Amino acid metabolism of the porcine blastocyst

The pattern of depletion and appearance of a mixture of amino acids by single porcine blastocysts incubated in two different media has been determined non-invasively using high performance liquid chromatography. Zygotes were produced by the in vitro fertilisation of in vitro-matured, abattoir-derived immature oocytes and cultured in medium NCSU 23 with or without amino acids. Embryos grown in the absence of amino acids up to the blastocyst stage were transferred to amino acid-containing culture medium for measurement of turnover (Experiment 1). Blastocysts grown in NCSU 23+amino acids were transferred into fresh droplets of the same medium (Experiment 2). Although the specific pattern of amino acid production and depletion varied between experiments, a general pattern emerged, with arginine being significantly depleted (p<0.001) and alanine consistently appearing in the media, in quantities that varied depending with culture conditions. The data suggest that arginine is important during porcine blastocyst development, most likely contributing to the formation of nitric oxide and polyamines and that alanine is produced as a means of disposing of excess amino groups. A model for the interactions of amino acids during porcine early embryo development is proposed. The profile of amino acid metabolism by porcine blastocysts is qualitatively and quantitatively similar to that given by human embryos during the morula:blastocyst transition suggesting that the porcine blastocyst is a good model for the human.     

Embryo metabolism during the expansion of the bovine blastocyst

Embryo metabolism was evaluated during re‐expansion of in vitro produced bovine blastocysts collapsed with cytochalasin D (CCD) and incubated in the presence and absence of ouabain, a specific inhibitor of the Na⁺, K⁺ pump. Day 8 expanded blastocysts were treated for 2 to 4 hr with 20 μg/ml CCD. Four conditions were tested: untreated embryos and embryos collapsed with CCD and allowed to re‐expand for 4 hr in the presence of 0 M, 1 nM, or 1 μM ouabain. Incubation of collapsed embryos for 4 hr in the presence of 1 nM or 1 μM ouabain significantly inhibited blastocyst re‐expansion. Glucose, pyruvate, and amino lactate uptake/release were not significantly affected by ouabain treatment and did not correlate with the degree of blastocyst re‐expansion. Few variations in the uptake/release of amino acids by the embryos were observed. Ouabain treatment significantly decreased oxygen uptake which directly correlated with the degree of blastocyst re‐expansion. For embryos allowed to re‐expand in the presence or absence of ouabain, a direct correlation was observed between the uptake of oxygen and of glucose. One mM cyanide or 2,4 dinitrophenol inhibited blastocyst re‐expansion although 0.01 and 0.1 mM were ineffective. This study indicates a role for oxidative metabolism in providing the energy necessary for blastocoel expansion in the bovine. Nevertheless, blastocyst expansion is relatively insensitive to inhibition of oxidative phosphorylation indicating the ability of the bovine blastocyst to adapt to hypoxic conditions. Mol. Reprod. Dev. 53:171–178, 1999. © 1999 Wiley‐Liss, Inc.     

Gene expression profiles of vitrified in vitro- and in vivo-derived bovine blastocysts

Vitrification is becoming a preferred method for pre‐implantation embryo cryopreservation. The objective of this study was to determine the differentially expressed genes of in vivo‐ and in vitro‐produced bovine embryos after vitrification. In vitro‐ (IVF) and in vivo‐derived (IVV) bovine blastocysts were identified as follows: in vitro‐produced fresh (IVF‐F), in vitro‐produced vitrified (IVF‐V), in vivo‐derived fresh (IVV‐F), in vivo‐derived vitrified (IVV‐V). The microarray results showed that 53 genes were differentially regulated between IVF and IVV, and 121 genes were differentially regulated between fresh and vitrified blastocysts (P < 0.05). There were 6, 268, 962, and 17 differentially regulated genes between IVF‐F × IVV‐F, IVF‐V × IVV‐V, IVF‐F × IVF‐V, and IVV‐F × IVV‐V, respectively (P < 0.05). While gene expression was significantly different between fresh and vitrified IVF blastocysts (P < 0.05), it was similar between fresh and vitrified IVV blastocysts. Significantly up‐regulated KEGG pathways included ribosome, oxidative phosphorylation, spliceosome, and oocyte meiosis in the fresh IVF blastocyst samples, while sphingolipid and purine metabolisms were up‐regulated in the vitrified IVF blastocyst. The results showed that in vitro bovine blastocyst production protocols used in this study caused no major gene expression differences compared to those of in vivo‐produced blastocysts. After vitrification, however, in vitro‐produced blastocysts showed major gene expression differences compared to in vivo blastocysts. This study suggests that in vitro‐produced embryos are of comparable quality to their in vivo counterparts. Vitrification of in vitro blastocysts, on the other hand, causes significant up‐regulation of genes that are involved in stress responses. Mol. Reprod. Dev. 79: 613–625, 2012. © 2012 Wiley Periodicals, Inc.     

Beneficial effects of serum supplementation during in vitro production of porcine embryos on their ability to survive cryopreservation by open pulled straw vitrification

The ability of porcine blastocysts produced in vitro, in the presence or absence of serum, to survive cryopreservation was investigated in this experiment. Porcine oocytes were matured, fertilized and cultured in vitro using serum-free culture systems. Starting at Day 4 of in vitro embryo culture (Day 0 = fertilization), the culture medium was supplemented with 10% fetal bovine serum (FBS). Embryos were cultured under these conditions until Day 6. Embryos cultured with only BSA supplementation served as serum-free controls. Day 6 blastocysts and expanded blastocysts of excellent quality were vitrified using the open pulled straw method. After warming, blastocysts were cultured in the presence of 10% FBS for an additional 18 h to recover. Portions of blastocysts from both groups, without cryopreservation, were also cultured under the same conditions to serve as non-vitrified controls. To further investigate the influence of FBS on the quality of embryos produced, the total cell numbers in Day 6 blastocysts from both groups were compared. In addition, the ratio of viable to total cells in fully recovered blastocysts at each group was examined. Blastocysts produced in the presence of FBS had an increased ability to survive cryopreservation and also had a higher cell number compared to those produced in serum-free systems (P < 0.05). The fully recovered blastocysts had a normal viable to total cell ratio, compared to non-vitrified controls. Overall, this experiment supports the hypothesis that serum supplementation during in vitro production of porcine embryos is beneficial to the ability of a blastocyst to survive cryopreservation.     

A comparative study on developmental capacity to blastocysts derived from 1-and 2(3)-cell bovine embryos after in vitro maturation and fertilization

The present study was conducted to compare the developmental capacity of 1-and 2(3)-cell embryos after 18 and 30 h of fertilization, and blastocyst cell number and in vitro survival after freezing and thawing of bovine blastocysts derived from the 1-and 2-cell embryos. Oocytes were matured and fertilized by conventional IVM/IVF methods. After 18 or 30 h of fertilization, 1-cell embryos (18 h-fertilization) or 1- and 2(3)-cell embryos (30 h-fertilization) were cultured for 8 or 10 d in synthetic oviduct fluid medium (SOFM) supplemented with 10% human serum (HS), minimum essential medium (MEM) essential or nonessential amino acids and glutamine. The separate culture of 1- and 2(3)-cell embryos after 30 h of fertilization showed higher (p < 0.01) cleavage, development to expanded and hatched blastocysts than culture of 1-cell embryos after 18 h of fertilization. Two-cell embryos of 30 h-fertilization group had higher developmental capacity to expanded and hatched blastocysts than 1-cell embryos at 18 or 30 h after insemination (Experiment I). However, there was no significant difference in the mean cell number of blastocysts derived from the culture of 1-cell and 2(3)-cell embryos, respectively (Experiment II). The in vitro survival or hatching after freezing and thawing of blastocysts was significantly affected by embryonic quality before freezing, but did not significantly differ with blastocysts derived from 1- and 2(3)-cell embryos after 18 or 30 h of fertilization. The results indicate that the culture of 2(3)-cell embryos after 30 h of fertilization is an effective method to produce more transferable embryos (blastocysts) in bovine IVM, IVF and IVC techniques.     

Influence of ovine oviducal amino acid concentrations and an ovine oestrus-associated glycoprotein on development and viability of bovine embryos

This study examined the effects of incorporating an ovine oviducal oestrus-associated glycoprotein (oEGP) and amino acids, at the concentrations present in the ovine oviduct around the time of oestrus, on in vitro production and subsequent viability of bovine embryos. The first experiment compared the influence of ovine oviducal concentrations of amino acids with MEM and BME amino acids. There was no treatment effect on cleavage rate (74.9% vs. 75.5%), but there was a higher (P < 0.05) blastocyst yield (30.4 vs. 25.2) and a shorter time (P < 0.05) to blastocyst formation (7.16 ± 0.64 vs. 7.27 ± 0.56 days) following use of oviducal concentrations of amino acids. Experiment 2 examined the influence of oEGP in combination with each of the amino acid treatments. oEGP had no effect on cleavage or blastocyst yield within amino acid treatments. Day of blastocyst formation significantly influenced nuclei numbers (P < 0.001) with higher numbers being obtained on day 7 than on either day 6 or day 8. There was also a significant (P < 0.01) interaction between day of blastocyst formation and amino acid treatment on blastocyst nuclei numbers. The third experiment studied the effects of the amino acid treatments on embryo viability. There was no effect of amino acid treatment of embryos on pregnancy rates (34.5 vs. 44.4%) following transfer of days 6 and 7 blastocysts to synchronized recipients. oEGP did not influence any of the parameters of bovine embryo development that were measured, suggesting that effects of this protein observed on ovine embryos are species specific. It is concluded that ovine oviducal amino acid concentrations are beneficial to blastocyst development in vitro but do not have any further beneficial effect following transfer of blastocysts to recipients. Mol. Reprod. Dev. 47:164–169, 1997. © 1997 Wiley-Liss, Inc.     

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.     

Development and subsequent cryotolerance of domestic cat embryos cultured in serum-free and serum-containing media

The objectives of this study were to examine the effects of the presence or absence of serum during the in vitro culturing period of domestic cat embryos on their developmental potential into blastocysts as well as their tolerance to cryopreservation using a slow-freezing method. In vitro-fertilized cat oocytes were incubated in a modified synthetic oviduct fluid (mSOF) containing 4 mg/mL bovine serum albumin (BSA) throughout culturing (BSA group) or in mSOF containing 4 mg/mL BSA for the first 3 days followed by mSOF containing 5% fetal bovine serum (FBS group). The developmental potential of the embryos to the blastocyst and expanded blastocyst stages was evaluated 7 days after in vitro fertilization. The blastocysts were frozen-thawed by the slow-freezing method and cultured for 3 days to examine their viability in vitro. There were no differences in the formation rates of blastocysts or expanded blastocysts, or number of cells in the embryos between the two groups. After cryopreservation, the hatching rates of the expanded blastocysts in the BSA group were significantly higher (P < 0.05) than those of the FBS group. The postthaw viability of blastocysts was lower than that of expanded blastocysts irrespective of culture medium. These results indicate that the developmental potential of cat embryos cultured in serum-free medium is comparable to those cultured in serum-containing medium. Furthermore, expanded blastocysts produced without serum exhibit better postthaw viability than those produced with serum.     

Triglyceride content of bovine oocytes and early embryos

A microfluorescence technique was used to measure the triglyceride content of a minimum of two bovine oocytes or preimplantation embryos up to the hatched blastocyst stage. Embryos were produced in vitro from abattoir-derived ovaries and grown in medium containing synthetic oviductal fluid, amino acids and BSA (SOFaaBSA medium); 10% fetal calf serum was added to some of the embryos at the four-cell stage. Before maturation, the triglyceride content of oocytes was 59 +/- 1.37 ng and it decreased (P < 0.05) after maturation to 46 +/- 0.85 ng. A decrease in triglyceride content (P < 0.05) was also observed after fertilization with the formation of the two-cell embryo (34 +/- 1.80 ng). In the absence of serum, the triglyceride content remained relatively constant from the two-cell to the hatched blastocyst stage. The triglyceride content of blastocysts produced in vivo was similar (33 +/- 0.70 ng) to that of blastocysts produced in vitro in the absence of serum. In contrast, the triglyceride content of embryos grown with 10% fetal calf serum increased steadily from the 9-16-cell stage to a value in hatched blastocysts (62 +/- 1.14 ng) almost double that in serum-free conditions. These results indicate that triglyceride may act as energy source during bovine oocyte maturation and fertilization and that the presence of serum causes excessive synthesis or accumulation of triglyceride in early embryos.     

Protein-free culture medium containing polyvinylalcohol, vitamins, and amino acids supports development of eight-cell hamster embryos to hatching blastocysts

In vitro development of eight-cell hamster embryos to hatching blastocysts requires the presence of amino acids and a group of water-soluble vitamins in the culture medium. The present studies investigated the effect of type of macromolecule on blastocyst hatching and on the requirement for vitamins. Embryos were cultured for 3 days in the presence of the synthetic macromolecule polyvinylalcohol (PVA) and of different types of bovine serum albumin (BSA), both with and without vitamins. The results showed th at eight-cell embryos develop to hatching blastocysts in the presence of vitamins and amino acids with PVA as the only macromolecule in the medium. The presence of certain types of BSA reduced but did not eliminate the need for vitamins. Glutamine alone was as efficient as a complete amino acid supplement in supporting blastocyst hatching. These results demonstrate for the first time that eight-cell hamster embryos can be cultured to hatching blastocysts in a chemically defined medium.     

Development, freezability and amino acid consumption of bovine embryos cultured in synthetic oviductal fluid (SOF) medium containing amino acids at oviductal or uterine-fluid concentrations

In this study, we examined the development, freezability and amino acid consumption of in vitro produced bovine embryos cultured in a chemically defined medium (SOF+polyvinyl alcohol), supplemented with 24 amino acids at concentrations measured in bovine oviductal or uterine fluid. Amino acids at concentrations in oviductal fluid tested by Elhanssan (EOAA) significantly improved development to the hatched blastocyst stage, compared to Sigma amino acid solutions BME and MEM (SAA). Amino acids at concentrations in uterine fluid tested by Li (LUAA) were not compared to SAA, and development in LUAA was not significantly different from development in EOAA. Amino acids at concentrations in uterine fluid tested by Elhanssan (EUAA) significantly reduced cleavage rate and blocked further embryo development. When the IVF embryos were cultured in EOAA for 48, 72, 96, or 120 h and then transferred to LUAA, blastocyst and hatched blastocyst rates were not significantly affected. The freezability of blastocysts cultured in EOAA for the first 72 h and then moved to LUAA was improved compared to that in SAA. During the 1-8-cell stages, embryos secreted all 23 amino acids (total, 6,368 pmol/embryo). During the 8-cell to morula stages, embryos continued to secrete 21 amino acids (total, 2,495 pmol/embryo), meanwhile embryos began to absorb Arg (70 pmol/embryo) and Gln (18 pmol/embryo). After the morula stage, embryos began to absorb 15 amino acids including Glu, Gly, Arg, and Gln (total, 2,742 pmol/embryo) and secreted eight amino acids (total, 1,616 pmol/embryo). Embryos absorbed only Arg (183 pmol/embryo) and secreted the other 22 amino acids (total, 3,697 pmol/embryo) when the culture medium was not changed during the entire culture period (zygote to blastocyst).     

Effect of estrous cow serum during bovine embryo culture on blastocyst development and cryotolerance after slow freezing or vitrification

The present study investigated the effect of estrous cow serum (ECS) during culture of bovine embryos on blastocyst development and survival after cryopreservation by slow freezing or vitrification. Embryos were derived from in vitro maturation (IVM) and in vitro fertilization (IVF) of abbatoir-derived oocytes. At Day 3, embryos were cultured in three different media: Charles Ronsenkrans medium + amino acids (CR1aa; without bovine serum albumin (BSA)) + 5% estrous cow serum (CR1-ECS), CR1aa + 3 mg/mL BSA (CR1-BSA) or CR1aa + 5% ECS + 3 mg/mL BSA (CR1-ECS-BSA). At 7.5 d post-insemination (PI), blastocyst yield and quality were evaluated; blastocysts and expanded blastocysts from each media were cryopreserved by Open Pulled Straw (OPS) vitrification method or slow freezing (1.5 M ethylene glycol, EM). Total blastocyst yield did not differ among CR1-ECS, CR1-BSA and CR1-ECS-BSA (30.9, 33.1 and 32.9%, respectively, P < 0.05). Embryo survival (hatching rate) was higher in vitrified versus slow-frozen embryos (43% versus 12%, respectively, P < 0.01), and in embryos cultured in CR1-BSA (40.3%) compared with those cultured in serum-containing media (CR1-ECS, 21.5% and CR1-ECS-BSA, 19.8%; P < 0.01). In conclusion: (a) it was possible to produce in vitro bovine embryos in serum-free culture medium without affecting blastocyst yield and quality; (b) serum-free medium produced the best quality embryos (in terms of post-cryopreservation survival); and (c) vitrification yielded the highest post-cryopreservation survival rates, regardless of the presence of serum in the culture medium.     

Promoting effect of amino acids added to a chemically defined medium on blastocyst formation and blastomere proliferation of bovine embryos cultured in vitro

This study was conducted to elucidate the role of amino acids added singly or in groups to a chemically defined culture medium in blastocyst formation and blastomere proliferation of bovine embryos. Embryos were generated by in vitro fertilization, and blastocyst formation and hatching, and blastomere number of blastocysts were subsequently monitored after the culture of embryos in synthetic oviduct fluid medium (SOFM). First, one of four non-essential amino acids (asparagine, aspartate, glutamate or serine) was added to SOFM and, compared with no addition, a significant (P <0.05) increase in blastocyst formation was found after the addition of asparagine, aspartate, or glutamate (35-42% versus 22%). Second, one of four essential amino acids (arginine, cystine, isoleucine or leucine) was added and arginine or isoleucine greatly improved blastocyst formation (30-36% versus 16%). Third, the addition of five stimulatory amino acids (aspartate, asparagine, glutamate, arginine and isoleucine) to SOFM significantly improved blastocyst formation compared with no addition (12% versus 21%) and such value was similar to that obtained after the addition of 19 amino acids consisting of MEM amino acid solutions (21-27%). However, five amino acids yielded fewer hatched blastocysts than 19 amino acids. Finally, although five amino acids yielded more cell number of blastocysts than no addition (93 versus 74 cells per blastocyst), it was lower than that from 19 amino acids (131 cells per blastocyst). In conclusion, either single or combined addition of asparagine, aspartate, glutamate, arginine and isoleucine stimulated blastocyst formation, while other amino acids might be necessary for further stimulating blastomere proliferation and blastocyst hatching.     

Bovine blastocyst development in vitro: timing, sex, and viability following vitrification

Selection of blastocysts based on their morphological characteristics and rate of development in vitro can skew the sex ratios. The aim of this study was to determine whether an embryo's developmental rate affects its survival after vitrification, and whether male and female embryos survive vitrification differently. In vitro fertilized bovine oocytes were cultured in potassium simplex optimized medium (KSOM) + 0.1% BSA for 96 h, and then into KSOM + 1% BSA (KSOM) or in sequential KSOM + 0.1% BSA for 96 h, and then into synthetic oviduct fluid (SOF) + 5% FBS (KSOM-SOF). In part 1 of this study, embryos cultured in each medium that had developed into blastocysts at approximately 144, 156, or 180 h were recovered from culture, graded, and then vitrified. After warming, blastocyst survival rates were immediately evaluated by reexpansion of the blastocoels. In the second part of the study, all blastocysts (n = 191) were sexed by polymerase chain reaction 48 h after warming. When cultured in KSOM medium, more 144-h blastocysts survived vitrification (68%) than blastocysts vitrified at 180 h (49%). Blastocysts derived at 156 h in KSOM-SOF survived vitrification better (87%) than blastocysts vitrified at either 144 h or 180 h, and subsequently hatched at a greater rate than those vitrified at 180 h. The overall blastocyst survival rates did not differ significantly whether embryos were cultured in KSOM or sequential KSOM-SOF. Blastocysts derived at 144 and 156 h in KSOM or KSOM-SOF were predominately male, and significantly more of them survived vitrification 48 h after warming. However, blastocysts cultured in KSOM-SOF, and then vitrified at 180 h were predominately female. Overall, blastocysts that survived vitrification, and subsequently hatched 48 h after warming, were male. In summary, embryos that reached the blastocyst stage earlier were predominantly males; these males had better morphology, endured vitrification, and subsequently hatched at a greater rate than did female blastocysts.     

Ultrastructural morphometry of bovine blastocysts produced in vivo or in vitro

The objective of this study was to compare the ultrastructure of bovine blastocysts produced in vivo or in vitro by using morphometric analysis. Blastocysts produced in vivo (multiple ovulations, MO) were obtained from superovulated Holstein cows. For blastocysts produced in vitro, cumulus-oocyte complexes aspirated from ovaries of Holstein cows were matured and fertilized in vitro. At 20 h postinsemination (hpi), zygotes were distributed into one of three culture media: 1) IVPS (in vitro produced with serum): TCM-199 + 10% estrous cow serum (ECS); 2) IVPSR (in vitro produced with serum restriction): TCM-199 + 1% BSA until 72 hpi, followed by TCM-199 + 10% ECS from 72 to 168 hpi; and 3) mSOF (modified synthetic oviductal fluid): mSOF + 0.6% BSA. At 168 hpi, six or seven grade 1 blastocysts from each of the four treatments (MO, IVPS, IVPSR, and mSOF) were fixed and prepared for transmission electron microscopy. Random micrographs of each blastocyst were used to determine the volume density of cellular components. Overall, as blastocysts progressed in development, the volume densities of cytoplasm and intercellular space decreased (P < 0.05) and the volume densities of mature mitochondria, nuclei, blastocoele, and apoptotic bodies increased (P < 0.05). Across treatments, the proportional volumes of nuclei and inclusion bodies were increased in inner cell mass cells compared with trophectoderm cells for mid- and expanded blastocysts. For blastocysts produced in vitro, the volume density of mitochondria was decreased (P < 0.05) as compared with that of blastocycts produced in vivo. The proportional volume of vacuoles was increased (P < 0.05) in blastocysts from the mSOF treatment as compared with blastocysts produced in vivo. For mid- and expanded blastocysts from all three in vitro treatments, the volume density of lipid increased (P < 0.05) and the volume density of nuclei decreased (P < 0.05) compared with those of blastocysts produced in vivo. In conclusion, blastocysts produced in vitro possessed deviations in volume densities of organelles associated with cellular metabolism as well as deviations associated with altered embryonic differentiation. However, the specific nature of these deviations varied with the type of culture conditions used for in vitro embryo production.     

Differences in the incidence of apoptosis between in vivo and in vitro produced blastocysts of farm animal species: a comparative study

The occurrence of pregnancies and births after embryo transfer (ET) of in vivo produced embryos is generally more successful compared to that of embryos produced in vitro. This difference in ET success has been observed when embryos of morphological equal (high) quality were used. The incidence of apoptosis has been suggested as an additional criterion to morphological embryo evaluation in order to assess embryo quality and effectively predict embryo viability. In this study, equine, porcine, ovine, caprine and bovine in vivo and in vitro produced morphologically selected high quality (grade-I) blastocysts were compared for the occurrence of apoptosis in blastomeres. The total number of cells per embryo and the number of cells with damaged plasma membranes, fragmented DNA and fragmented nuclei per embryo were assessed in selected blastocysts by combining Ethidium homodimer (EthD-1), terminal dUTP nick end labeling (TUNEL) and Hoechst 33342 staining. In general, the level of blastomere apoptosis was low. A higher level of apoptosis was observed in in vitro produced equine, porcine and bovine blastocysts compared to their in vivo counterparts. Interestingly, 4 of the initially selected 29 bovine in vitro produced blastocysts exhibited extensive signs of apoptosis affecting the inner cell mass (ICM), which is not compatible with a viable conceptus. Repeated occurrence of this observation may explain the lower ET outcome of in vitro produced bovine embryos compared to in vivo produced embryos. It is concluded that, although in morphologically high quality blastocysts of several farm animal species a significant difference exists in the percentages of apoptotic cells between in vivo and in vitro produced embryos, the incidence of apoptosis at the blastocyst stage is at such a low level that it cannot reflect the substantial differences in embryo viability that have been described between in vivo and in vitro produced blastocysts following ET.     

Development and subsequent cryotolerance of domestic cat embryos cultured in serum-free and serum-containing media

The objectives of this study were to examine the effects of the presence or absence of serum during the in vitro culturing period of domestic cat embryos on their developmental potential into blastocysts as well as their tolerance to cryopreservation using a slow-freezing method. In vitro-fertilized cat oocytes were incubated in a modified synthetic oviduct fluid (mSOF) containing 4 mg/mL bovine serum albumin (BSA) throughout culturing (BSA group) or in mSOF containing 4 mg/mL BSA for the first 3 days followed by mSOF containing 5% fetal bovine serum (FBS group). The developmental potential of the embryos to the blastocyst and expanded blastocyst stages was evaluated 7 days after in vitro fertilization. The blastocysts were frozen-thawed by the slow-freezing method and cultured for 3 days to examine their viability in vitro. There were no differences in the formation rates of blastocysts or expanded blastocysts, or number of cells in the embryos between the two groups. After cryopreservation, the hatching rates of the expanded blastocysts in the BSA group were significantly higher (P < 0.05) than those of the FBS group. The postthaw viability of blastocysts was lower than that of expanded blastocysts irrespective of culture medium. These results indicate that the developmental potential of cat embryos cultured in serum-free medium is comparable to those cultured in serum-containing medium. Furthermore, expanded blastocysts produced without serum exhibit better postthaw viability than those produced with serum.     

Development of OPS vitrified pig blastocysts: effects of size of the collected blastocysts, cryoprotectant concentration used for vitrification and number of blastocysts transferred

Unhatched blastocysts from Large White hyperprolific gilts (n=103) were identified, measured and vitrified using the Open Pulled Straw (OPS) technique to evaluate the effects of the collected blastocyst size and cryoprotectant concentrations used for vitrification, and the number of embryos transferred per recipient. Vitrified/warmed blastocyst viability was estimated in vitro, as the percentage of embryos developing after 72h, and in vivo, on pregnancy Day 30. In the in vitro study, we compared the use of three cryoprotectant concentrations (16.5, 18, or 20% DMSO+16.5, 18, or 20% EG+0.4M sucrose). Survival rates differed significantly between the control (98.3%) and the three cryoprotectant concentrations (67, 62.3, and 57%, respectively). Blastocyst size at vitrification determined the further in vitro development of embryos (26% survival for blastocysts 126-144microm versus 100% for blastocysts >199microm). For the in vivo study, blastocysts were vitrified using cryoprotectant concentrations of 16.5 or 18% DMSO+EG and transferred surgically in groups of 20 or 30 per recipient (n=40). Recipients were slaughtered on pregnancy D30. No significant differences were detected in gestation rates (50-70%) and embryo survival rates (14.7-25%), although survival was higher (P=0.0003) when 20 blastocysts were transferred compared to 30 (24.7% versus 15.5%). Our findings indicate that best results, in terms of subsequent in vivo embryo survival, were achieved after transferring 20 embryos at the blastocyst or expanded blastocyst stage, previously vitrified using cryoprotectant concentrations of 16.5 or 18%.     

Is apoptosis in bovine in vitro produced embryos related to early developmental kinetics and in vivo bull fertility?

Although several studies have indicated a paternal effect on bovine embryo development, no conclusive data exist on the effect of in vivo bull fertility on apoptosis. Therefore, it was the main objective of this study to compare the apoptotic cell ratio (ACR) in embryos originating from bulls with different in vivo fertility. However, since it is has been demonstrated before that bulls with different in vivo fertility differ in timing of first cleavage, it was necessary to investigate first the effect of timing of development on apoptosis in vitro in order to get an unbiased insight in the contribution of in vivo bull fertility on apoptosis in bovine blastocysts. In the first experiment, bovine embryos (n = 939) were allocated to different groups according to cleavage rate at 30, 36 and 48 hpi and blastocysts were selected at 7 and 8 dpi. The blastocyst rate at 7 dpi was significantly lower in embryos which had first cleaved at 48 hpi than in embryos from the 30 and 36 hpi group (P < 0.05). The ACR after TUNEL in day 7 blastocyst was significantly lower in the 30 hpi group in comparison with the 36 and 48 hpi group (P < 0.05) and lower in day 7 blastocysts than in day 8 blastocysts. In the second experiment, sperm of eight bulls with different non return rates was used for in vitro bovine embryo production (n = 3820 oocytes). Cleavage rates (30, 36 and 48 hpi) and blastocyst rate (7 dpi) were determined. Only very low negative correlations could be found between in vivo and in vitro bull fertility and ACR did not differ between groups derived from sires with either low or normal fertility (P > 0.05). Further research in serum free conditions is needed to confirm that the lower ACR in early cleaved embryos could be mediated by the cooperative interaction of embryos of good quality cultured in group. In vivo bull fertility could hardly be correlated with in vitro blastocyst yield and could not be correlated with appearance of apoptosis.     

Nonelectrophoretic PCR-sexing of bovine embryos in a commercial environment

Techniques for sex determination of bovine embryos have evolved from karyotyping of older preimplantation embryos some 25 years ago to the current variety of widely used polymerase chain reaction (PCR) protocols. Although highly accurate, most PCR protocols for sex determination have included an electrophoresis step. The present work is a retrospective study utilizing a unique PCR protocol to sex bovine embryos without use of electrophoresis in a commercial embryo transfer program. Both in vivo and in vitro-derived embryos were produced by conventional techniques and biopsied between 7 and 8 days of age with a steel blade attached to a mechanical micromanipulator. Males constituted 49.0% of 3964 in vivo and 53.0% of 1181 in vitro-derived embryos subjected to PCR. Based on ultrasound fetal sexing and on calvings, the accuracy of sex determination was 98.7% for male embryos and 94.4% for females, with no samples producing an undetermined outcome. Pregnancy rates following transfer of biopsied Grade 1 embryos were lower than control, intact embryos as follows: 8, 6 and 16% points for in vivo, in vitro and in vivo frozen embryos, respectively. Pregnancy rates were similar for all stages of in vivo-derived embryos, whereas the pregnancy rate was significantly lower for in vitro-derived morulae compared to all stages of blastocysts. The sex ratio was significantly skewed in favor of females among in vitro-derived morulae, and in favor of males among in vitro expanded blastocysts. The sex ratio of in vivo expanded blastocysts was significantly skewed in favor of female embryos. No seasonal variation in either pregnancy rate or sex ratio was detected. There was no evidence that DNA contamination influenced the PCR assay during the duration of the study. The assay was sensitive to single blastomeres from male embryos, whereas it was not sensitive to Percoll-centrifuged or accessory sperm cells.