Development Of Ovine Embryos Research Articles

Low Serum Concentration in Ovine Embryo Culture Media

Background: Fetal bovine serum (FBS) is rich in nutrients, growth factors, hormones, and other essential compounds necessary for cell growth. However, its use has several disadvantages. Platelets in particular contain the majority of the potent mitogenic factors found in serum. Objectives: The present study aimed to evaluate the effect of platelet lysate (PL) on in vitro embryo production by replacing FBS, to determine if this modified Charles Rosenkrans medium (mCR2aa) can support ovine embryo development compared to the commercially available BO-IVC medium. Methods: In the first experiment, varying concentrations of FBS (0%, 2.5%, 5%, and 10%) and PL (0%, 2.5%, 5%, and 10%) were investigated in the mCR2aa culture medium. In the second experiment, the optimized mCR2aa medium was compared to the BO-IVC medium. Ovine oocytes were matured and fertilized in vitro using BO-IVM Bioscience medium and Brackett and Oliphant medium, respectively. Subsequently, the resulting zygotes were cultured in either mCR2aa medium supplemented with amino acids or BO-IVC medium, according to the experimental design, under a humidified atmosphere of 5% CO2, 5% O2, and 90% N2 at 38.5ºC. Results: The results indicated that using 5% PL in the mCR2aa culture medium reduced the serum percentage to 2.5%. Conclusions: It was not possible to completely eliminate serum from the mCR2aa medium solely by substituting it with PL. Further studies are needed to explore other serum properties such as chelating agents and antioxidant activities to fully optimize the mCR2aa medium without serum.

Effect of Stainless Steel Tank Incubation System on Development of Ovine Embryos

Effect of Stainless Steel Tank Incubation System on Development of Ovine Embryos

Interleukin-6 stimulates in vitro development of late-stage ovine embryos

The effect of interleukin-6 (IL-6) supplementation during the different phases of in vitro embryo culturing (IVC) on embryo development and embryonic gene expression was studied in ovine. IL-6 was added to IVC medium during the late phases (72–192 h; 5, 10, and 25 ng/ml IL-6) or entire period (0–192 h; 10 ng/ml IL-6) of IVC to determine its effect on embryo development. Further, the effect of IL-6 (10 ng/ml) supplementation at the 72 h of IVC on gene expressions associated with JAK/STAT signalling and pluripotency in 8–16 cell embryos (1 h post-supplementation) and compact morulae (48 h post-supplementation), and apoptosis and primitive endoderm (PrE) development in compact morulae was investigated. The supplementation of 10 ng/ml IL-6 during the late phases of IVC significantly (P < 0.05) increased blastocyst formation (35.2 ± 1.52%) compared to the control (21.1 ± 1.11%), and 5 ng/ml (25.9 ± 2.98%) or 25 ng/ml (16.5 ± 0.73%) IL-6 groups. Conversely, IL-6 (10 ng/ml) treatment throughout the IVC period significantly (P < 0.05) decreased the rate of cleavage (55.4 ± 1.57%) and blastocyst formation (14.5 ± 1.28%) compared to the control group (65.8 ± 1.35% and 21.5 ± 0.97%, respectively). In 8–16 cell embryos and compact morulae, the IL-6 treatment significantly (P < 0.05) affected the expression of genes associated with JAK/STAT signalling and pluripotency. Further, the treatment significantly (P < 0.05) downregulated BAX and CASP3, and upregulated GATA6 expression in compact morulae. In conclusion, IL-6 supplementation affected the in vitro development of ovine embryos in a dose- and time-dependent manner. The beneficial effect of IL-6 on the development of late-stage embryos was mediated through the changes in gene expressions associated with JAK/STAT signalling, pluripotency, apoptosis and PrE development.

Timing of cleavage divisions determined with time-lapse imaging is linked to blastocyst formation rates and quality of in vitro-produced ovine embryos

Time-lapse (TL) imaging provides a practical and safe tool to constantly monitor the development of in vitro-derived embryos. TL may help develop novel methods of predicting the timing of embryo cleavage that will lead to optimizing blastocyst cryopreservation or transfer. The primary objective of the present study was to employ TL imaging to examine associations among the division kinetics of ovine embryos, their quality and rates of development to the blastocyst stage. Oocytes were collected by ovary scarification from 78 Longwool ewes slaughtered in the breeding season (November–March). Cumulus oocyte complexes (COCs) were matured for 24 h in TCM 199 media containing 0.1 IU/mL LH/FSH and 10% FBS. In-vitro fertilization was carried out by co-incubation of semen and COCs for 19 h. Presumptive zygotes were placed in microwells, in droplets of Cult medium (Gynemed, Lensahn, Germany). Digital images of developing embryos were captured every 10 min by Primo Vision TL system (EVO+; Vitrolife, Göteburg, Sweden). The following time intervals were recorded: from IVF to the attainment of two-cell (t2), three-cells (t3) or four-cell (t4) stage, to morula detection (tM), blastulation (tSB) and blastocyst formation (tB). Lastly, the duration of the second cell cycle (cc2; t3-t2) and complete synchronous cell division (s2; t4-t3) were calculated, and the incidence of developmental anomalies noted. Out of 147 embryos selected for TL observations, 55 (37.4%) developed to the blastocyst stage (normally developing embryos, NE) and 92 (62.6%) failed to reach the blastocyst stage (arrested embryos, AE; P < 0.05). Mean t2, tM, s2 and cc2 were all less (P ≤ 0.02) in NE compared with AE. Approximately 61.9% of embryos exhibited developmental anomalies (35.5% in the NE group and 78.2% in the AE group; P < 0.05) and AE exceeded (P < 0.05) NE in the proportion of FRG (blastomeric fragmentation), IRR (blastomeres of irregular size after cleavage), DC (direct cleavage) and MA (multi-morphological aberrations). Of all NE, 63.6% were classified as good quality and 36.4% as poor quality blastocysts (P < 0.05). Good quality ovine blastocysts attained t2, t3, t4, tSB and tB stages earlier (P ≤ 0.03) than poor quality blastocysts and none of the poor quality blastocysts was seen to hatch. To recapitulate, the present results indicate that the kinetics of early ovine embryo development are significant predictors of their potential to develop to the blastocyst stage and the markers of blastocyst quality. Time-lapse imaging may serve as a useful technique for predicting the outcome and enhancing efficacy of in vitro embryo production in sheep.

Differential influence of ovine oviduct ampullary and isthmic derived epithelial cells on in vitro early embryo development and kinetic

Several critical physiological procedures occurred in oviduct during early embryo development. It is now well documented that in vivo there exist a great cross talk between early embryos and oviductal epithelial cells. For many years, the use of somatic cells for coculture with embryos has been investigated to improve in vitro embryo production (IVEP). The objectives of this study was to investigate; (i) whether ampullary and isthmic derived epithelial cells have different effects on IVEP, (ii) if by using different segments (2 equal segments) of isthmus in a sequential coculture system (2×2 Days culture period) we could improve IVEP outcomes, and (iii) whether the different coculture systems accelerate the kinetics of blastocyst development. Results of the present study confirmed the positive and highly specialized effects of isthmic compared to ampullary derived epithelial cells on in vitro early development of ovine embryos (45.42%±4.01 vs. 36.62%±4.75 expanded blastocysts, 18.26%±1.00 vs. 10.22%±2.75 hatched blastocysts). The usage of isthmic derived epithelial cells as feeder cells in a coculture system compare to ampullary led to a significant (P<0.05) increase in the total cells number of blastocysts (290.88±68.76 vs. 190.22±55.65). The kinetics of blastocyst development showed a significant increase in coculture system by using isthmic epithelial cells as the feeder cells compare to ampullary derived epithelial cells (25.62%±3.05 vs. 12.42%±1.00 blastocysts at day 6). Our results showed that by using different segments of isthmus in a sequential coculture system as the feeder cells did not improve the IVEP parameters concerning development or kinetic. In conclusion, we found for the first time that oviductal epithelial cells play the highly specialized role in vitro, same as their in vivo counterparts, in terms of development rate and kinetic.

Enhanced in vitro developmental competence of sheep embryos following sericin supplementation of the in vitro maturation and in vitro culture media

The objective was to determine the effects of various sericin concentrations (0, 0.1, 0.5, 1, and 2.5%) during in vitro oocyte maturation (IVM) and in vitro embryo culture (IVC) on ovine embryo development. Pubertal Sanjabi ewes’ oocytes were aspirated from slaughterhouse derived ovaries and matured in vitro. Semen was collected from a healthy fertile ram, capacitated and the in vitro matured oocytes were fertilized using capacitated spermatozoa. The IVM and IVC media were supplemented with 0, 0.1, 0.5, 1 and 2.5% of sericin. Addition of 0.5% of sericin during IVM and IVC significantly increased cleavage and blastocyst rates (85.48±1.57 and 50.07±3.04, respectively) compared with 0 and 0.1% of sericin. However, a too high concentration (2.5%) of sericin during IVM and IVC decreased cleavage and blastocyst rates compared with low concentrations (P<0.05). In conclusion, appropriate concentrations (0.1 and 0.5%) of sericin promoted ovine blastocyst formation in vitro, although a high concentration of sericin suppressed embryo development.

Effects of ghrelin on developmental competence and gene expression of in vitro fertilized ovine embryos

The objective was to determine the effects of various ghrelin concentrations (0, 10, 50, and 250 ng/mL) during in vitro oocyte maturation (IVM) and in vitro embryo culture (IVC) on ovine embryo development and expression patterns of genes involved in transcription regulation (OCT4), glucose transport (GLUT1), pregnancy recognition (IFNT), and ghrelin receptor (GHSR-1a). Rates of cleavage and blastocyst formation were decreased when oocytes were matured with 250 ng/mL ghrelin compared with 0, 10, or 50 ng/mL ghrelin (P < 0.05). Addition of 50 ng/mL ghrelin during IVC or during IVM and IVC significantly increased blastocyst rates (35.3% and 36.7% vs. 26.9% and 26.8%, respectively) and total cell numbers per blastocyst (110.4 and 108.3 vs. 81.2 and 77.4) compared with 0 and 10 ng/mL ghrelin. However, a high concentration (250 ng/mL) of ghrelin during IVM and IVC decreased cleavage, blastocyst rate, and total cell number of blastocyst compared with low concentrations (P < 0.05). Relative abundances of GLUT1 and IFNT transcripts were higher in blastocysts treated with 50 ng/mL ghrelin during IVC compared with other concentrations (P < 0.05). Expression of GHSR-1a was higher when 10 ng/mL ghrelin was added during IVM (0.079) or during IVM and IVC (0.053) compared with other treatments. However, addition of ghrelin at higher concentrations (50 or 250 ng/mL) reduced relative abundances of GHSR-1a transcripts (0.032 and 0.039, P < 0.05). In conclusion, appropriate concentrations of ghrelin promoted ovine blastocyst formation in vitro and increased expression of GLUT1, IFNT, and GHSR-1a genes in blastocysts, although a high concentration of ghrelin suppressed embryo development.

130 EFFECT OF HYALURONIC ACID SUPPLEMENTATION ON OVINE EMBRYO DEVELOPMENT IN VITRO AND ON SURVIVAL AFTER VITRIFICATION

Studies have shown that supplementation with hyaluronic acid (HA), a glycosaminoglycan found in mammalian follicular, oviduct, and uterine fluids, improves in vitro development and post-thaw survival of bovine embryos. In this study, we examined the effect of HA supplementation on ovine embryo development and on survival after vitrification using the minimum volume cooling (MVC) cryotop method. Abattoir sourced ovine oocytes were in vitro matured and fertilized as per routine procedures (Walker et al. 1996 Biol. Reprod. 55, 703–708). In Experiment 1 (5 replicates), presumptive zygotes were randomly allocated to IVC medium supplemented with 0.8 mg mL–1 BSA, amino acids, and 0, 0.5, 1.0, 1.5 or 2.0 mg mL–1 HA. Cleavage rates were recorded and blastocyst development evaluated on Day 7 (Day 0 = day of IVF). In Experiment 2 (3 replicates), presumptive zygotes were placed in in vitro culture (IVC) medium with or without 1.0 mg mL–1 HA. Embryos were vitrified using the MVC cryotop method (Kelly et al. 2004 Reprod. Fert. Dev. 16, 172) on either Day 5 (morula–blastocyst stages), Day 6 (compact morula–hatching blastocyst stages), or Day 7 (blastocyst–hatching blastocyst stages). Vitrified embryos were thawed 7 days later and placed into IVC medium. Embryo survival (assessed by blastocoele re-expansion) and hatching rates were recorded on Day 8. Variables were assessed using procedure CATMOD in SAS (SAS Institute Inc., Cary, NC, USA). In Experiment 1, the addition of HA did not affect cleavage or blastocyst formation rates but hatching rates were significantly (P &lt; 0.05) improved at concentrations of 0.5 to 1.5 mg mL–1 (Table 1). In Experiment 2, HA supplementation (1.0 mg mL–1) compared with control medium did not affect cleavage (96.8 and 97.1%, respectively) or blastocyst formation rates (68.6 and 70.2%, respectively). HA significantly (P &lt; 0.05) improved survival after thawing of embryos vitrified on Day 5 (100 v. 85.6%, n = 85 and 90). However no effect was observed when embryos were vitrified on either Day 6 (97.8 v. 97.8%, n = 91 and 92) or Day 7 (96.7 v. 97.9%, n = 92 and 94). Within day, HA supplementation did not affect hatching rate compared with control medium (Day 5, 54.1 v. 53.2%; Day 6, 62.9 v. 65.6%; Day 7, 71.9 v. 62.0%, respectively). These results demonstrate that HA supplementation of IVC medium significantly improves hatching rate of ovine embryos and we speculate that this improvement may correlate with comparable improvements in pregnancy rates after transfer. Hyaluronic acid binds to CD44, a glycoprotein expressed on the surface of preimplantation ovine embryos and shown to play a role on embryo development (Luz et al. 2012 Genet. Mol. Res. 11, 799–809). Hyaluronic acid plays a role in cell migration and we suggest that, in the early blastocyst, it affords an advantage to the trophectoderm cells. Table 1.Effect of hyaluronic acid (HA) supplementation in IVC medium on cleavage, blastocyst, and hatching rates

Impact of contrasting fish oil concentrations in the diet on ovine embryo development &lt;i&gt;in vivo&lt;/i&gt; and of corresponding diet-specific derivative sera during &lt;i&gt;in vitro&lt;/i&gt; culture

Impact of contrasting fish oil concentrations in the diet on ovine embryo development &lt;i&gt;in vivo&lt;/i&gt; and of corresponding diet-specific derivative sera during &lt;i&gt;in vitro&lt;/i&gt; culture

Effect of Pre‐Treatment of Ovine Sperm on Male Pronuclear Formation and Subsequent Embryo Development Following Intracytoplasmic Sperm Injection

The objective of this study was to determine the effects of various methods of sperm pre-treatment on male pronuclear (MPN) formation and subsequent development of ovine embryos derived from in vitro-matured oocytes and intracytoplasmic sperm injection (ICSI). The effect of treatment of injected oocytes with dithiothreitol (DTT) on embryo development was also assessed. In Exp. 1, the injected oocytes with non-treated sperm were activated with three different procedures. The cleavage and blastocyst rates in those activated with DTT was lower (p<0.05) than those activated with either ionomycin (Io) +6-dimethylaminopurine (6-DMAP) or DTT + I + 6-DMAP. In Exp. 2, the effects of sperm pre-incubated with DTT, sodium dodecyl sulphate (SDS) or DTT + SDS as well as two-time frozen/thawed sperm (without cryoprotectant) on MPN formation and oocyte activation were examined. The non-treated sperm served as controls. The MPN formation in DTT + SDS group was higher (p<0.05) than other groups except for freeze-thaw group. No difference in the rate of activated ICSI oocytes was observed among groups. In Exp. 3, the effect of pre-treatment of sperm on subsequent development of ICSI embryos and blastocyst cell numbers were examined. The rates of cleavage and blastocyst formation as well as the blastocyst cell numbers were similar among the pre-treated and control groups. In conclusion, pre-treatment of sperm with DTT + SDS positively affected MPN formation, although the subsequent development capacity of the resulting embryos remained limited. Moreover, DTT was not effective on oocyte activation compared with Io + 6-DMAP after ICSI.

212 EFFECTS OF VASCULAR ENDOTHELIAL GROWTH FACTOR ON THE EARLY DEVELOPMENT AND POLYSPERMY RATE OF OVINE EMBRYOS PRODUCED IN VITRO

To investigate the effect of vascular endothelial growth factor (VEGF) on the early development and polyspermy rate of ovine embryos in vitro, 2 experiments were conducted with human recombinant VEGF165 supplemented to the media during maturation, fertilization, and culture in vitro, respectively. Ovaries were collected from ewes at a local slaughterhouse. All oocytes surrounded by a multilayer of cumulus cells were collected and rinsed 3 times in maturation medium (control medium and treatment medium, respectively). A total of 100 oocytes in each group were cultured in 4-well plates (Nunc) containing 800 μL of maturation medium at 38.5°C in an atmosphere of 5% CO2 with saturated humidity. Four replicates of each experiment were conducted. Statistical analyses were conducted by ANOVA with SPSS 12.0 software (SPSS Inc., Chicago, IL, USA). Data are expressed as means, and P &lt; 0.05 was considered significant. In Experiment 1, to investigate the effect of VEGF on the early development of ovine embryos in vitro, VEGF was used at 5 ng mL–1 (treatment group A) and 10 ng mL–1 (treatment group B) in maturation medium (TCM-199 + BSA), HSOF fertilization medium, and SOF culture medium. The results showed that the maturation rate was increased significantly (P &lt; 0.01), from 75.76% in the control treatment to 83.98 and 80.23% in treatment group A and treatment group B, respectively. The cleavage rate was increased from 75.85% in the control group to 79.39% in treatment group A (P &gt; 0.05). The development rates of morulae (45.03%) and blastocysts (23.54%) in treatment group A were significantly higher (P &lt; 0.01) than those in the control group (38.94 and 18.09%, respectively). In addition, the development rates of blastocysts in treatment group B (21.05%) were lower than those in treatment group A (P &gt; 0.05) and higher than those in the control group (P &gt; 0.05). In Experiment 2, to investigate the effect of VEGF on the polyspermy rate of ovine embryos in vitro, 5 ng mL–1 of VEGF was used in TCM-199 + BSA maturation medium in this experiment. The results showed that the fertilization rate after 18 h of IVF was increased significantly (P &lt; 0.01), from 75.75% in the control group to 83.86% in the treatment group, and that the polyspermy rate was decreased significantly (P &lt; 0.01), from 12.64% in the control group to 7.68% in the treatment group. These results indicate that VEGF significantly improved the maturation and fertilization rates of ovine oocytes and, consequently, the rate of embryo development in vitro, especially when the medium was supplemented with 5 ng mL–1 of VEGF. The VEGF obviously decreased the polyspermy rate and bated the phenomenon of polyspermy in the process of ovine oocyte IVF. The present study was supported by the National Natural Science Foundation of China (No. 30371035).

In Vitro Development of Ovine Embryos Following Maturation Under Limited CO2

An experiment was conducted to examine the influence of CO2 during in vitro oocyte maturation on the in vitro ovine embryo development. Three treatments of CO2 were subjected to the oocyte development. Those were 2h gasses prior to maturation in incubator (T1); without CO2 either prior to or over maturation (T2) and CO2 exposure both prior to and over 22h maturation (T3). A total of 324 oocytes were used. Putative zygotes were cultured for seven days and evaluated for their developmental stage. Presence of CO2 (T3) increased the proportion of oocytes reaching Metaphase II ( 66.50 + 3.5%; p 0.05). This study suggests that it is possible to mature ovine oocytes in the absence of CO2 without loss its potensial development. It may therefore be an effective method of maturing ovine oocytes during transportation to IVP (in vitro production) laboratory.

The effect of melatonin on the secretion of progesterone in sheep and on the development of ovine embryos in vitro.

Two experiments were carried out in order to determine whether melatonin can improve secretion of progesterone in vivo, and its effect on embryonic development in vitro. In the first experiment, blood samples were collected from 5 ewes at 15 min intervals for 2 h at 7 and 10 days after withdrawal of progestagen pessaries. The first hour constituted a control period, which ended with an intravenous administration of 3 microg/(kg bw)(0.75) melatonin. All the ewes on day 7 and three of the ewes on day 10 showed a progesterone response to melatonin challenge, defined as an increase in the plasma progesterone concentration in at least two consecutive samples during the post-treatment period above the mean+2SD of the values in the pre-treatment period. A paired t-test revealed a significant effect of melatonin on the overall plasma progesterone concentrations before and after the challenge, both on day 7 (pre, 0.61 +/- 0.11; post, 0.73 +/- 0.13 ng/ml; p<0.01) and day 10 (pre, 1.16 +/- 0.19; post, 1.30 +/- 0.20 ng/ml; p<0.05). Ninety-one thawed embryos (46 morulae and 45 blastocysts) were used in the second experiment, being cultured with or without 1 microg/ml melatonin. If the embryos were blastocysts when the culture started. melatonin increased the percentage that had hatched after 24 h of culture (p<0.01), and there was a lower percentage of degenerated embryos at the end of the incubation period (p<0.05). It may be concluded that melatonin treatment in sheep can increase both fertility and prolificacy by improving luteal function and embryonic survival.

Ovine embryo development in culture systems causing fetal oversize

Ovine embryo development in culture systems causing fetal oversize

Leukaemia inhibitory factor in endometrium during the oestrous cycle, early pregnancy and in ovariectomized steroid-treated ewes.

Leukaemia inhibitory factor (LIF), a pleiotropic cytokine, is essential for blastocyst implantation in mice and maintains the development of ovine embryos in culture. The expression of LIF was examined by northern blot analysis in endometrial tissue from cyclic (days 4-16) and pregnant (days 4-20) ewes, and the corresponding protein was immunolocalized. Expression of mRNA encoding LIF remained relatively constant throughout the oestrous cycle and was present during early pregnancy. A decrease in mRNA encoding LIF was observed during early pregnancy (on days 12-14) and expression was highest on days 16-20. Immunoreactive LIF was present in the cellular compartments of the endometrium throughout the oestrous cycle and early pregnancy, with maximal immunostaining in the caruncular and intercaruncular luminal epithelium, and moderate staining in the glandular epithelium and intercaruncular stroma. Immunoreactive LIF was also detected in the trophoblast cells of day 17 blastocysts. Separately cultured endometrial epithelial and stromal cells from pregnant animals both expressed mRNA encoding LIF. Ovariectomized steroid-treated ewes were studied to establish whether steroid hormones had a role in regulating endometrial LIF. Ewes treated with oestradiol alone showed lower concentrations of immunoreactive LIF in the endometrium in comparison to ovariectomized, control animals, while treatment of ovariectomized animals with both oestradiol and progesterone had a greater inhibitory effect on LIF immunolocalization. These studies demonstrate the presence of mRNA encoding LIF and protein throughout the oestrous cycle and early pregnancy and suggest that steroid hormones may be involved in their regulation.

The effects of an ovine oviductal estrus-associated glycoprotein on early embryo development

Oviduct specific estrus-associated glycoproteins (EGP) secreted around the time of estrus are known to be present in several species. Many of these glycoproteins, including that of the sheep (oEGP), have been shown to bind to the zona pellucida and to become associated with individual blastomere membranes, indicating a role for these proteins in early embryo development. Given that the culture of ovine embryos can result in a number of developmental abnormalities as well as a reduction in viability, the present study examined the effects of oviducal fluid and gel filtration fractions of oviducal fluid on the in vitro development of ovine embryos. The first fraction of oviducal fluid (F1) contained predominantly oEGP, while the second (F2) contained albumin and was included in the study as a control. The culture medium was synthetic oviduct fluid (SOF) supplemented with 20% human serum (HS). The study consisted of 2 experiments; the first examined the effects of oviducal fluid, F1 and F2 in combination with 0, 10 and 20% HS; while the second, which aimed to investigate the effect of F1 during the first 72 h of culture, examined the influence of oviducal fluid and the fractions in the presence of either a low concentration of serum or no serum for the first 72 h. The presence of Fl significantly decreased the proportion of zygotes undergoing first cleavage, increased the mean number of nuclei per blastocyst and increased the mean time taken for blastocyst formation. Unfractionated oviducal fluid appeared to be detrimental to embryo development while F2 had no influence. The use of a low concentration of serum during the first 72 h of culturewas not beneficial to development. Removing F1 from the culture medium after the initial 72 h of culture did not reverse the effects of this protein, implying that it is required only during the first 2 to 3 d of development. It is postulated that oEGP is involved in a selection mechanism at the zygote stage and may improve the viability of in vitro cultured embryos.

Development of ovine embryos in synthetic oviductal fluid containing amino acids at oviductal fluid concentrations.

The effects of supplementing synthetic oviductal fluid (SOF) with amino acids, at oviductal fluid concentrations, on the development of ovine in vitro-matured/in vitro-fertilized embryos was examined in three experiments. In the first, embryo development in SOF, SOF + 2% human serum (HS), SOF + 20% HS, and SOF + BSA, with and without amino acid supplementation, was examined. Development of zygotes to the blastocyst and hatching blastocyst stages was highest in medium containing 20% HS (64.8% and 54.4%, respectively) irrespective of amino acid supplementation. However, supplementation was significantly beneficial in all other media, with up to 42.1% of zygotes developing into hatching blastocysts. In these media, supplementation also significantly increased the mean number of nuclei per newly formed blastocyst (up to a mean of 70.8) and reduced the time during which blastocysts formed. Experiment 2 was an examination of the effect on embryo development of three amino acid preparations (oviduct amino acid concentrations vs. Eagle's Basal Medium (BME) essential + Minimum Essential Medium (MEM) nonessential vs. MEM essential + MEM nonessential concentrations) and the presence or absence of BSA. Both the amino acid and BSA treatments significantly influenced the percentage of zygotes that developed to the hatching blastocyst stage but not to the blastocyst stage. The preferred medium contained amino acids at oviductal fluid concentrations and BSA (54.5% hatching rate). The amino acid treatments did not significantly influence the mean number of nuclei per newly formed blastocyst, but the addition of BSA had a significant effect (70.7 +/- 1.14 vs. 75.7 +/- 1.13). In experiment 3, embryo development to Day 13 was examined after culture in SOF containing amino acids at oviductal fluid concentrations. Embryos were cultured in the presence of either BSA, polyvinyl alcohol (PVA), or no additional supplement and were transferred to recipient ewes on either Day 0 (after in vitro fertilization), 3, or 5. The addition of BSA or PVA had no significant effect, but significantly more embryos developed to Day 13 following transfer on Day 0 (60.0%) than on either Day 3 or 5 (overall 45.4%). It is concluded that SOF containing oviductal fluid concentrations of amino acids 1) facilitates the development of a high percentage (57.5%) of blastocysts, 2) improves embryo morphology compared with that observed in medium containing HS, 3) significantly improves hatching rates compared with those obtained in SOF containing commercially available preparations of amino acids, and 4) produces embryos with relatively high levels of viability to Day 13 of pregnancy.

Lamb birth weight is affected by culture system utilized during in vitro pre-elongation development of ovine embryos.

It has previously been reported that ovine embryos cultured in Synthetic Oviduct Fluid medium supplemented with 20% human serum (SOF+HS) develop into lambs with a high birth weight. We have investigated this phenomenon by culturing ovine zygotes in SOF+HS or a serum-free version of Synthetic Oviduct Fluid with BSA and amino acids (SOFaaBSA) in place of serum. Zygotes were either obtained from superovulated and naturally mated ewes or produced in vitro. Embryos were subsequently transferred to synchronized recipient ewes (n = 63). An additional group of ewes (n = 16) served as flock fertility and lambing controls. Development of zygotes to stages suitable for transfer (i.e., good to excellent compact morulae or blastocysts) was not affected by medium (SOFaaBSA = 53 +/- 5% vs. SOF+HS = 59 +/- 5%) but was affected by source (in vivo-derived = 74 +/- 5% vs. in vitro-derived = 35 +/- 5%, p < 0.001). Embryos incubated in SOF+HS were morphologically different from those incubated in SOFaaBSA, having abundant lipid droplets. Pregnancy rate (65%) and embryo survival (48%) of recipients determined by ultrasonography on approximately Day 60 of pregnancy did not differ between medium treatments or source of embryo. Mean weight of lambs from embryos cultured in SOF+HS (4.2 +/- 0.2 kg) was significantly heavier than that of controls (3.4 +/- 0.2 kg, p < 0.01) or of lambs from embryos cultured in SOFaaBSA (3.5 +/- 0.2 kg, p < 0.05). Furthermore, mean gestation length was longer in recipients receiving embryos incubated in SOF+HS (147 +/- 1 days) than in SOFaaBSA (145 +/- 1 day, p < 0.05). Reasons for this birth weight and gestation length difference are unclear, but our data suggest that different culture conditions can produce embryos with differing morphology, apparent chemical composition, and rate of development, resulting in lambs with differing gestation length and birth weight.

Two culture systems showing a biphasic effect on ovine embryo development from the 1-2 cell stage to hatched blastocysts

This study compared the effect of using either CZB or TCM 199 media on both the development of 1-2 cell ovine embryos from superovulated ewes to the blastocyst stage (Experiment 1), and the hatching process of ovine blastocysts developed in vitro (Experiment 2). For the first 5 d, the CZB medium showed higher rates of embryo development than the TCM 199 medium (p < 0.001). The embryos reaching the > 16 cell stage were 79 vs 52% and 74 vs 20% with or without an oviductal monolayer, respectively, and those reaching the blastocyst stage were 71 vs 46% and 46 vs 13% with or without cells. The CZB medium was less able to support the hatching process of the blastocysts obtained in the first experiment than was the TCM-199 medium + 10% FCS (fetal calf serum) with cells (31 vs 92%; p < 0.001) or without cells (13 vs 66%; p < 0.001). No blastocysts completely escaped from the zona pellucida (ZP) in the CZB medium compared with 80 and 61% in the TCM 199 medium with or without cells, respectively. In Experiment 3, 47% of the blastocysts migrated through the artificial opening of the ZP and hatched completely. After 24 h of culture in the CZB medium, however, they showed blastocoelic cavity breakdown. During the preliminary cleavages, the ovine embryos developed better in CZB medium than in TCM 199, but the latter was more efficient in promoting the hatching process of the blastocysts.

The effect of amino acids at oviductal fluid concentrations on the development of ovine embryos in a defined medium

The effect of amino acids at oviductal fluid concentrations on the development of ovine embryos in a defined medium