Improved in vitro development of porcine embryos with different energy substrates and serum

The effect of replacing 5.5 mM glucose in North Carolina State University (NCSU)-23 medium with 0.5 mM pyruvate/5.0 mM lactate on porcine IVF embryo development was investigated in Experiment 1. Culturing embryos with pyruvate/lactate for 7 days or with pyruvate/lactate from Days 0 to 2, and then glucose from Days 2 to 7 improved cleavage rates. In Experiment 2, embryos were cultured for 7 days in pyruvate/lactate containing NCSU-23 medium supplemented with 0.05% PVA, 0.4% BSA or 10% fetal bovine serum (FBS). The BSA supplement increased the rates of cleavage, blastocyst formation, and the number of total cells in blastocysts. In Experiment 3, embryos were cultured in pyruvate/lactate containing NCSU-23 medium supplemented with 0.4% BSA for 7 days (BSA-PL), 0.4% BSA from Days 0 to 4 and then 10% FBS from Days 4 to 7 (BSA-PL-->F ) or 0.4% BSA from Days 0 to 7 with addition of 10% FBS (BSA-PL + F ) at Day 4. More blastocysts in BSA-PL--> F and hatching or hatched blastocysts in BSA-PL-->F and BSA-PL+F were obtained. Total cell number in blastocysts derived from BSA-PL-->F and BSA-PL+F were increased. Our results demonstrated that supplementing pyruvate/lactate containing NCSU-23 medium with 0.4% BSA for 4 days and replacing it with 10% FBS for another 3 days improved porcine IVF embryo development.     

Effect of bovine blastocyst size at embryo transfer on day 7 on conceptus length on day 14: Can supplementary progesterone rescue small embryos?

Conceptus size on Day 14 after multiple embryo transfer of Day 7 in vitro–produced blastocysts varies greatly within animal. One explanation for this variation may be related to blastocyst cell number at the time of transfer. The aim of this study was to examine the effect of Day 7 blastocyst cell number on Day 14 conceptus size and to examine the effect of progesterone (P4) supplementation on embryo development after the transfer of Day 7 blastocysts containing a low total cell number. The estrous cycles of crossbred beef heifers were synchronized using an 8-day progesterone (P4)–releasing intravaginal device (PRID) with the administration of a prostaglandin F_2α analog on the day before device removal. Only those heifers recorded in standing estrus (Day 0) were used. Heifers were randomly assigned to one of four treatment groups: (1) control: large blastocysts (high total cell number), (2) control: small blastocysts (low total cell number), (3) small blastocysts plus a single intramuscular injection of 3000 IU human chorionic gonadotropin (hCG) on Day 2 after estrus, or (4) small blastocysts plus insertion of a vaginal P4 insert (PRID, 1.55 g P4) between Days 3 and 5 after estrus. In vitro–produced blastocysts were transferred to each heifer on Day 7 (n = 10 blastocysts per heifer), and conceptuses were recovered at slaughter on Day 14. Daily blood samples were collected from Day 0 to 14 to measure serum P4 concentrations. Data were analyzed using the PROC MIXED procedure of SAS. Total cell number on Day 7 was significantly lower in small versus large blastocysts (72.4 ± 3.93 vs. 144.8 ± 3.90, P < 0.05). Conceptus recovery rate was 53.8% overall (140 of 260) and was highest in the large blastocyst group (68.3%, 41 of 60) compared with the other groups (45.7%–55.0%). Concentrations of serum P4 were similar in the two unmanipulated recipient groups but were significantly elevated (P < 0.05) by Day 8 in the hCG-treated heifers and on Days 4 and 5 in the PRID group (P < 0.003). In the absence of supplemental P4, Day 14 conceptuses resulting from the transfer of small blastocysts (2.48 ± 0.54 mm) were smaller than those from large blastocysts (3.32 ± 0.52 mm). Administration of hCG on Day 2 approximately doubled conceptus length on Day 14 (4.94 ± 1.15 mm; P < 0.05), whereas insertion of a PRID from Days 3 to 5 increased conceptus length approximately fivefold (13.09 ± 2.11 mm; P < 0.05) compared with controls. In conclusion, results indicate that supplemental P4 is capable of “rescuing” poor-quality blastocysts, presumably via the now well-described actions on the endometrium and consequent effects on uterine lumen fluid composition.     

The sex ratio of bovine embryos produced in vitro in serum-free oviduct cell-conditioned medium is not altered

The sex ratio of bovine blastocysts produced in vitro in serum-free oviduct cell-conditioned medium was investigated. Bovine embryos reaching the blastocyst stage were removed from culture medium on Days 6, 7, 8 and 9 and were identified as small, mid-sized or expanded blastocysts. One third (29/91) of the blastocysts appeared on Day 6. Twelve from them were small blastocysts (5 males), 7 were mid-sized blastocysts (4 males) and 10 were expanded blastocysts (5 males). On Day 7, 33 blastocysts were obtained: 8 small (5 males), 9 mid-sized (3 males) and 16 expanded (13 males) blastocysts. Finally, on Days 8 and 9, 29 blastocysts were obtained: 12 small (9 males), 9 mid-sized (6 males) and 8 (3 males) expanded blastocysts. Sexing of the 91 blastocysts was performed by using an original polymerase chain reaction (PCR) protocol generating discreet internal control signals from both female and male samples and Y-specific smears from the male samples. Proportions of male embryos on Days 6, 7 and on Days 8+9 were 48, 64 and 62%, respectively. These values did not differ significantly among days and did not differ from 50%. Fifty-nine percent of small blastocysts, 52% of mid-sized blastocyst and 62% of expanded blastocysts were male. No difference between these values or with respect to 50% could be observed. These results show that bovine blastocysts produced in serum-free oviduct cell-conditioned medium do not have an altered sex ratio.     

Estradiol production by preimplantation blastocysts and increased serum progesterone following estradiol treatment in llamas

Estradiol is a potential candidate for the blastocyst signal responsible for maternal recognition of pregnancy in the llama (Lama glama). Two experiments were conducted to determine if the llama blastocyst produces estradiol during the presumed period of maternal recognition of pregnancy and if exogenous estradiol can extend the luteal phase. In Experiment 1, llamas were superovulated with eCG and mated 7 days later (Day 0=day of mating). Blastocysts were collected nonsurgically on Days 7, 9, or 11 or at necropsy on Days 13 and 15 post-mating and cultured for 48h. Conditioned medium was recovered, replaced with fresh medium at 24-h intervals, and assayed for estradiol-17beta. Estradiol production (pg/blastocyst) over the 48-h culture increased (P<0.05) by day of gestation where more estradiol (P<0.05) was produced by Day 11 compared to Day 7 blastocysts, Day 13 compared to Days 7-11 blastocysts, and Day 15 compared to Days 7-13 blastocysts. A dramatic increase was observed between Days 11 and 13 when estradiol production by Day 13 blastocysts increased (P<0.05) more than 50-fold. In Experiment 2, 30 females were induced to ovulate with hCG (Day 0=day of hCG injection). Starting on Day 7 and continuing through Day 15, animals received daily injections i.m. of 0 (n=11), 5 (n=7), or 10mg (n=12) estradiol benzoate (EB) dissolved in isopropylmyristate. Sera were collected immediately prior to each injection and on Days 16, 17, 18, 20, and 22 and analyzed for progesterone. Progesterone concentrations were greater (P<0.05) on Days 14, 15, 16, and 17 in llamas treated with 10mg EB compared to llamas treated with 0mg EB. These results demonstrate that llama blastocysts produce estradiol and exogenous estradiol can enhance and transiently extend luteal progesterone production. Estradiol produced by the preimplantation llama blastocyst may play a role in maternal recognition of pregnancy and early luteal support.     

Successful piglet production after transfer of blastocysts produced by a modified in vitro system

Porcine in vitro production (IVP) systems, including in vitro maturation (IVM) and in vitro fertilization (IVF) of oocytes and their subsequent in vitro culture (IVC), have been modified by many researchers, but are still at a low level because of a low developmental rate of embryos to the blastocyst stage and their poor qualities. Our objectives were to establish reliable IVP procedures for porcine blastocysts and to examine the ability of the blastocysts to develop to term after transfer to recipients. Porcine cumulus-oocyte complexes were matured in vitro under 5% O(2) or 20% O(2), fertilized in vitro under 5% O(2), and subsequently cultured under 5% O(2) in 1) IVC medium supplemented with glucose (IVC-Glu) from Day 0 (the day of IVF) to Day 6; 2) IVC-Glu from Days 0 to 2, then IVC medium supplemented with pyruvate and lactate (IVC-PyrLac) from Days 2 to 6; 3) IVC-PyrLac from Days 0 to 2, then IVC-Glu from Days 2 to 6; and 4) IVC-PyrLac from Days 0 to 6. There were no significant differences in blastocyst formation rates on Day 6 between the 5% O(2) and 20% O(2) conditions (19.9% and 14.0%, respectively). However, the quality of blastocysts, as evaluated by the total cell number, was better after IVM under 5% O(2) than under 20% O(2) (mean cell number, 43.5 and 37.8, respectively). When IVP embryos were cultured in IVC-PyrLac from Days 0 to 2 and subsequently in IVC-Glu from Days 2 to 6, the rate of blastocyst formation (25.3%) and cell number (48.7) were higher than the rates (5.8% to 18.1%) and numbers (35.4 to 37.1) with the IVC-Glu then IVC-Glu, the IVC-Glu then IVC-PyrLac, and the IVC-PyrLac then IVC-PyrLac regimens, respectively. We then prepared conditioned medium (CM) from culture of porcine oviductal epithelial cells for 2 days in IVC-PyrLac and evaluated its effect on development to the blastocyst stage. Cultivation in CM for the first 2 days, followed by IVC-Glu for a further 4 days, had a significantly greater effect in increasing the number of cells in the blastocyst (58.3) than did in IVC-PyrLac (48.4). Finally, we evaluated the ability of blastocysts, generated by IVM under 5% O(2) and IVC in CM, to develop to term. When Day 5 expanding blastocysts (mean cell number, 49.7) were transferred to an estrus-synchronized recipient (50 blastocysts per recipient), the recipient remained pregnant and farrowed eight normal piglets. Furthermore, when Day 6 expanded blastocysts (mean cell number, 80.2) were transferred to two estrus-synchronized recipients, both gilts remained pregnant and farrowed a total of 11 piglets. These results suggest that an excellent piglet production system can be established by using this modified IVP system, which produces high-quality porcine blastocysts. This system has advantages for the generation of cloned and transgenic pigs.     

Effect of the in vitro culture system on the kinetics of blastocyst development and sex ratio of bovine embryos

Bovine blastocysts were produced using 6 different systems: 5 commonly used in vitro culture systems (synthetic oviduct fluid medium - SOF- without fetal calf serum, SOF supplemented with 10% serum for the entire culture period, SOF supplemented with 10% serum from Day 4 of culture, M199 coculture with bovine oviduct epithelial cells, M199 coculture with granulosa cell monolayer) and 1 in vivo culture system involving collection of blastocysts from superovulated bovine donors at Day 7. Zygotes obtained from IVM/IVF were assigned randomly to 1 of the 5 systems tested and were cultured for 9 d (Day 0= day of insemination). Cleavage, development to the blastocyst stage and blastocyst sex ratio were assessed in all treatments. In addition, the effect of the IVC system on the kinetics of blastocyst development and sex ratio was assessed on Days 6, 7, 8, and 9. The presence of fetal calf serum in SOF not only resulted in faster development (19.1% of blastocysts in SOF supplemented with serum vs 7.1% in absence of serum at Day 6; P < 0.05) and increased blastocyst production (47.5% of blastocysts in SOF supplemented with serum vs 34.4% in absence of serum; P < 0.05) but it also enhanced overall male survival. The coculture systems produced fewer blastocysts than culture in SOF (27.6 to 28.3% in coculture vs 47.5% in SOF supplemented with serum; P < 0.05), but similar to SOF without fetal calf serum, they had no effect on blastocyst sex ratio.     

Evaluation of different culture systems on the in vitro production of bovine embryos

The objective of this study was to determine the development potential and quality of in vitro produced bovine embryos cultured individually or in groups. After IVM and IVF, presumptive zygotes were cultured in groups or individually, either in drops or in the modified "well of the well" (mWOW) system. In Experiment 1, four culture systems were utilized: T1: drop in group (control); T2: mWOW in groups; T3: mWOW individually; and T4: drop individually. Cleavage and blastocyst rates at Days 6, 7 and 8 and total cell number of Day 6 blastocysts were similar (P > 0.05) for all treatments. However, in Day 7 blastocysts, total cell number was lower (P < 0.05) in embryos cultured individually in a small drop than those cultured in the mWOW. In Experiment 2, blastocysts of T1, T2 and T3 were allocated into two groups, control and vitrified. After warming, the vitrified embryos were cultured for 72 h. At 48 h, the development of the Days 6 and 7 embryos was similar (P > 0.05) for all treatments in the control group. For the vitrified embryos, lower hatching rates (P < 0.05) were observed in the T3 group. In conclusion, embryos cultured in groups in the mWOW system had the same blastocyst rates but better quality (measured by their survival after vitrification) than those cultured individually in the mWOW system.     

Cryopreservation of in vitro-derived bovine blastocysts microinjected with foreign DNA at the pronuclear stage

Days 6 and 7 bovine blastocysts derived from in vitro-fertilized and DNA-injected zygotes (day of IVF = Day 0) were cryopreserved either by conventional two-step freezing or by vitrification. Foreign DNA used for microinjection was the green fluorescent protein gene under the control of the immediate early promoter of human cytomegalovirus. All blastocysts were produced by an in vitro system and were harvested on Days 6 and 7. The proportion of DNA-injected zygotes developing into blastocysts on Days 6 and 7 (total 8%) was lower than that of nontreated zygotes (total 19%; P < 0.01). After cryopreservation in 1.5 M ethylene glycol, the survival rates of DNA-injected blastocysts assessed by re-expansion at 24 h of culture (Day 6: 59%, Day 7: 71%) were comparable with those of nontreated blastocysts (Day 6: 76%, Day 7: 71%). The post-thaw hatching rate within 72 h of culture of DNA-injected Day 7 blastocysts (38%) was not different from that of nontreated Day 7 blastocysts (40%), but the hatching rate of DNA-injected Day 6 blastocysts (23%) was lower than that of nontreated Day 6 blastocysts (47%; P < 0.05). After vitrification in 7.2 M ethylene glycol, 0.0026 M Ficoll-70 and 0.3 M sucrose, the survival and hatching rates of DNA-injected Day 7 blastocysts (61 and 28%, respectively) were similar to those of nontreated Day 6 (71 and 33%, respectively) and Day 7 (75 and 36%, respectively) blastocysts. However, the post-warming survival rate of DNA-injected Day 6 blastocysts was only 30%, and none of the blastocysts hatched (P < 0.01). The mean cell number of DNA-injected Day 6 blastocysts (100.3 +/- 36.4 cells) was lower than that of nontreated Day 6 blastocysts (130.5 +/- 37.1 cells; P < 0.01), while those of DNA-injected and nontreated Day 7 blastocysts were not different (111.2 +/- 42.8 and 119.6 +/- 31.4 cells, respectively). These results indicate that Day 7 IVMFC bovine blastocysts derived from DNA-injected zygotes can be successfully cryopreserved by conventional two-step freezing or vitrification.     

Impact of pro-oxidant agents on the morula-blastocyst transition in bovine embryos

Exposing day 5 bovine morulae to reactive oxygen species induces a delayed degeneration of some blastocysts on day 8 post-insemination (pi) but without affecting the blastocyst rates. The aim of this study was to characterize the resisting and the degenerating population of blastocysts. The kinetics of degeneration of the embryos exposed to the two pro-oxidant agents: 2,2'-azobis (2-amidinopropane) dihydrochloride (AAPH) and buthionine sulfoximine (BSO) was evaluated using time-lapse cinematography. With both agents the first signs of degeneration appeared at day 7.5 pi but the duration of the degeneration process was shorter in presence of AAPH than BSO (4.2 vs. 12.5 hr, ANOVA, P < 0.05). The resisting blastocysts derived from morulae with a larger diameter (mean diameter: 161 vs. 154 microm, ANOVA, P < 0.05) and showed an earlier cavitation (135 vs. 142 hpi, P < 0.05) than the degenerating ones. The profile of protein neosynthesis at day 7 was not affected by the treatment. The proportion of male embryos was more important in the resisting than in the degenerating population (70 vs. 55%, chi2, P < 0.05) especially when the stress was induced by AAPH. The quality of the resisting embryos, measured by the total cell number and the rate of apoptosis, did not seem to be affected when compared to control embryos. In conclusion, resistance to oxidative stress seems related to the kinetics of development and/or the sex of the embryos. Resisting embryos apparently display a quality similar to untreated embryos.     

Relationship between in vitro fertilisation of ewe oocytes and the fertility of ewes following cervical artificial insemination with frozen-thawed ram semen

No laboratory test exists that can reliably predict differences among rams in field fertility after artificial insemination (AI) with frozen-thawed semen. In vitro fertilisation (IVF) has been proposed as a method of predicting these differences. The objectives of this study were to evaluate whether IVF system could discriminate among rams of different fertility in vivo after AI using frozen-thawed semen. Also, to examine effects of lowering sperm concentration on discrimination power between rams used for IVF. The aim of Experiment 1 was to evaluate the effect of altering the sperm concentration from 2 x 10(6) to 0.03125 x 10(6) spermatozoa/mL on subsequent cleavage rate and blastocyst rate in vitro. In Experiment 2, six rams (three High and three Low in vivo fertility; average pregnancy rates of 37.6% and 21.8%, respectively) were compared for their fertilising ability in IVF. Spermatozoa from each of the six rams were added to ewe oocytes using a concentration of either 2 x 10(6) or 0.0625 x 10(6)/mL. There were six replicates with 25 oocytes per well and two wells per ram per replicate. Cleavage rate was monitored at 48 h post-insemination (p.i.) and blastocyst rate determined on Days 6-8 p.i. In Experiment 1, cleavage rate increased with increasing sperm concentration and blastocyst rate was not affected by sperm concentration on any day. When the six rams were tested using 2 x 10(6) spermatozoa/mL, no significant differences were found between High and Low fertility groups for cleavage rate or blastocyst rate on Days 6, 7, or 8 p.i. (P>0.05). When the experiment was repeated using 0.0625 x 10(6) spermatozoa/mL, no differences were found between High and Low group rams for blastocyst rate on any of Days 6, 7 or 8 p.i. (P>0.05). However, there was a significant difference between High and Low fertility rams for percentage of oocytes cleaved (16.4, S.E. 2.02%; P<0.01) and the correlation between fertility in vivo and cleavage rate in vitro was significant (P=0.013). Replicate of IVF was a source of significant variation for both cleavage rate and blastocyst rate and conditions need to be further controlled. However, we suggest that using a low concentration of spermatozoa (0.0625 x 10(6)/mL) for IVF may be a useful method for predicting field fertility of frozen-thawed ram semen.     

Cross-validation of techniques for measuring lipid content of bovine oocytes and blastocysts

The main objective was to test and validate a fluorescence approach to quantify lipid content of individual bovine oocytes and blastocysts. For Experiment 1, denuded oocytes were evaluated, as well as in vitro-produced blastocysts in a factorial design: cows versus feedlot heifers; three additives during Days 2.5-7.5 of culture (Control; 10% FCS; 0.3 μM phenazine ethosulfate (PES), an electron acceptor that oxidizes NADPH); and two blastocyst stages (early versus expanded). All blastocysts were graded subjectively for darkness (1 = clear … 4 = dark). In Experiment 2, denuded oocytes were used to measure lipid content in a factorial design of: cows versus heifers and four subjective darkness grades (1 = clear … 4 = dark). To quantify lipids, oocytes and 7.5 d blastocysts were fixed and then stained with 1 μg/mL Nile Red dye in mPBS overnight. A digital photograph of the equatorial part of the oocyte and embryo was taken at 200×, and fluorescence intensity (Arbitrary Fluorescence Units, AFU) was measured with Image Pro software. Reverse images of the same photographs were used to count numbers of cytoplasmic lipid droplets of various sizes (LC). The linear regression equation of LC with AFU in oocytes had an r² = 0.84, and for blastocysts r² = 0.91. The LC and AFU also had similar coefficients of variation from the ANOVA for blastocysts (38 vs 44%, respectively). Treatment differences were of similar magnitude with both procedures: lipid content in oocytes and blastocysts from heifers and cows was similar (P > 0.1); PES reduced lipid accumulation, and FCS increased it relative to the Control for AFU (18.6 vs 46.6 vs 36.9 units, respectively), and LC (1763 vs 4081 vs 3310, respectively; all, P < 0.01). Early blastocysts resulted in more lipid accumulation per unit area than expanded ones based on AFU (41.5 vs 26.6) and LC (3519 vs 2583; both P <0.01). There was a strong relationship (P < 0.01) between subjective oocyte and blastocyst darkness and lipid content. The less labor intensive fluorescence staining was a reliable technique for quantifying lipid droplets in oocytes and blastocysts.     

Prevalence of apoptosis and inner cell allocation in bovine embryos cultured under different oxygen tensions with or without cysteine addition

Supraphysiological oxygen tension during embryo culture can generate reactive oxygen species (ROS), which can induce apoptosis. Antioxidants such as thiol compounds (cysteine, cysteamine) can be used to prevent ROS damage to the embryo. The purpose of this study was to evaluate the prevalence of apoptosis during bovine embryo development and to evaluate the effect of the presence or absence of cysteine 0.6 mM in modified synthetic oviduct fluid (mSOF) on in vitro produced cattle embryos cultured under two different oxygen tensions (5% O2 versus 20% O2). Effects were assessed by checking embryo development at Days 7, 8 and 9 and by evaluating Day 9 hatched blastocysts for differentiation by means of differential staining and for apoptosis by means of TUNEL-assay. Apoptotic cells were present in 94% of Day 7 blastocysts and in 100% of Days 8 and 9 blastocysts. Cysteine addition affected Day 8 blastocyst rates in a negative way (P < 0.05) regardless of the oxygen tension. In fact, cysteine addition to the mSOF culture medium had a negative effect upon embryo development in terms of blastocyst rates, hatching rates and apoptotic cell ratio. Embryos cultured under 5% O2 in the presence of cysteine, however, possessed significantly higher numbers of ICM cells. This finding corroborates the theoretical assumption that antioxidants are beneficial for ICM development.     

Analysis of DNA fragmentation of in vitro cultured bovine blastocysts using TUNEL

Programmed cell death (apoptosis) characteristically affects the single cells of blastocysts whereas necrosis affects cluster of cells in both the inner cell mass (ICM) and the trophectoderm (TE). This study uses the trophectodermrminal deoxynucleotidyl transferase (TdT) mediated dUTP nick-end labeling (TUNEL) assay as a way of evaluating the proportion of apoptotic cells and, thus, bovine blastocyst quality during in vitro culture at Days 6,7, and 8. Furthermore, parthenogenetic blastocysts were compared to in vitro fertilized blastocysts at Day 7. Confocal microscopy was used to generate three-dimensional reconstructions of the blastocysts. Apoptosis was observed in both early (Day 6) and late (Day 8) developing blastocysts. The dead cell index (DCI, total number of apoptotic nuclei/total number of nuclei) tend to increase as the in vitro culture time increases, and apoptosis is proportionately higher in the ICM than in the TE. The ratio of ICM to TE cells remains relatively constant even as the blastocysts cell number increases (Day 6 = 11.9 +/- 2.2, Day 7 = 11.2 +/- 0.5, Day 8 = 11.7 +/- 0.4). The overall cell number is significantly reduced in parthenogenetic blastocysts compared to Day 7 in vitro produced blastocysts (P = 0.037). The parthenogenetic blastocysts also show an increase of apoptosis over Day 7 controls. The decrease in cell number in the parthenogenetic blastocysts may be due to the increase of apoptotic nuclei observed. Based on these results we found the TUNEL assay to be a useful method for evaluating in vitro culture conditions of pre-implantation bovine embryos.     

Optimization of cryopreservation of buffalo (Bubalus bubalis) blastocysts produced by in vitro fertilization and somatic cell nuclear transfer

The objective of this study was to optimize cryopreservation conditions for buffalo in vitro produced (IVP) embryos. The in vitro fertilized (IVF) and somatic cell nuclear transferred (SCNT) blastocysts were vitrified with either 40% ethylene glycol (EG), 25% EG + 25% dimethylsulfoxide (DMSO), or 20% EG + 20% DMSO + 0.5 m sucrose, and the IVF blastocysts produced from abattoir-derived ovaries were also slow-frozen with either 10% EG or 0.05 m trehalose dehydrate + 1.8% EG + 0.4% BSA. Cryosurvival rates of blastocysts harvested on various days or at various developmental stages were also examined. In this study: (1) vitrification with 20% EG + 20% DMSO + 0.5 m sucrose had the best cryopreservation efficiency; (2) IVF and SCNT blastocysts had similar cryotolerance (P > 0.05); (3) after thawing, slow-frozen blastocysts reexpanded earlier than the vitrified blastocysts (P < 0.01); (4) cryosurvival rate of expanded blastocysts was higher than that of early blastocysts (P < 0.05); (5) cryosurvival rates of Days 5 to 7 blastocysts (Day 0 = day of IVF or SCNT) were higher than those of Days 8 to 9 blastocysts (P < 0.01); and (6) after embryo transfer, pregnancy rates for fresh and cryopreserved blastocysts were not different (P > 0.05). In conclusion, vitrification of Days 6 to 7 expanded blastocysts with 20% EG + 20% DMSO + 0.5 m sucrose was optimal for cryopreservation of buffalo IVP embryos.     

A paucity of structural integrity in cloned porcine blastocysts produced in vitro

The structural integrity of blastocyst stage embryos, consisting of the inner cell mass (ICM) and trophectoderm (TE) cells, is a prerequisite for normal development after implantation in mammals. In this study, allocation of nuclear transfer (NT)-derived porcine blastocysts to the ICM and to the TE cells was examined and compared with IVF- and in vivo-derived embryos. NT-derived embryos had a lower developmental competence to the blastocyst stage than IVF-derived embryos (P < 0.05). Total cell number of NT-derived blastocysts was inferior to that of IVF-derived embryos (P < 0.05), although no difference was detected between the two groups in the ratio of ICM to total cells. However, in vivo-derived blastocysts had a higher proportion of ICM to total cells compared with in vitro-produced embryos (P < 0.01). To investigate what proportions of in vitro-produced porcine embryos represent normal structural integrity, differentially-stained blastocysts were individually classified into three presumptive groups (I: <20%; II: 20-40%; III: >40%) according to the ratio of ICM to total cells. Low proportions of NT- (12.5%, 7/56) and IVF-derived blastocysts (15.8%, 9/57) were assigned to Group II, presumptively having a normal range of structural integrity, whereas, almost all in vivo-derived embryos (97.5%, 39/40) were allocated to Group II. In conclusion, limited structural integrity may lead to the poor survival to term of NT- or IVF-derived porcine embryos produced in vitro.     

Role of prostaglandins in development of porcine blastocysts

Rapid elongation of porcine blastocysts between Days 11 to 12 of pregnancy coincides with an increase in uterine luminal content of prostaglandins. The present study evaluated the effect of two prostaglandin synthesis inhibitors (indomethacin and flunixin meglumine) on elongation of porcine blastocysts from spherical to filamentous forms between Day 11 to 12 of pregnancy. Gilts were hemi-hysterectomized on Day 11 of pregnancy. The excised uterine horn was flushed with 0.9% saline and diameter of blastocysts recovered were measured. Immediately following surgery, pregnant gilts were assigned to receive either: 1) vehicle every 4 h, 2) flunixin meglumine (banamine) every 4 h, or 3) indomethacin every 12 h. The remaining uterine horn was removed and flushed after the time of blastocyst elongation estimated for each gilt on basis of blastocyst development in the first horn. Uterine flushings were analyzed for total calcium, protein, acid phosphatase activity, estrone, estradiol-17 beta and prostaglandin F. Pretreatment blastocyst diameter was similar for all groups and ranged from 1 mm to 20 mm. Treatment of gilts with either banamine or indomethacin effectively inhibited (P less than 0.001) the increase in uterine luminal content of PGF. Total calcium, estrone and estradiol-17 beta were not influenced by treatment. Total uterine luminal protein and acid phosphatase activity were reduced (P less than 0.05) in banamine treated gilts compared to those receiving vehicle or indomethacin treatments. Although total PGF recovered in uterine flushings was reduced during the period of blastocyst elongation, treatment with PGF synthetase inhibitors failed to block rapid elongation of blastocysts from the spherical to filamentous forms.     

Noncytopathogenic bovine viral diarrhea virus (BVDV) reduces cleavage but increases blastocyst yield of in vitro produced embryos

The growing application of in vitro embryo production systems that utilize slaughterhouse tissues of animals of unknown health status conveys the risk of disease transmission. One pathogen of concern in this regard is bovine viral diarrhea virus (BVDV), and the objective of this study was to investigate the effect of BVDV on in vitro embryonic development. A bovine in vitro embryo production system was experimentally infected with BVDV at 2 stages: prior to in vitro maturation by incubating cumulus-oocyte complexes (COC) with virus (strain Pe515; titer 10(6.2) tissue culture infective dose (TCID)50/mL) or vehicle for 2 h, and then during in vitro culture by the use of BVDV infected granulosa cells. Exposure to BVDV throughout in vitro production reduced cleavage rates (P = 0.01) but increased (P = 0.05) the number of embryos that reached the 8-cell stage when expressed as a percentage of cleaved oocytes. Blastocyst yield was increased by the presence of virus when expressed as a proportion of oocytes (P = 0.0034) or of those cleaved (P < 0.0001). The percentage of total blastocyst yield on Days 7, 8 and 9 for the control and virus treatments was 20, 51, 29 and 29, 41, and 29%, respectively, indicating that the rate of blastocyst development was nonsignificantly faster in the virus-treated group (P = 0.06). These results indicate that the presence of non-cytopathogenic BVDV in an in vitro production system may reduce cleavage rates but allow those cleaved to develop to blastocysts at a higher rate.     

Effects of porcine granulocyte-macrophage colony-stimulating factor on porcine in vitro-fertilized embryos

This study investigated the effects of porcine granulocyte-macrophage colony-stimulating factor (pGM-CSF) on the developmental potential of porcine in vitro-fertilized (IVF) embryos in chemically and semidefined (with BSA) medium. In experiment 1, zygotes were treated with different concentrations of pGM-CSF (0, 2, 10, 100 ng/mL). The results indicated that 10 ng/mL pGM-CSF significantly (P < 0.05) increased blastocyst development and total cell number (15.1% and 53.5, respectively) compared with the control (6.1%, and 38.8, respectively). Comparing blastocyst formation, early and expanded blastocyst formation was significantly higher in the 10 ng/mL-pGM-CSF group than in the control on Days 6 and 7 of the culture period. However, there was no significant difference in cleavage rate. Experiment 2 demonstrated that pGM-CSF influenced the percentage of blastocyst formation and total cell number when pGM-CSF was added during Days 4 to 7 (14.6% and 53.9, respectively) or Days 0 to 7 (15.2% and 54.0, respectively) compared with the control (7.8% and 43.1, respectively) and compared with Days 0 to 3 (8.7% and 42.5, respectively). Similarly, early blastocyst formation rates were significantly higher at Days 4 to 7 than in the control, and expanded blastocyst formation was significantly higher at Days 4 to 7 or Days 0 to 7. No significant difference in cleavage rates appeared among the groups. In experiment 3, in the presence of BSA, pGM-CSF also increased the percentage of embryos that developed to the blastocyst stage and the total cell number (20.3% and 59.8, respectively) compared with the control (14.9% and 51.4, respectively), whereas there was no significant difference in cleavage rate. Experiment 4 found that the total cell number and the number of cells in the inner cell mass (ICM) were significantly increased compared with the control when zygotes were cultured in either porcine zygotic medium (PZM)-3 or PZM-4 supplemented with 10 ng/mL pGM-CSF. The number of trophectoderm (TE) cells was significantly higher in PZM-3 medium supplemented with pGM-CSF than in the control, and the number tended to increase (P = 0.058) in PZM-4 medium supplemented with pGM-CSF. The ratio of inner cell mass to trophectoderm cells was significantly higher in PZM-4 supplemented with 10 ng/mL pGM-CSF, but not in PZM-3. In experiment 5, it was found that the male pronuclear formation rate, monospermic penetration and sperm/oocyte were 95.4%, 37.2%, and 2.4, respectively. Together, these results suggest that pGM-CSF may have a physiological role in promoting the development of porcine preimplantation embryos and regulating cell viability and that addition of pGM-CSF to IVC medium at Days 4 to 7 or 0 to 7 improves the developmental potential of porcine IVF embryos.     

Paternal influence on the time of first embryonic cleavage post insemination and the implications for subsequent bovine embryo development in vitro and fertility in vivo

The objectives of this study were: (1) to evaluate the effect of sire on the time from insemination to first cleavage following insemination in vitro and the relationship of this parameter to field fertility and (2) to establish the relationship between the kinetics of cleavage in vitro and oocyte developmental competence for bulls of known field fertility. Frozen semen from six bulls with 150-day non-return rates ranging from 57-78% was used. In experiment 1, after insemination with semen from one of the six bulls, presumptive zygotes were transferred to IVC in droplets of synthetic oviduct fluid. Droplets were examined at 24, 27, 30, 33, 36, 42, and 48 hr after insemination and the number of cleaved oocytes was recorded. Blastocyst yield was recorded on Days 6-, 7-, and 8-post insemination. In experiment 2, culture droplets were examined at 30, 36, and 48 hr after insemination. At each time point, the number of cleaved embryos was recorded and these embryos were transferred into new droplets and were cultured separately for the duration of the experiment. The proportion of embryos developing to the blastocyst stage was recorded for each of the groups for each bull. The best predictor of field fertility was a model containing 33-hpi-cleavage percentage only (r = 0.689, P < 0.0001). There was also a significant correlation between blastocyst yield and non-return rate, with Day 7 blastocyst yield having the highest correlation (r = 0.356), although this was relatively low in comparison. In experiment 2, irrespective of sire, a significantly higher proportion of those early-cleaving oocytes (before 30 hpi) developed to blastocysts than those cleaving later. In most cases, a higher proportion of blastocysts derived from early-cleaving oocytes hatched from the zona pellucida suggesting that such blastocysts are of superior quality to those derived from late-cleaving oocytes. In conclusion these data confirm our earlier observations that earliest cleaving zygotes are more competent in terms of development to the blastocyst stage than those that cleave later. This phenomenon is independent of the sire used. However, we have demonstrated that the kinetics of early embryonic development as measured by the timing of the first cleavage division post insemination vary between different bulls and that these differences can be used to discriminate between bulls of high and low bull field fertility.     

In vitro development up to hatching of bovine in vitro-matured and fertilized oocytes with or without support from somatic cells

To verify the importance of somatic cells upon in vitro embryo development, in vitro-matured (IVM) and -fertilized (IVF) bovine oocytes were cultured in TCM 199 supplemented with estrous cow serum (10% v/v) and 0.25 mM sodium pyruvate (ECSTCM) under the following treatments: 1) ECSTCM alone; 2) together with bovine oviduct epithelial cells (BOEC); 3) with cumulus cells (CC); 4) in fresh BOEC conditioned ECSTCM; or 5) in frozen-thawed BOEC conditioned ECSTCM. Culturing zygotes encased in cumulus cells significantly reduced the cleavage rate (P<0.05). There was no difference between culture systems in the proportions of embryo development through the 8-cell stage (P=0.42) up to the morula/blastocyst stages (P=0.50) at Day 7 post insemination. However, co-culture with BOEC yielded the highest percentage (21.2% of zygotes; P<0.05) of quality Grade-1 and Grade-2 embryos with the number of blastomeres per embryo (114.4) comparable to that of 7-day-old in vivo-developed embryos of similar grades (102.5), and higher (P<0.05) than those of the other treatments. The ratio of blastocysts to total morulae/blastocysts obtained from frozen-thawed conditioned medium was lower (P<0.05) than that from ECSTCM or after co-culture with BOEC at Day 7 post insemination. On average, 7.5 to 17.5% of the zygotes developed to blastocyst, expanded blastocyst and hatched blastocyst stages by Day 10 post insemination, depending upon the culture system. The difference between treatments, however, was not significant (P=0.68). The results indicate that chronological development up to hatching of bovine IVM-IVF embryos is not favored by somatic cells; however, the presence of viable oviduct epithelial cells in culture significantly improves the quality of 7-day-old embryos.