Equilibrium vitrification of mouse embryos at various developmental stages using low concentrations of cryoprotectants

We previously developed a new vitrification method (equilibrium vitrification) by which two-cell mouse embryos can be vitrified in liquid nitrogen in a highly dehydrated/concentrated state using low concentrations of cryoprotectants. In the present study, we examined whether this method is effective for mouse embryos at multiple developmental stages. Four-cell embryos, eight-cell embryos, morulae, and blastocysts were vitrified with EDFS10/10a, 10% (v/v) ethylene glycol and 10% (v/v) DMSO in FSa solution. The FSa solution was PB1 medium containing 30% (w/v) Ficoll PM-70 plus 0.5 M sucrose. The state of dehydration/concentration was assessed by examining the survival of vitrified embryos after storage at –80°C. When four-cell embryos and eight-cell embryos were vitrified with EDFS10/10a in liquid nitrogen and then stored at –80°C, the survival rate was high, even after 28 days, with relatively high developmental ability. On the other hand, the survival of morulae and blastocysts vitrified in liquid nitrogen and stored at –80°C for four days was low. Therefore, morulae and blastocysts cannot be vitrified in a highly dehydrated/concentrated state using the same method as with two-cell embryos. However, when blastocysts were shrunken artificially before vitrification, survival was high after storage at –80°C for four days with high developmental ability. In conclusion, the equilibrium vitrification method using low concentrations of cryoprotectants, which is effective for two-cell mouse embryos, is also useful for embryos at multiple stages. This method enables the convenient transportation of vitrified embryos using dry ice.     

Optimization Protocol for Storage of Goldfish (Carassius auratus) Embryos in Chilled State

A series of five experiments were conducted to explore suitable conditions for storing of goldfish embryos in a chilled state. The factors studied were embryo stage, storage temperature, physiological saline solutions and goldfish artificial coelomic fluid (GFACF) medium, antibiotics (penicillin and streptomycin), antioxidants (vitamin E, vitamin C), buffer (Hepes, Tris) and BSA (bovine serum albumin). First, goldfish embryos at eight developmental stages were incubated in aerated and dechlorinated tap water at 0°C for 24 h. Result shows that early developmental stages were most sensitive to chilling. Heartbeat‐stage goldfish embryos were chilled at 0, 4 or 8°C for up to 72 h in water, and chilled storage was possible only for up to 18, 24 and 48 h at 0, 4 and 8°C, respectively, without a decrease in viability. Chilling of goldfish embryos at 8°C in GFACF medium and Dettlaff's solution instead of water and other physiological saline solutions prolonged their viability (p < 0.01). Nevertheless, viability of chilled embryos in GFACF medium was slightly, but non‐significantly, higher than in Dettlaff's solution. Supplementation of the GFACF medium with antibiotics, Hepes or BSA increased the viability of chilled embryos, but the tested vitamin E analogue Trolox, vitamin C or Tris concentration had no effect on embryo viability. The outcome of this series of experiments shows that heartbeat‐stage goldfish embryos could be chilled for 60 h in GFACF supplemented with 25 mm Hepes, 100 U/ml penicillin, 10 μg/l streptomycin and 1 g/l BSA in such a way that embryonic development does not proceed, and viability is not lost.     

The effects of cooling and vitrification of embryos from mares treated with equine follicle-stimulating hormone on pregnancy rates after nonsurgical transfer

Equine embryos can remain viable for 12 to 24 hours when cooled and stored at 5°C.¹ Cryopreservation of embryos would allow for long-term preservation of genetic material and more efficient management of embryo recipients. This study compared pregnancy rates after transfer of equine embryos vitrified within 1 hour of collection or cooled for 12 to 19 hours before vitrification. Mares (N = 40) were superovulated using equine follicle-stimulating hormone (eFSH). Embryos were recovered 6.5 days after ovulation or 8 days after human chorionic gonadotropin. Forty morulae or early blastocysts with a grade of 1 to 2 and <300 mm in diameter were randomly assigned to 1 of 2 treatments: Group 1 (n = 20), washed 4 times in a commercial holding medium and then vitrified; Group 2 (n = 20), washed 3 times and then stored in the same holding medium at 5°C to 8°C in a passive cooling device for 12 to 19 hours before being vitrified. To thaw, embryos were warmed by holding the straw in air at room temperature for 10 seconds and then submerged in a water bath (20°C to 22°C) for an additional 10 seconds. The contents of the straw were transferred directly into a recipient that had ovulated 4 to 6 days previously. There were no differences (P > .05) in embryo diameter, grade, or morphology score between treatment groups before vitrification. Pregnancy rates (day 16) were not different (P > .05) between embryos vitrified immediately after collection (15 of 20; 75%) and embryos cooled for 12 to 19 hours before vitrification (13 of 20; 65%). Based on these results, small equine embryos (<300 mm) can be stored at 5°C to 8°C for 12 to 19 hours before vitrification without a significant loss of viability.     

Effect of embryo age on the viability of equine embryos after cooled storage using two transport systems

The aim of this study was to compare the viability of 7- and 8-day-old equine embryos cooled and stored for 6 or 24 hours in two different transport systems. Embryos (n = 97) were recovered on day 7 or 8 and assigned to 10 groups (n = 10/group). Embryos within the same age group (D7 or D8) were evaluated immediately after collection (Group-0h) or after storage in an Equitainer at 5°C for 24 hours in 5 ml Emcare Holding Solution (EHS) (Group-E-24h) or 5 ml Ham's F10 (Group-H-24h) or in a refrigerator at 5°C in 500 ml Emcare Flushing Solution (EFS) for 6 hours (Group-B-6h) or 24 hours (Group-B-24h). After collection or storage, embryos were incubated in 1 μg/ml DAPI to determine the percentage of dead cells per embryo (DAPI positive, fluorescent cells). Subsequently, embryos were fixed in 4% paraformaldehyde and re-stained with DAPI to determine the total number of cells. The percentage of dead cells in group-0h and B-6h was similar and significantly lower than for embryos stored for 24 hours in groups B-24h, E-24h, and H-24h. The percentage of dead cells was similar for embryos stored in an Equitainer (groups E-24h and H-24h) and was significantly higher for embryos stored 24 hours in EFS (Group B-24h). Within each storage system (0h, B-6h, B-24h, E-24h, and H-24h) no significant difference in the percentage of dead cells was observed between 7- and 8-day-old embryos. Storage in 500 ml EFS at 5°C for 6 hours resulted in embryos of better quality than after the traditional 24-hour storage in an Equitainer, suggesting that this simplified system offers a good alternative for short-term storage and transport.     

Evidence of Damage in Cryopreserved and Fresh Bovine Embryos Using the Tunel Technique

The objective of the present study was to evaluate the quality of bovine embryos cryopreserved in different years in Chiapas, Mexico. The embryos were obtained from a government institution (FIMEGEN) dedicated to promoting embryo transfer among dual-purpose cattle farmers. Forty-three embryos frozen in 1988, 1989, 2000 and 2002 were analysed with the Tunel technique to detect programmed cell death (apoptosis). Eleven fresh embryos were used as controls. Analysis of variance was used in embryos stored in the different years with averages tested using Tukey's test. Student's t-test was employed to compare fresh and frozen cells. Embryos with shorter storage time presented a lower number (p < 0.001) of Tunel-positive cells compared with embryos stored for longer time. On the contrary, when comparing the number of apoptotic cells between frozen and fresh embryos a higher number of positive cells (p < 0.05) were found in the former. The present results suggest that the cryopreservation per se caused damage that compromises the viability of the embryo. Another explanation for the lower pregnancy rate found in the tropics could be irreversible damage caused by poor storage technique in these large operations.     

Establishment of an advanced chemically defined medium for early embryos derived from in vitro matured and fertilized bovine oocytes

A chemically defined medium would be useful for analyzing promoters or inhibitors in in vitro culture (IVC) of bovine embryos. However, an IVC system for bovine embryos in a chemically defined medium has not been fully established. The present study was carried out to establish an advanced chemically defined medium for bovine embryos that supports a high rate of embryo development to the blastocyst stage. In the first experiment, we examined the effects of addition of Medium RD (RPMI1640 and Dulbecco's MEM, 1:1 v/v) to mKSOM/aa on developmental competence. The addition of 10% RD to mKSOM/aa with BSA improved the rate of development to the blastocyst stage; however, 10% RD-mKSOM/aa with PVP, which is a chemically defined medium, caused a reduction in the percentage of hatching blastocysts. In the second experiment, embryos were cultured in the chemically defined medium of 10% RD-mKSOM/aa containing 11.7, 23.4, 46.8, 70.2 or 96.8 μM inositol. Inositol at the concentration of 70.2 μM improved the rate of development to the hatching blastocyst stage. In the third experiment, the optimal RD concentration in the IVC medium was evaluated. Embryos were cultured in the chemically defined medium supplemented with 10, 20 or 30% (v/v) RD. The rate of development to the blastocyst stage was highest with 20% RD. In the fourth experiment, the effects of N-acetylglucosamine (GlcNAc) as an IVC medium supplement on developmental competence were examined. The rate of development to the blastocyst stage with 1.0 mM GlcNAc was significantly higher than that without GlcNAc, but the rate of development with 1.2 mM GlcNAc was not different from that without GlcNAc. We also evaluated the ability of blastocysts produced in RD-mKSOM/aa to develop to normal calves after being transferred into recipients. Ten of the 16 recipients became pregnant, with 9 delivering normal calves. These results indicate that 20% RD-mKSOM/aa containing 70.2 μM myo-inositol and 1 mM GlcNAc is useful as a chemically defined medium for IVC of bovine embryos.     

Programming of postnatal phenotype caused by exposure of cultured embryos from Brahman cattle to colony-stimulating factor 2 and serum

Alterations in the environment of the preimplantation embryo can affect competence to establish pregnancy and phenotype of resultant calves. In this study, the bovine embryo produced in vitro was used to evaluate postnatal programming actions of the embryokine colony-stimulating factor 2 (CSF2) and serum, which is a common additive of culture media. Oocytes were collected by ovum pick up from Brahman donors and fertilized with semen from Brahman bulls. Embryos were randomly assigned to one of the three treatments: vehicle, CSF2 10 ng/mL, or 1% (v/v) serum. Treatments were added to the culture medium from day 5 to 7 after fertilization. Blastocysts were harvested on day 7 and transferred into crossbred recipients. Postnatal body growth and Longissimus dorsi muscle characteristics of the resultant calves were measured. The percent of cleaved embryos becoming blastocysts was increased by serum and, to a lesser extent, CSF2. Treatment did not affect survival after embryo transfer but gestation length was shortest for pregnancies established with serum-treated embryos. Treatment did not significantly affect postnatal body weight or growth. At 3 mo of age, CSF2 calves had lower fat content in the Longissimus dorsi muscle and less subcutaneous fat over the muscle than vehicle calves. There was a tendency for cross-sectional area of the muscle to be smaller for serum calves than vehicle calves. Results confirm the importance of the preimplantation period as a window to modulate postnatal phenotype of resultant calves. In particular, CSF2 exerted actions during the preimplantation period to program characteristics of accumulation of intramuscular and subcutaneous fat of resultant calves. The use of a low serum concentration in culture medium from day 5 to 7 of development can increase the yield of transferrable embryos without causing serious negative consequences for the offspring.     

Follicullar Development and Embryo Recovery Following 3 versus 8 FSH Injections in Heifers

Ovarian follicular dynamics and embryo yield were studied during 2 different FSH regimens for superovulation of cattle. Twenty heifers were given intramuscular injections of FSH (total of 35 mg NIH) either once daily for 3 days (Group 3×1) or twice daily for 4 days (Group 4×2). At 72 h after the first FSH injection, each animal was injected with 0.75 mg cloprostenol. Inseminations were performed at 12 h and 24 h after the onset of heat. Transrectal ultrasonography was performed on the day of the first FSH injection, the day of cloprostenol injection, the day of insemination and finally on the day of embryo recovery (day 6 or 7 after heat). The numbers of small (2–4 mm), medium (5–9 mm) and large (>10 mm) size follicles were recorded. The total number of corpora lutea, eggs and transferable embryos were recorded on the day of embryo recovery. No differences were found between the 2 groups in either of the parameters studied (p>0.05). It can be concluded that treatment with this FSH preparation once daily for 3 days gives a folliculogenic and superovulatory response similar to a treatment regimen where it is given twice daily for 4 days.     

Effect of linoleic acid-albumin on post-thaw survival of in vitro-produced bovine embryos at the 16-cell stage

The effect of addition of linoleic acid-albumin (LAA) to culture medium before freezing on the survival rate of bovine 16-cell embryos after freezing-thawing was investigated. Embryos were incubated in CR1aa containing LAA (0.25 mg/ml) for 4 days after insemination. A conventional slow cooling method was used, in which embryos were cooled at a rate of 0.3 degrees C/min to -30 degrees C in medium supplemented with 1.5 M ethylene glycol and 0.2 M trehalose. The developmental rate to the blastocyst stage of thawed embryos that had been cultured with LAA-containing medium before freezing was higher than that of these cultured without LAA (P<0.05). However, with fresh, non-frozen, embryos that were incubated under the same culture conditions (with and without LAA), no such difference was found.     

A method for the cytogenetic detection of early embryos in heifers

The objective of the study was to work out a simple and reliable method of the cytogenetic examination of bovine embryos. Whole embryos free of the zona pellucida, or parts of these embryos, were cultivated for five hours in a medium with 0.05 microgram colcemide per 1 millilitre. The embryos were hypotonized for 10 to 15 minutes in a mixture of medium and distilled water (1:2 ratio) at the temperature of 37 degrees C. Stepwise fixation was used: first in a mixture of acetic acid, methyl alcohol and water (1:4:5), followed by dropping a mixture of acetic acid and methyl alcohol (1:1) onto the embryo already on the microscope slide during microscopic control. The chromosomes were stained with a 3% solution of Giemsa dye for 10 minutes.     

In vitro exposure of bovine morulae to Ureaplasma diversum.

Ureaplasma diversum has been associated with infertility in the cow experimentally and in naturally occurring cases. However, the pathogenic mechanism is undetermined. The purpose of this study was to determine whether ureaplasmas are pathogenic for bovine morulae in vitro. Twenty-one morulae were recovered from three superovulated, mature, Holstein cows six or seven days postestrus. The embryos were divided into three groups (A,B,C) and incubated for 16 hours at 37 degrees C in humidified air with 10% CO2. Group A was incubated in embryo culture medium alone, Group B was incubated in culture medium with sterile ureaplasma broth added and Group C was incubated in culture medium containing 1.7 X 10(6) colony forming units Ureaplasma diversum strain 2312. After incubation, the morulae were examined using an electron microscope. Structures morphologically identical to U. diversum were present on the outer surface of the zonae pellucidae of all the morulae exposed to the organism and none were present on the unexposed control embryos. No other morphological differences were observed in either the ureaplasma-exposed embryos or the two groups of control embryos. Ureaplasma diversum was isolated from three of the five embryos incubated in culture medium with sterile ureaplasma broth added. These three embryos were recovered from one donor cow which cultured positive for U. diversum from the vulva and flush fluid. This finding suggests that the contaminating organisms entered the embryo culture wells either in the embryo collection medium or attached to the embryos.(ABSTRACT TRUNCATED AT 250 WORDS)     

猪植入前胚胎体外培养条件的优化

探讨了更换胚胎培养液及添加FBS、高渗透压和不同浓度VE对猪卵母细胞体外受精(IVF)和孤雌激活(PA)胚胎体外发育的影响,进一步优化了猪植入前胚胎体外培养体系。试验一：在第2天、第4天更换新的培养液(换液组),在换液基础上第4天更换为添加10%FBS的培养液(FBS组)。试验二：胚胎分别在0.05 mol/L蔗糖(蔗糖组)和138 mmol/L氯化钠(氯化钠组)的PZM-3(300～320 mOsmol)中培养2 d后移至PZM-3(288 mOsmol)中培养5 d。试验三：在培养液中分别添加50、100和200 μmol/L VE。对照组均在PZM-3(288 mOsmol)中培养7 d。结果表明：试验一,IVF和PA胚胎FBS组囊胚率显著高于对照组和换液组(P<0.05);试验二,IVF胚胎氯化钠组卵裂率、囊胚率均显著高于对照组与蔗糖组(P<0.05);试验三,IVF胚胎添加100 μmol/L VE组囊胚率显著高于对照组(P<0.05)。结果提示,在换液的基础上添加FBS有利于猪IVF和PA胚胎的体外发育;氯化钠调节的高渗透压可以促进猪IVF胚胎的早期发育;添加100 μmol/L VE可以改善猪IVF胚胎的体外发育体系。     

The use of embryonic stem cell derived bioactive material as a new protein supplement for the in vitro culture of bovine embryos

Embryonic stem (ES) cells are expanded versions of the inner cell mass cells that compose the early mammalian blastocyst. Components derived from ES cells may contain various bioactive materials (BM) helpful for early preimplantation embryo growth. In this study, we examined the effect of human ES cell derived BM (hES-BM) on in vitro culture of bovine embryos. When bovine parthenogenetic day 2 embryos were cultured in 10% hES-BM, a significantly higher embryo development rate (44.3%) and increased cell numbers were observed relative to control medium containing 3 mg/ml BSA (19.5%; P<0.01). Among the various concentrations (5, 10 and 15%) and days of treatment (2 or 4 days) tested, 10% hES-BM treatment for 4 days provided the best culture environment to support the growth of bovine embryos in vitro (P<0.05). Little difference was observed between 10% hES-BM and 10% FBS treatment in the examined parthenogenetic or in vitro fertilized embryos, although the hES-BM group developed at a slightly better rate. However, the ICM cell numbers were significantly higher in the hES-BM group in irrespective of embryo origin (P<0.05). In addition, the relative levels of pluripotency (Oct4, × 1.8 fold; Nanog. × 3.3 fold), embryogenesis (Stat3, × 2.8 fold; FGF4, × 18.8 fold; E-cad, × 2.0 fold) and growth (Glut5, × 2.6 fold) genes were significantly higher in the 10% hES-BM group than in the 10% FBS group (P<0.05), while the levels of other genes (Bax, Bcl2, MnSOD and Connexin43) were not different. This is the first report examining the positive effects of hES-BM on bovine embryo development in vitro. Based on our results, we conclude that hES-BM can be used as a new protein supplement for bovine preimplantation embryo development.     

Effect of group culture and embryo-culture conditioned medium on development of bovine embryos

We investigated the effect of group culture on bovine embryo development, and also investigated the effect of embryo-culture conditioned medium on developmental competence of individually cultured bovine embryos. Slaughterhouse-derived bovine oocytes were matured and fertilized in vitro. The presumptive zygotes were cultured individually or cultured in groups of 2 to 5 embryos with a constant culture density (5 mul/embryo). After 7 days of culture, the rates of embryos developed to the blastocyst stage were significantly higher (P < 0.05) in group cultures of more than 3 embryos/drop than for embryo culture of 1 or 2 embryos/drop. These results suggest a beneficial effect of group culture may be exerted by possible growth promoting factors secreted by embryos. In the next experiment, we investigated the effect of timing of fresh medium replacement on the development of embryos cultured in groups. The blastocyst formation rate was lower when culture medium was replaced freshly on days 2-4 after fertilization than on days 5-6. The blastocyst formation rates of single-cultured embryos were significantly (p < 0.05) increased by the addition of conditioned medium derived from multiple-embryo culture. These results indicate that group culture promotes embryo development and that embryo culture-derived conditioned medium is effective for supporting development of single cultured embryos.     

Effect of heat-stress on bovine embryo development in vitro.

Chronic elevation of uterine temperature has long been known to increase embryo mortality in dairy cattle. Short-term elevation in temperature of mouse embryos to 43 degrees C (acute) has been shown to induce intracellular production of heat-shock proteins. In this study, in vitro development of bovine embryos was assessed during short-term (60 h) coculture with oviduct epithelial cells at 38.6 degrees C (T1), 40 degrees C (T2), 38.6 degrees C after a prior pulse treatment (20 min) at 43 degrees C with 5% CO2 (T3), or 38.6 degrees C after a prior pulse treatment (20 min) at 43 degrees C with 100% CO2 (T4). During incubation, embryos cocultured at 40 degrees C had a greater (P < .05) mean embryo development score at 36 h than embryos cocultured at 38.6 degrees C. At 60 h of incubation, embryo development scores were greater (P < .05) for embryos cultured at 38.6 degrees C than for those cocultured at 40 degrees C. The number of embryos hatched at 60 h was similar after coculture at 38.6 degrees C (T1) or a prior pulse treatment with 5% CO2 and 43 degrees C (T3), but the embryo development score at 60 h was greater (P < .05) for the pulse-treated embryos. Embryos in T4 had greater (P < .05) embryo development scores than did T1 embryos from 36 through 60 h. Pulse treatment (T4) resulted in a greater (P < .05) number of hatched embryos at 60 h than T1, T2, and T3. These results indicate a detrimental effect of a chronic elevation in temperature that was evident shortly after embryo hatching.(ABSTRACT TRUNCATED AT 250 WORDS)     

Survival Rate and Ultrastructure of Vitrified Bovine in vitro and in vivo Developed Embryos

The capacity of different vitrification media and methods was tested onto in vivo and in vitro produced bovine morula/blastocysts and their ultrastructure and survival studied post-thawing. Two vitrification solutions were finally selected, named 40 ES (40% ethylene glycol in PBS containing 0.5 M sucrose) and 35 EFS (composed of 35% (v/v) ethylene glycol in PBS containing 0.5 M/l sucrose and 30% (w/v) Ficoll 70). The straws were either precooled or not precooled in nitrogen vapour, plunged and stored in LN₂ for 10–25 days, and then thawed in a 20° C waterbath. The content of the straws was rediluted in 1M sucrose solution in PBS and later cocultured with BOEC for 48 h. The overall survival rates for in vitro and in vivo embryos were 36% (12 of 33) and 20% (3 of 15) after 24 h and 21% (7 of 33) and 33% (5 of 15 ) after 48 h. The survival rates for precooled embryos were significantly higher than for not precooled (48% vs 13% after 24 h and 44% vs 4% after 48 h) when tested across vitrification media. The in vitro-produced embryos presented an ultrastructure similar to the pre-freeze state, irrespective of the vitrification media used. The in vivo developed embryos showed a rather modified post-thaw ultrastructure, with clear signs of osmotic changes at both the trophoblastic and embryonic cells. The results indicated that in vitro and in vivo developed bovine embryos can survive vitrification using ethylene glycol as a cryoprotectant.     

The influence of medium on viability of bovine morulae produced in vitro after their biopsy

The aim of this study was to determine the influence of two different media on the viability of in vitro produced biopsied bovine morulae. Bovine morulae were produced in vitro, then biopsied and cultured in the Ham's F10 and IVM media. Cultured and control morulae were stained with Hoechst 33342 and propidium iodide. Morulae were classified morphologically for excellent, good and degenerated quality. 42.86% of biopsied morulae cultured in the Ham's F10 medium and only 11.11% (only one) of these embryos cultured in the IVM were of excellent quality. Embryos of good quality were about 2 times less numerous in Ham's F10 medium (28.57%) than in IVM medium (55.5%) (P < or = 0.05). 28.57% of biopsied morulae cultured in Ham's F10 medium and 33.33% of these embryos cultured in the IVM degenerated (P > or = 0.05). The media had no significant influence on the number of total and viable blastomeres of morulae cultured in vitro after biopsy (P > or = 0.05). But the quantity of restored (excellent and good quality) embryos was higher when they were cultured after biopsy in Ham's F10 medium than in IVM. These statistically significant results (P < or = 0.05) show that the Ham's F10 medium is better for the restoring of biopsied bovine embryos produced in vitro than IVM.     

In vivo culture of IVM/IVF embryos in bovine oviducts by transvaginal endoscopy

This study was conducted to establish a new approach for in vivo culture of in vitro produced embryos in the bovine oviduct by transvaginal endoscopy. Embryos were in vitro matured, fertilized and cultured for 1-4 days and assigned to groups consisting of 10-30 embryos. Embryos were transferred unilaterally into oviducts of 24 heifers by the means of transvaginal endoscopy. After 3-6 days of in vivo incubation embryos were re-collected. Experiment I aimed to evaluate the capability of embryos to migrate to the uterus. The uterine horns of four animals were flushed first, followed by a combined flushing of both oviducts and uterine horns resulting in collection rates of 31 and 34%, respectively. In experiment II, the transfer of embryos into the oviduct close to ovulation (day 1-2--experiment IIA) or at a more advanced cyclic stage (day 3--experiment IIB) succeeded in the collection of 46 and 34% of the transferred complexes, of which 13 and 37% showed the blastocyst stage. This is the first report of successful recovery of transferable blastocysts by transvaginal endoscopy after tubal in vivo culture in the homologous species of originally in vitro produced embryos.     

Evaluation of the relationship between serum paraoxonase-1 activity and superovulation response/embryo yield in Holstein cows

In this study, the effect of serum paraoxonase-1 (PON-1) activity on superovulation response and embryo yield was evaluated. The study material comprised 50 Holstein cows aged 3–4 years on postpartum day 90–120 with a body condition score of 3–3.25. A progesterone-based estrus synchronization protocol was initially administered to the selected donors. For this purpose, progesterone source was inserted intravaginally (day 0) and gonadotropin-releasing hormone injection was performed (day 6). Seven days after the insertion of progesterone device, follicle-stimulating hormone injections (total dose of 500 µg in decreasing doses for 4 days) were administered for superovulation. On the morning of the ninth day, prostaglandin (PG) F2α was administered, and the progesterone device was removed from the vagina in the evening on the same day. Two days after PGF2α administration, fixed-time artificial insemination was performed in the morning and in the evening. On the day of artificial insemination, blood samples were taken from the donors to determine the serum PON-1 activity. Uterine flushing was performed seven days after insemination. The results revealed that the serum PON-1 activity (mean ± SD, 562.71 ± 140.23 U/l) of the cows that responded to superovulation (donors with total corpus luteum count of ≥3 in both ovaries) was higher than those (389.91 ± 80.51 U/l) that did not (P<0.05). On the day of insemination, a positive correlation was determined between serum PON-1 activity and the counts of total corpus luteum (r=0.398), total oocyte/embryo (r=0.468), transferable embryo (r=0.453), and Code I embryos (r=0.315, P<0.05). Unlike the Code I embryos, there was no significant correlation between serum PON-1 activity and the number of Code III embryos. Moreover, no significant difference in the number of Code III embryos between the two PON-1 groups was observed. However, embryo yield and quality were found to have increased with increased PON-1 activity. Therefore, it was concluded that serum PON-1 activity may be associated with superovulation response, embryo yield and quality in donor cows.     

Toxicity potential of absorbed-retained ethylene oxide residues in culture dishes on embryo development in vitro

Four hundred eight-cell mouse embryos were cultured in vitro in either polystyrene (plastic) dishes (Exp. 1) or watch glasses (Exp. 2) to analyze the toxicity potential of absorbed-retained ethylene oxide (EtO). Culture dishes were gas-sterilized with Anprolene equipment and allowed to aerate (21 C) for varying durations. Post-sterilization EtO residues, as determined by weight measurement, were eluted from polystyrene dishes at an exponential rate. After 1 wk of aeration, .625 mg EtO was retained/g of polystyrene product. To determine the effect of EtO residue on in vitro culture, embryos were collected from superovulated donor mice (C57BL/6N), pooled in a modified Dulbecco's phosphate buffered saline (PBI) medium and then randomly allotted to dishes subjected to various aeration durations. Embryos were cultured in Whitten's medium +3 mg/ml bovine serum albumin (BSA) under a humidified 5% CO2 in air atmosphere at 37 C. Gross assessments of embryo development were made using standard morphology and quality grading systems and a fluorescein diacetate staining assay. In Exp. 1, embryo development was retarded at the 8- to 16-cell stage when less than or equal to 12 h aeration of polystyrene dishes was permitted. Aeration for 24 and 36 h resulted in suboptimal embryo development with fewer (P less than .01) blastocysts and greater degeneration rates at 48 h of in vitro culture compared with the control (manufacturer-packaged dishes, no EtO) and 1-wk aeration treatment groups.(ABSTRACT TRUNCATED AT 250 WORDS)