Effects of co-culture and embryo number on the in vitro development of bovine embryos

It is generally accepted that culturing embryos in groups or with somatic cells improves both the yield and quality of the blastocysts obtained. The aims of this study were 1) to compare the yield and quality of the embryos obtained after culture in several number conditions and in several culture systems and 2) to assess the effect of co-culture started at various stages of embryo development. Under cell-free culture conditions (modified synthetic oviduct fluid [mSOF] supplemented with 10% fetal calf serum [FCS] 48 h post insemination, the rate of Day 10 blastocysts was lower when embryos were cultured in small groups (1 to 6 per drop) than in large groups (4 versus 23% ; P < 0.01). There was no group effect when embryos were co-cultured either with Buffalo rat liver (BRL) cells in TCM 199, or in a culture system allowing the progressive development of cumulus cells in mSOF, even if co-culture started at 66 or 114 h post insemination. However, embryos cultured singly had lower cell numbers than embryos cultured in large groups when co-culture started at 114 h post insemination. This suggests that 1) somatic cells improve the development of singly cultured bovine embryos up to the blastocyst stage after the 9-16 cell stage; 2) co-culture affects blastocyst cell number of singly cultured embryos by acting roughly between the 5-8 and the 9-16 cell stage; and 3) cooperation between embryos could replace the effect of co-culture either on the yield of blastocysts or on blastocyst cell number. Blastocysts appeared significantly earlier in co-culture with cumulus cells in mSOF than in co-culture with BRL cells in TCM 199 (detection of the blastocysts: 7.3 +/- 0.1 d post insemination with cumulus cells versus 8.1 +/- 0.1 d with BRL cells; P < 0.001) and had a significant higher number of cells (143 +/- 9 versus 85 +/- 11; P < 0.001). This system thus seems suitable for the culture of small numbers of embryos resulting from in vitro maturation and fertilization of oocytes from individual donor cows.     

Porcine oviductal cells support in vitro bovine embryo development

This study was designed to investigate the developmental competency of in vitro-matured and in vitro-fertilized bovine embryos co-cultured with a) medium alone, b) bovine oviductal cells (BOC), c) bovine conditioned medium (BCM), d) porcine oviductal cells (POC), and porcine conditioned medium (PCM). Follicular oocytes collected from cattle at local slaughterhouses were matured and fertilized in vitro. Epithelial cells were scraped from the luminal surface tissue of either bovine or porcine oviducts collected after ovulation, cultured in TALP + 10% heat-treated fetal calf serum, and the conditioned media were collected following a 3- to 5-d incubation period. After 18 to 22 h of sperm-ova co-incubation, the fertilized and/or cleaved ova were randomly assigned to 1 of 5 co-culture groups. The results revealed that the efficiency of medium alone in supporting embryo development from the 16- to 32-cell stage up to the blastocyst stage was significantly (P<0.01) lower than of embryos co-cultured with either bovine or porcine epithelial cells, or with conditioned media from such cells. Epithelial cell co-culture, regardless of cell source, was more effective (P<0.01) than culture with conditioned medium. Co-culture in medium containing or conditioned by porcine cells was more effective in supporting bovine embryo development than co-culture with bovine-derived cells or conditioned medium. These data support the concept that oviductal cells produce a soluble component which enhances embryo development to the blastocyst stage in vitro and that the effect is not species-specific.     

Effects of acetoacetate and D-beta-hydroxybutyrate on bovine in vitro embryo development in serum-free medium

It is known that the ketone bodies acetoacetate and D-beta-hydroxybutyrate can be metabolized by the early bovine embryo for in vitro development. In the present work, we report experiments leading to the culture of bovine embryos in the absence of serum. In vitro-produced bovine zygotes were cultured in modified synthetic oviduct fluid medium supplemented with acetoacetate derivatives, acetoacetate and D-beta-hydroxybutyrate. Acetoacetate and its derivatives prevented blastocysts from forming in the absence of serum during the whole culture period. However, from Days 6 to 8 of culture in the absence of serum, acetoacetate did not affect development as compared to controls containing lactate and pyruvate or no substrate. Interestingly, D-beta-hydroxybutyrate stimulated blastocyst and expansion development, and allowed lipid mobilization. In feeder cells coculture, embryos produced with D-beta-hydroxybutyrate showed improved hatching. Embryos cultured in D-beta-hydroxybutyrate were viable upon transfer to recipients, although no pregnancies were confirmed later by ultrasonic scanning. The protective effect of serum upon embryos cultured in medium containing acetoacetate is apparently not required in the presence of D-beta-hydroxybutyrate.     

Effects of glucose and protein sources on bovine embryo development in vitro

Oviductal factors may be obtained by ultrafiltration of conditioned medium, added to a simple media and used in bovine embryo culture. In this study, we aimed to analyze the development of bovine embryos produced with oviductal factors compared to those cultured in the presence of BSA or serum, the effects of glucose in presence of these protein supplements, and the ability of oviductal factors to support embryo development during the entire culture period. In vitro produced bovine zygotes from slaughterhouse ovaries were cultured in modified-synthetic oviduct fluid (mSOF) alone or supplemented with (1) oviductal factors, (2) BSA and (3) FCS. Oviductal factors showed embryotrophic activity, although with blastocyst rates lower than those in BSA and FCS. Glucose (1.5 mM) added at Day 2 of culture did not affect development in the presence of oviductal factors. The number of cells in expanded blastocysts was unaffected by the presence of glucose or any of the protein supplements used. Both BSA and FCS, respectively, improved blastocyst rates of Day 6 embryos produced with oviductal factors. The effect of oviductal factors was masked by the presence of BSA during the entire culture. FCS promoted an earlier appearance of blastocysts. It is concluded that the effect of glucose on in vitro embryo development depends upon the source of protein. Oviductal factors are not an appropriate supplement for embryos beyond Day 6 of culture in SOF, although blastocyst rates of such embryos may be increased by culturing them in the presence of FCS or BSA.     

Nuclear maturation and development of IVM/IVF canine embryos in synthetic oviductal fluid or in co-culture with buffalo rat liver cells

In vitro embryo production in the domestic bitch can provide valuable insights for conservation of endangered canids. In the present study, canine oocytes underwent in vitro maturation (IVM) in simple or complex media, with production of in vitro matured and fertilized (IVM/IVF) canine embryos. Cumulus–oocyte complexes (COCs) were harvested from ovaries by slicing and subjected to IVM in four media (SOF, TCM 199, Ham-F10, and DMEM/F12). After culture for 48 h, oocytes were stained and examined for nuclear maturation. There were no significant differences in the mean (±S.D.) percentage of nuclear maturation (metaphase II) of oocytes cultured in SOF (18.6 ± 7.6%), TCM 199 (18.3 ± 4.5%), Ham-F10 (13.9 ± 8.2%), or DMEM/F12 (11.9 ± 4.2%). For assessment of embryo development, oocytes were matured for 48 h in synthetic oviductal fluid (SOF), fertilized with frozen-thawed sperm, and presumptive zygotes were cultured for 7 d, either in SOF or as co-cultures with BRL cells in TCM 199. Percentages of IVM/IVF oocytes that developed to the 2-cell, 3–4-cell, and 5–7-cell stages were higher (P < 0.05) following culture in SOF versus BRL cell co-cultures (33.6 ± 1.2% vs 13.7 ± 1.2%, 24.7 ± 0.5% vs 8.7 ± 1.1%, and 15.1 ± 2.2% vs 4.3 ± 1.3%, respectively). However, none of the embryos developed beyond the 8–16-cell stage. In conclusion, simple or complex media successfully induced resumption of meiosis and nuclear maturation of canine oocytes. Furthermore, SOF supported in vitro development of IVM/IVF canine embryos to the 8–16-cell stage.     

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).     

Factors affecting bovine embryo development in synthetic oviduct fluid following oocyte maturation and fertilization in vitro

Employing a total of 3465 bovine oocytes this study was aimed at improving the efficiency of bovine embryo production under defined and undefined conditions. Following in vitro maturation (IVM) and in vitro fertilization (IVF), oocytes were allocated to various culture treatments using synthetic oviduct fluid (SOF). In our 3 experiments we showed that: 1) the addition of fetal calf serum (FCS 10% v/v) to SOF droplets after 20 to 24 h significantly improved blastocyst yields on Day 6 (21 vs 12%; P < 0.01), but not at later stages and resulted in significantly higher Day-8 blastocyst cell numbers (148 +/- 61 vs 92 +/- 35; P < 0.05); 2) the removal of bovine serum albumin (BSA) from the standard SOF medium resulted in significantly reduced blastocyst yields on Days 6, 7 and 8, respectively (17 vs 8%; 28 vs 18%; 31 vs 21%; P < 0.05); 3) the presence or absence of cumulus cells surrounding the presumptive zygote in culture in SOF had no effect on cleavage rate, percentage of 5-8 cell embryos or blastocyst yields (Day 6,7 or 8); 4) the culture of presumptive zygotes in SOF in an atmosphere of 5% CO2 in air (20% O2) resulted in significantly reduced development compared with culture in 5% CO2, 5% O2, 90% N2 in terms of blastocyst yield on Days 6, 7 and 8 and on Day 8 hatching rate, respectively (5 vs 22%; 9 vs 33%; 13 vs 48%; 50 vs 8%; P < 0.001) and 5) embryo density (1 embryo per 1 or 3 microl SOF) or replacing the culture medium every 48 h had no effect when SOF was supplemented with serum; however, under serum-free conditions, changing of the media resulted in a slightly improved Day-6 blastocyst yield such that renewal of serum-free medium mimicked the effect of serum addition.     

The effect of oviductal epithelial cell co-culture during in vitro maturation on sow oocyte morphology,fertilization and embryo development

In vitro embryo production in the sow is challenged by poor cytoplasmic maturation, low sperm penetration and low normal fertilization, leading to the development of poor quality blastocysts containing a small number of nuclei. In prepubertal gilt oocytes, the presence of porcine oviductal epithelial cells (pOECs) during maturation increases cytoplasmic maturation and blastocyst development. These aspects, as well as blastocyst quality, may be improved when adult sow oocytes are matured with pOEC. Therefore, the effect of the presence of pOEC on sow oocyte morphology, fertilization and the progression of embryo development was evaluated. The pOEC were cultured in M199 for 18 h, then cultured in NCSU23 for 4 h before the oocytes were added. Oocytes from 2 to 6 mm follicles were matured in 500 microl NCSU23, with eCG and hCG, for 24 h, and then cultured with or without pOEC, in NCSU23 without hormones, for 18 h. In vitro fertilization took place in modified Tris-buffered medium, for 6 h, and the presumptive zygotes were then cultured for 162 h in NCSU23. Morphology of the IVM oocytes was compared to that of immature oocytes and in vivo matured MII oocytes from slaughtered sows in estrus. The in vitro matured oocytes had a greater diameter and a wider perivitelline space than the immature and in vivo matured MII oocytes (P < 0.01). Penetration, polyspermy and pronucleus formation did not differ between the pOEC and Control groups, although the total penetration rate was higher for the Control oocytes (26% versus 39%; P < 0.01). Fewer blastocysts developed in the pOEC group than in the Control group (19% versus 27%; P < 0.01), but blastocyst growth was accelerated, leading to a higher percentage of hatched blastocysts (3% versus 10%; P < 0.01). Finally, the average blastocyst cell number was higher in the pOEC group (47 versus 40; P < 0.05) and a greater percentage of blastocysts contained a superior number of nuclei. In conclusion, the addition of pOEC during the second half of in vitro maturation resulted in fewer blastocysts formed, but of those blastocysts that did form the quality was improved.     

Comparison of conditioned medium and direct co-culture of human granulosa cells on mouse embryo development

Although numerous investigations have demonstrated the beneficial effects of co-culture system of different somatic cells on in vitro development of embryos, the effects of conditioned-media of co-culture cells have not been well documented. The objective of this study was to compare the effects of human granulosa cells co-culture system and its conditioned medium on the developmental rate of mouse embryos in vitro. Two sets of experiments were undertaken: in the first one 317 mouse one-cell embryos were cultured in human granulosa cell co-culture system (GC). Ham's F10 medium conditioned with granulosa cells (CM) and non-conditioned Ham's F10 for 120 h. In the second experiment. 391 late two-cell embryos were cultured in the 3 fore-mentioned culture treatments for 72 h. Embryos were obtained from NMRI mice. Granulosa cells were collected from patients undergoing an IVF program during oocyte pickup. In the first set of experiments, 23.6, 14.5 and 11.1% of one-cell embryos passed two-cell block and continued growing to 4-cell in GC, CM and HF, respectively. This index in GC was significantly different from two other treatments. Also significantly more embryos reached blastocyst stage in GC compared with two other treatments. The blastocyst rate was not significantly different between CM and HF. In the second set of experiments the proportion of blastocyst stage was significantly higher in CM than that in HF and lower than that in GC. In conclusion, although human granulosa cell-conditioned medium has beneficial effects on mouse embryo development, it was not as effective as co-culture of these cells.     

Effects of oxygen concentration and oviductal epithelial tissue on the development of in vitro matured and fertilized bovine oocytes cultured in protein-free medium

Examination was made of the effects of oxygen concentration and supplementation of bovine oviductal epithelial tissue (BOET) on the development of bovine in vitro matured and fertilized (IVM/IVF) oocytes in a protein-free medium. The IVM/IVF embryos were cultured in protein-free tissue culture medium 199 supplemented with or without BOET under 5% CO(2) in air (20% O(2)) or 5% CO(2), 5% O(2) and 90% N(2) (5% O(2)). We found that blastocyst development without BOET at 5% O(2) was the same as that with BOET at 20% O(2) (30 vs 33%); BOET suppressed blastocyst development at 5% O(2) (4%). Blastocysts cultured without BOET at 5% O(2) developed into normal fetuses after transfer to recipient heifers. Examination was also made of oxygen pressure in the medium cultured with or without BOET at 20% O(2) or 5% O(2) by a blood gas analyzer. Oxygen pressure in the medium cultured with BOET at 20% O(2) was lower than that without BOET (111.0 +/- 13.3 vs 149.2 +/- 1.3 mmHg). These results indicate that bovine IVM/IVF embryos can develop to the blastocyst stage in a protein-free medium without somatic cell support at low oxygen concentration (5%) and that the beneficial role of BOET for embryonic development may be to reduce oxygen concentration in the medium.     

Origin of oestrus-associated glycoproteins in bovine oviductal fluid.

The aim of the present study was to determine whether the synthesis of an oestrus-associated protein found in bovine oviductal fluid varies with oviductal region, stage of cycle or day of pregnancy. Explant culture was performed using oviducts recovered from naturally cycling animals either at oestrus or 12-14 days after oestrus. Three oviductal regions, the preampulla, ampulla and isthmus, were cultured individually in the presence of 20 muCi [35S]methionine in serum-free medium for 6 h at 37 degrees C. Synthesis of oestrus-associated protein was assessed by one-dimensional SDS-PAGE, fluorography and densitometry of radiolabelled bands. Significantly more oestrus-associated protein was synthesized by the ampullar region of the oviduct, although it was detected in explant culture media from both the isthmic and preampullar regions. A polyclonal antibody produced against oestrus-associated protein was used to localize the protein in paraffin-embedded sections of oviductal explant cultures and other bovine tissues. Localization of the protein in oviductal tissue sections varied with stage of cycle (oestrus > luteal > pregnant) and region of oviduct (ampulla > preampulla/isthmus). These findings indicate an effect of oviductal region and hormonal state (cycling versus pregnant) on the synthesis and secretion of the oestrus-associated protein. Lectin affinity studies indicated that galactosyl(beta 1,3)N-acetylgalactosamine and N-acetylglucosamine residues are associated with the oestrus-associated protein.     

Effect of protein supplementation and presence of an antioxidant on the development of bovine zygotes in synthetic oviduct fluid medium under high or low oxygen tension

The aim of this study was to investigate the effect of protein supplementation of culture medium and the presence of a putative antioxidant on bovine zygote development under 5% (low) and 20% (high) O2. In Experiment 1, presumptive zygotes (n=992) were cultured in synthetic oviduct fluid (SOF) alone or supplemented with 3 mg/mL PVP, 3 mg/mL BSA (SOFB), and/or 10% FCS (SOFBF) in 5% CO2, 5% O2, 90% N2. In Experiment 2, zygotes (n=1916) were cultured in SOF, SOFB or SOFBF with or without taurine under high and low O2. In Experiment 1, presence of BSA or BSA plus FCS significantly increased the speed of development compared to SOF or SOF+PVP. Blastocyst quality was also improved, as evidenced by increased hatching rate and cell numbers. In Experiments 2, taurine had no effect on development irrespective of oxygen concentration or protein supplementation. In conclusion, the presence of protein in the culture medium and culture under reduced O2 significantly improved embryo development. Taurine had no effect on development.     

Effect of high molecular weight factors present in bovine oviduct-conditioned medium on in vitro bovine embryo development

To investigate the presence of embryotrophic factors in bovine oviduct-conditioned medium (BOCM), the high molecular weight fraction (> 10 KDa) from BOCM was added to 3 chemically defined embryo culture media (TCM199, DMEM/F12 and modified synthetic oviduct fluid [mSOF]). Zygotes were obtained by in vitro maturation and fertilization of oocytes. Conditioning of TCM199 with oviduct cells increased both cleavage to the 5- to 8-cell stage (59 vs 37%) and further development to the blastocyst stage (19 vs 4%). The low molecular weight fraction (< 10 KDa) of BOCM maintained development to the 5- to 8-cell stage but did not allow development to the blastocyst stage. Adding the high molecular weight fraction to the inactive low molecular weight fraction restored bovine embryo development up to the blastocyst stage. This embryotrophic effect of the high molecular weight fraction was not observed when this fraction was added to TCM199 or DMEM/F12 medium. Whereas adding this fraction to mSOF medium significantly (P<0.05) increased embryo development up to the blastocyst stage (36%) in comparison with that of mSOF (15%) or BOCM (14%). These results show that BOCM contains high molecular weight factors promoting embryo development up to the blastocyst stage. Some chemically defined media mask the effect of these embryotrophic factors.     

Effects of resazurin on bovine oocyte fertilization and embryo development in vitro

Resazurin is a redox dye (7-hydroxy-3H-phenoxazin-3-one-10-oxide) used for assessing potential fertility of spermatozoa and functional status of eukaryotic cells. In this study, the fertilizing capacity of spermatozoa treated with resazurin and effects of resazurin on bovine embryo development in vitro was examined. Abattoir-derived bovine oocytes were collected and subjected to in vitro maturation (IVM), fertilization (IVF) and culture (IVC). In Experiment 1, bovine oocytes (n=2767) were fertilized with spermatozoa exposed to resazurin (17.6 μg/ml) for 0, 15, 30, 60 min, respectively. There was no significant (P>0.05) difference with respect to oocyte cleavage, morula and blastocyst production between treatments. In Experiment 2, oocytes (n=1671) were treated with resazurin (1.8 μg/ml) during IVM, IVF, IVC, respectively, or during the entire IVM, IVF and IVC procedures. There was no significant (P>0.05) difference in cleavage rates. However, the proportion of embryos that developed into blastocysts, expanded and hatched blastocysts in those groups in which oocytes/embryos were treated with resazurin during IVC or IVM/IVF/IVC was significantly (P<0.05) less than those exposed to resazurin during IVM only, or during IVF only. We conclude that resazurin did not have significant adverse effects on fertilizing capability of bovine spermatozoa; however, extended treatment of embryos with resazurin may be detrimental to embryonic development.     

Effects of amino acids and α-amanitin on bovine embryo development in a simple protein-Free medium

Five experiments, utilizing 3741 embryos produced in vitro, were designed to test the effects of Eagle's nonessential amino acids, and combinations of Eagle's essential amino acids and the RNA polymerase inhibitor α-amanitin on the development of preimplantation bovine embryos in a modified protein-free KSOM medium. Embryos were cultured in 5% O₂:5% CO₂:90% N₂ at 39°C for the first 40–44 hr in modified KSOM, and embryos with ≥4 cells were cultured in modified KSOM-PVA with different amino acids in experiments 1–4, and with the addition of α-amanitin in experiment 5. In experiment 1, addition of 0.5× of the essential amino acids, with different concentrations of nonessential amino acids significantly increased hatching of blastocysts and decreased blastocyst degeneration, but increasing the nonessential amino acids from 1× to 5×, did not stimulate embryo development. In experiments 2–4, increasing only the glycine concentration, or adding each of the 12 essential amino acids singly or several in combination to the medium containing nonessential amino acids, did not significantly improve embryo development. Taurine (0.4 mM) in the modified KSOM medium reduced blastocyst degeneration. In experiment 5, α-amanitin (20 μM) completely inhibited further embryo development when it was added at several stages from 4-cell embryos to morulae. The study with protein-free KSOM plus amino acids provided a completely defined simple medium for culturing bovine embryos, with evidence that continuous mRNA activity and presumed protein synthesis was obligatory to meet the complex and continuous requirements for proteins by the developing blastocyst. Mol. Reprod. Dev. 46:278–285, 1997. © 1997 Wiley-Liss, Inc.     

Effects of bovine oviductal proteins on bull spermatozoal function

The effects of bovine oviductal proteins on bull sperm viability, acrosome reaction and motility were studied. Motile frozen/thawed spermatozoa from Percoll gradients were incubated with 1.0 mg/mL oviductal proteins (>8 kDa) extracted by ammonium sulphate precipitation from oviductal extract (OE) or serum-free oviductal epithelial cell-conditioned media (CM), treated in the presence (CM+) or absence (CM-) of 1 microg/mL 17beta-estradiol. Inclusion of oviductal proteins had a significant beneficial effect on sperm viability (76.3 to 80.6%+/-5.3) compared with the control (without oviductal proteins; 57.8%+/-5.3) immediately after the commencement of incubation. After 5 h, viability was significantly higher for CM- and OE treatments than for the control, although no differences were observed at 24 h. Acrosomal status only differed among treatments after 24 h, when higher percentages of acrosome- reacted spermatozoa were found in the control (46.0%+/-2.5) than in the oviductal protein treatments (33.1 to 38.2%+/-2.5). No differences in percentages of motile spermatozoa occurred within the first hour of incubation, although inclusion of CM proteins decreased sperm velocities, beat cross frequency, linearity, and straightness but increased values for mean angular displacement. These findings suggest that proteins secreted by oviductal epithelium promote viability, delay the acrosome reaction and suppress the motion of spermatozoa.     

The effects of bovine serum albumin, bovine uterine fluid, and heat-treated bovine serum on in vitro mouse embryo development

Two-cell mouse embryos were cultured in Whitten's medium with one of three supplements: bovine serum albumin (WM + BSA), heat-treated bovine serum (WM + HTBS) or bovine uterine fluid (WM + BUF). Protein concentrations for cultures of WM + BSA were 50.2, 100.5, 251.2, 502.5, and 1005.0 mug/ml and for WM + HTBS were 70.4, 105.1, 269.0, 524.5 and 1193.9 mug/ml. Protein concentrations ranged from 56.9 to 739.1 mug/ml for 22 WM + BUF samples. Embryo development in all media was significantly correlated with the log total protein concentration. When compared to WM + BSA, development was not significantly inhibited or stimulated in any WM + BUF cultures or in WM with 70.4, 524.5 and 1193.9 mug/ml HTBS. Development was enhanced in WM with 105.1 and 269.0 mug/ml HTBS (P<0.05). The results suggest that at the protein concentrations used, culture media supplemented with BUF and BSA support similar mouse embryo development. Culture medium supplemented with HTBS supported embryo development more than medium with BSA. Uterine factors in the bovine capable of enhancing or inhibiting early embryo development were not detected.     

Macromolecular source as dependent on osmotic pressure and water source: effects on bovine in vitro embryo development and quality

This study evaluated the protective effect of protein, as dependent on osmolarity, and the quality of water sources used to prepare embryo culture media. In Experiment 1, two concentrations of NaCl were used to obtain culture media with normal (280 mOSM) and low (245 mOSM) osmolarity, each supplemented with either bovine serum albumin (BSA) or polyvinyl alcohol (PVA). Low osmolarity improved blastocyst rates in the presence of BSA (P < 0.01) and tended to do it in medium containing PVA (P < 0.07). Furthermore, low osmolarity allowed PVA to increase inner cell mass (ICM) numbers and ICM/total cell rate (P < 0.05), while trophectoderm (TE) and total cell counts tended to decrease (P < 0.08). In Experiment 2, culture media were prepared with two water sources (Milli-Q and Sigma-W3500-) in combination with BSA or PVA. Both water sources yielded similar embryo development rates, but in the presence of BSA, Milli-Q water produced embryos with increased ICM/total cells rates (P < 0.05). On the contrary, Sigma water tended to increase trophectoderm cell counts (P < 0.08). In conclusion, the present study showed that low osmolarity is beneficial to embryo development and combinations of macromolecule and osmolarity influence trophectoderm differentiation. Both Milli-Q and Sigma supported embryo development at comparable rates, although in the presence of BSA, blastocysts obtained in the medium prepared with Milli-Q water had superior quality in terms of ICM/total cells rates.     

Effects of post-ovulatory food deprivation on oviductal sperm concentration,embryo development and hormonal profiles in the pig

Nutrition is one of the multiple factors that modulate reproduction in animals. The effect of 48 h food deprivation on reproductive and metabolic hormonal changes in relation to cleavage rates was studied. Insemination of 15 sows was performed 20–10 h prior to expected ovulation and ova were recovered at slaughter 65–91 h post ovulation. Blood samples were collected every second hour, beginning from the time of insemination until slaughter, for measurements of progesterone, cortisol, the prostaglandin F_2α metabolite (15-keto-13,14-dihydro-PGF_2α) and insulin levels. The embryos from the food-deprived sows (D-group) had fewer accessory spermatozoa in their zona pellucida (ZP) compared with the control sows (C-group). A lower cleavage rate of the embryos in the D-group compared with the C-group was detected. Plasma progesterone, cortisol and prostaglandin F_2α metabolite levels were significantly higher in the D-group compared with the C-group. Food deprivation is associated with changes in reproductive and metabolic hormones that might lead to changes in the oviductal environment, culminating in a lower cleavage rate of the embryos and presence of fewer viable spermatozoa in the reservoir.