronized for the first time before being implanted with biopsied embryos. Significant decreases in such rates were found in recipients synchronized for the second or third times or those with an abortion history at the first or second synchronization-implantation treatment (P &lt; 0.01). Better pregnancy rates (45.6%, 41/90; 46.1%, 76/165; and 45.5%, 5/11) were obtained for recipients implanted with biopsied embryos at Days 7.5, 8.0, and 8.5 post-heat detection, respectively, compared to 16% at Day 7 (3/18, P &lt; 0.05). It is concluded that mechanical treatment (cutting) does not reduce the survival of biopsied embryos; however, cryopreservation reduces their ability to survive in vivo. The analyses also suggest that holding embryos in vitro should not be longer than 5 h unless more favorable in vitro conditions can be provided. To achieve better results of implantation of biopsied embryos, embryo transfer should be performed during 7.5–8.5 days post-estrus, and the healthy recipients synchronized for the first time should be used.

47 CRYOPRESERVATION OF BOVINE GERM CELL USING ANTIFREEZE POLYAMINO-ACID (CARBOXYLATED POLY-L-LYSINE)

2017 ◽  
Vol 29 (1) ◽  
pp. 131
Author(s):  
T. Fujikawa ◽  
C. Kubota ◽  
T. Ando ◽  
S. Imamura ◽  
M. Tokumaru ◽  
...  
Keyword(s):  
Survival Rate ◽  
Germ Cell ◽  
Embryo Transfer ◽  
Conception Rate ◽  
Control Group ◽  
Vitro Fertilization ◽  
Polyamino Acid ◽  
Significant Difference

Carboxylated poly-l-lysine (CPLL) is an ampholytic polymer compound, and it is obtained by converting 65% amino groups to carboxyl groups after synthesising ε-poly-l-lysine aqueous solution and succinic anhydride. CPLL has cryoprotective property similar to antifreeze protein, and addition of CPLL into cryopreservation medium improves the post-thaw survival rate of cells and embryos. In this research, we examined the effectiveness of CPLL as a bovine germ cell cryoprotective material. In experiment 1 (in sperm), the conventional cryopreservation medium used for control group was consisted of 6.5% (vol/vol) glycerin, and the cryopreservation medium used for CPLL group was consisted of 3.25% (vol/vol) glycerin and 0.5% CPLL (wt/vol). The post-thaw survival and motility were assessed by using Sperm Motility Analysis System (DITECT Corp., Tokyo, Japan). There was no significant difference for post-thaw survival rate and motility (control v. CPLL; 98.8% v. 96.6% and 69.7% v. 62.2%, respectively). Artificial insemination was carried out in 65 cows (control v. CPLL; 34 v. 31), and the conception rate of the CPLL group was higher than that of the control group (80.6% v. 67.6%; P = 0.23). In experiment 2 (embryos), the conventional cryopreservation medium used for control group was consisted of 5% (vol/vol) ethylene glycol and 6% (vol/vol) propylene glycol in PBS. In the CPLL group, 7% (wt/vol) CPLL was added to the conventional medium. In vitro fertilization embryos were cryopreserved at Day 7 and Day 8. There was no significant difference in survival rate at 0, 24, and 48 h and hatched rate until 72 h after thawing (control v. CPLL: 93.6% v. 93.2%, 69.0% v. 64.7%, 56.1% v. 56.3%, 12.9% v. 10.2%, respectively). Embryos obtained by superovulation treatment and in vivo fertilization at Day 7 were cryopreserved using above 2 media, and transferred non-surgically into synchronized recipient cows (1 embryo per animal). Embryo transfer (ET) was carried out in 81 cows (control v. CPLL: 31 v. 50), and recipients were diagnosed for pregnancy ultrasonically 50 days after embryo transfer. Conception rate of CPLL group was higher than control group (50.0% v. 29.0%; P = 0.063). In both experiments, the significant differences between control group and CPLL group were determined by chi-squared test. The effectiveness of CPLL in cells and embryos has been reported; however, there is no report using CPLL in bovine germ cells. In this research, CPLL improved the conception rate of AI and ET, probably due to its low toxicity and protection of the cell membrane. These results suggest that CPLL is available as a new cryoprotective material for bovine sperm and embryo in slow freezing methods.

138 Effects of different treatments of donors on the efficiency of embryo production and conception in bovine ovum pickup-in vitro production

2019 ◽  
Vol 31 (1) ◽  
pp. 194
Author(s):  
A. Katae ◽  
Y. Kaneda ◽  
M. Sugawara ◽  
T. Nishisouzu ◽  
O. Dochi ◽  
...  
Keyword(s):  
High Efficiency ◽  
Single Injection ◽  
Calf Serum ◽  
Conception Rate ◽  
Bovine Embryo ◽  
In Vitro Production ◽  
Newborn Calf Serum ◽  
Culture Dish ◽  
Embryo Production

An in vitro-produced bovine embryo has a low conception rate compared with that of an in vivo embryo. The present study was conducted to examine the effects of different treatments delivered to donors before ovum pickup (OPU) sessions to improve the conception rate of in vitro-produced bovine embryos. In total, 351 OPU sessions were performed on 138 Holstein and 213 Japanese Black cows from January to December 2017. Donors were divided into 4 groups based on their pretreatment before OPU: (1) single injection of 2.5 AU of FSH 40h before OPU; (2) CIDR insertion on Day 0, injection of 2mg of oestradiol benzoate on Day 1, 4 injections of FSH (each 2.5 AU) every 12h beginning from Day 5 to 7, followed by removal of CIDR and OPU on Day 9; (3) injection of 50μg of gonadotropin-releasing hormone 72h before OPU; or (4) no pretreatment. The collected cumulus-oocyte complexes were matured for 22h in 25mM of HEPES buffered TCM-199 supplemented with 5% newborn calf serum and 0.02 AU mL−1 FSH. After 6h of gamete co-culture (5.0×106 sperm mL−1), the presumptive zygotes were washed and the remaining cumulus cells were denuded by pipetting. The presumptive zygotes were then cultured in KSOMaa supplemented with 5% newborn calf serum for 9 days in a micro-well culture dish (Dai Nippon Printing, Tokyo, Japan). Blastocyst formation rates were analysed 9 days after insemination, and the formed blastocysts were transferred to oestrous synchronized recipients on the seventh or eighth day after oestrus. The data were analysed by Chi-squared test with Yates correction. The average numbers of collected oocytes were 57.7±17.4 (n=136), 25.3±12.8 (n=20), 28.8±12.5 (n=18) and 24.3±12.9 (n=177) in groups 1 to 4, respectively. Groups 1 and 2 showed significantly (P&lt;0.01) high percentages of Grade-1 oocytes (52.1 and 49.6%, respectively) compared with groups 3 and 4 (37.3 and 39.9%, respectively). The proportion of blastocysts in groups 1 (38.6%) was significantly different compared with that in groups 2 (32.1%) and 4 (35.3%), but the difference was insignificant in the case of group 3 (36.4%). The conception rates in groups 1 (43.5%, n=868) and 2 (59.1%, n=44) were significantly (P&lt;0.05) higher than those in groups 3 (35.1%, n=57) and 4 (34.9%, n=768). These results suggest that although the efficiency of embryo production did not differ largely between donors pretreated with 4 FSH injections and those without any pretreatment, the conception rate in donors pretreated with 4 FSH injections was significantly higher than that in donors without pretreatment. Moreover, donors pretreated with a single injection of FSH showed significantly high efficiency of embryo production and conception rate than donors without pretreatment.

scholarly journals Sex-sorted sperm in farm animals: scale of use, the effectiveness of insemination, risk factors

2017 ◽  
Vol 73 (5) ◽  
pp. 272-279
Author(s):  
Bartłomiej M. Jaśkowski ◽  
Magdalena Woźna ◽  
Marek Gehrke
Keyword(s):  
Risk Factors ◽  
Embryo Transfer ◽  
Conception Rate ◽  
Farm Animals ◽  
Lower Percentage ◽  
Technical Parameters ◽  
Cattle Herds ◽  
The One

The aim of the study was to present the scale of use, risk factors and possibilities, which sorter semen gives in biotechnics used in reproduction of cattle. Modern sorters allow for the evaluation of 6 million X and Y spermatozoa per hour. Sex-sorted semen, which is commercially used, contains 2.1 x 106 of spermatozoa. It is used mostly in AI of milk heifers, mainly in large cattle herds. Sorted semen containing Y spermatozoa is sold less often in the world than the one with X spermatozoa. The percentage of the desired sex of the young is higher than 90. The pregnancy rate after application of sorted semen is about 20–25% lower than after insemination of non-sorted semen and depends on a number of factors. The main factors are: breed of female, service number, the herd of origin, the depth of semen deposition, the bull producing semen, ambient temperature and technical parameters during sperm sorting. A number of methods have been developed to improve conception rate, including timed artificial insemination (TAI) and synchronization of heat and ovulation. Results of donor inseminations with the use of sorter semen are presented, with the lower percentage of embryos suitable for the transfer and embryos of the highest quality highlighted. Previous studies do not indicate a reduction of the conception rate after the transfer of embryos obtained in vitro and in vivo after fertilization using sorted semen. It remains difficult to justify a significant increase in the frequency of stillbirths of bulls after using sorted sperm. Similarly, 16% of stillbirths of bulls were observed after embryo transfer, when donors were inseminated with sorter semen. The percentage of stillbirths of bulls after embryo transfer with the use of conventional semen is 9%. The sorted semen is not often used for inseminations in pigs, sheep and goats.

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