Embryo Yield Research Articles

Refinement of shed-microspore culture protocol to increase normal embryos production in hot pepper ( Capsicum annuum L.)

A low percentage of normal-looking embryos (20%) is still a problem in an efficient shed-microspore culture of hot pepper ( Capsicum annuum L.) and it is more a serious problem in other androgenesic culture systems of pepper. Therefore, several factors were investigated to refine the protocol in order to increase the percentage of normal-looking embryos. The most important factors which improved the protocol and resulted in a significantly higher percentage of normal-looking embryos produces (>50%), were delayed enrichment of the liquid upper layer medium with 2.5 μM Zeatin and 5 μM IAA, and reduced incubation temperature from 28 °C to 21 °C, both after 3 weeks of culture. Addition of 1% activated charcoal in the solid lower layer of the medium enhanced the total embryo yield only, while an application of abscisic acid and increased osmolality medium had a detrimental effect on embryo production. The use of doubled haploid lines as anther donor plants has clearly decreased the variability and improved the statistical analysis of treatment effects. A higher percentage of normal embryos are produced with this refined protocol, and is therefore more suitable for implementation in the breeding programs of hot pepper.

The effect of exogenous gonadotropins on ovarian function in goats actively immunized against inhibin

The aim of this investigation was to compare the ovarian response to superovulatory treatments in does before and after inhibin immunization, with a view to optimizing the superovulatory potential of the caprine ovary. To avoid interference by the ovarian cycle, the experiment was conducted out-of-season. At the onset of the experiment 48 does were subjected to treatment with an sc implant of the progestogen norgestomet, combined with a gonadotropin; eight does each received a single injection of 1200 IU eCG, 400 IU eCG or 2 mL physiological saline (control) or six injections (at 12 h intervals) constituting 16 or 5.4 AU pFSH. The does were mated and subjected to embryo collection 6 to 7 d later. Throughout the experiment ovarian function (by ultrasonography) and plasma levels of inhibin antibodies and progesterone were monitored. Of 40 does treated during the first part of the experiment, 48% showed estrus. The ovarian response in does treated with a high or low dose of eCG or a low dose of pFSH was barely in excess of the ovarian response in the saline-treated controls, whereas a superovulatory dose of pFSH (16 AU) gave a satisfactory response of, on average, 14.5 ovulations (yielding 8.8 flushed ova and embryos). Immediately after the does had been subjected to embryo collection they were actively immunized against inhibin by administering two injections of a recombinant α-subunit of ovine inhibin at four week intervals. All immunized does produced antibodies with the maximal titer reached two weeks after the second injection. Groups of immunized does were subjected to the same gonadotropin treatments as before (avoiding allocation of individuals to the same treatments). This time all does showed estrous symptoms. The ovulatory response to the various treatments, including the saline controls, was virtually identical, the overall average being 21.8 follicles and 9.1 ovulations. The average embryo yield per doe was 5.7. The results imply that inhibin acted as the key factor in determining the ovulatory response since no impact of any of the supplementary gonadotropins was noted in inhibin-immunized does. This finding gives rise to the notion that inhibin antibodies may act primarily by an intraovarian paracrine action rather than by reducing the suppressive action of inhibin on pituitary FSH release. Further, these findings confirm earlier reports that eCG is less suitable than FSH for inducing superovulation in goats, and indicate that active immunization against inhibin may be considered a viable alternative to using exogenous gonadotropin for inducing superovulation in goats.

Effects of day 0 protocol for superovulation on embryo yields and ovarian responses in Kilis and Angora goats

Effects of day 0 protocol for superovulation on embryo yields and ovarian responses in Kilis and Angora goats

고추의 소포자 배양 시 배지 첨가와 진탕이 배의 생산에 미치는 영향

The influences of the agitation as well as the addition of medium during culture on the production of embryos were invested in isolated microspore culture of hot pepper (Capsicum annuum L.). When the culture medium was added during initial liquid culture step of liquid-double layer culture, the embryo yield and quality greatly increased. The most effective time point for medium addition was 5 days after the culture commenced. On the other hand, the effect of medium addition at later double layer culture step in liquid-double layer culture on the embryo production was less compared to that of medium addition during the initial liquid culture step. Agitating the culture for 1 week during later double layer culture step in liquid-double layer culture effectively increased the production of normal cotyledonary embryos. In the case of liquid culture, agitating the culture for 1 week from 7 days after the culture commenced was also effective for embryo development. However, when the total agitation time was longer (2 to 3 weeks) during liquid-double layer culture or liquid culture, the embryos developed abnormally in both cases. The normal cotyledonary embryos obtained in this study successfully developed to plants when transferred to regeneration media. These regenerated plants were either diploid or haploid, and there was a difference in the number of chloroplasts between guard cells of diploid and haploid. These results can be used as an important data for developing an efficient microspore culture system with high quality embryo production in hot pepper.

Effect of culture condition and cell-permeable superoxide dismutase on levels of reactive oxygen species (ROS) production in “in vitro” produced sheep embryos

This study was carried out to evaluate the pattern of reactive oxygen species (ROS) production variation during in vitro oocyte development and maturation and embryonic development in sheep, and to investigate whether embryo culture conditions and the presence of cell-permeable superoxide dismutase mimetic [Manganese (III) meso-tetrakis (4-benzoic acid) porphyrin (MnTBAP)] can influence the ROS production pattern. Oocytes at the germinal vesicle (GV), germinal vesicle breakdown (GVBD) and metaphase II (MII) stages and fertilized embryos at different stages of development (2, 4, 8 and 16-cell, morula, blastocyst and hatching) in two embryo culture media [modified synthetic oviductal fluid (mSOF) or tissue culture medium (TCM199)] were used for assessment of the ROS levels, using a 2,7-dichloro dihydro flourescein diacetate (DCHFDA) probe. A dose of 200 μM MnTBAP was added to the mSOF before, after or before/after embryo arrest (4th embryonic cell cycle) or to the TCM199 media throughout the culture period. ROS levels in immature and unfertilized oocytes were significantly lower, when compared to all fertilized embryonic stages ( P < 0.05). Maximum ROS levels were detected in the morula stage embryos with TCM199 (77.9 ± 1.7) and for the 16-cell stage embryos in mSOF (75.2 ± 2.6). Blastocyst formation was concomitant with a sharp decrease in the ROS production level. The addition of MnTBAP did not significantly decrease the ROS levels and embryonic development. In conclusion it can be said that (i) fertilization triggers an increase in ROS levels, which peaked around the embryo arrest time window, (ii) embryo culture conditions do not confer significant changes in the ROS production pattern, accompanied by a subtle change in surge time, and (iii) MnTBAP does not affect the ROS levels and embryo yield rate.

Ovarian response and embryo yield of Angora and Kilis goats given the day 0 protocol for superovulation in the non-breeding season

The aim of this study was to compare ovarian response and embryo yield of Day0 protocol in Angora goats (AG) and indigenous Kilis goats (KG) in the non-breeding season. A total of 16 Angora goats (AG group) and 11 Kilis goats (KG group) were used in this study. In the synchronization process, after controlled internal drug release withdrawal, when estrus signs were observed, natural mating was performed. Ovarian response was determined by synchronized laparotomy 6days after natural mating, and number of corpora lutea (CL) was recorded. Embryos were collected and morphologically evaluated by stereomicroscope. Synchronization rates did not differ between AG (88%, 14/16) and KG group (91%, 10/11). In AG and KG groups, the proportion of CL on the right (44% and 53%, respectively) and left (56% and 47%, respectively) ovaries were similar. The CL number per animal did not differ significantly between the two breeds and was determined as 4.4 ± 0.90 in AG group and 6.4 ± 1.44 in KG group. Transferable embryo yields were significantly higher in AG group (31/42, 74%) compared to KG group (16/46, 35%) in the non-breeding season (P < 0.01). In conclusion, it is suggested that the day0 protocol can be used for goat superovulation in the non-breeding season; however, transferable embryo yields are affected by the breed.

Effects of Guaiazulene on In Vitro Bovine Embryo Production and on mRNA Transcripts Related to Embryo Quality

Reactive oxygen species (ROS) are between the major contributors for the reduced rate of in vitro bovine embryo production. It is believed that they can cause abnormal meiosis of oocytes, developmental arrest or cell death of embryos. Reports on the effectiveness of various antioxidants on embryo yield are rather conflicting mainly due to the nature and the concentration of the substances used. Here we report the effects of guaiazulene--an exogenous antioxidant, without known properties that could interfere with the biological process of IVF--on embryo development and on the quality of the produced blastocysts. Bovine cumulus oocyte complexes (COCs) were aspirated from abattoir ovaries and COCs were matured in TCM199 with FCS and EGF at 39 °C under an atmosphere of 5% CO(2) in air, with maximum humidity. After 24 h oocytes were inseminated with frozen/thawed semen and co-incubated for further 24 h. Zygotes were cultured in groups of 25 in 25 μl of SOF with 5% FCS at 39 °C under an atmosphere of 5% CO(2) , 5% O(2) in air with maximum humidity. In the first experiment the maturation medium was modified with addition of 0.1 mM of G (n = 497), or 0.01 mM of guaiazulene (n = 468), 0.05% DMSO--the guaiazulene diluent (Control(+), n = 467), and 459 oocytes were used as Control(-). In the second experiment, the culture medium was modified with the addition of 0.1 mM of guaiazulene (n = 344), 0.01 mM of guaiazulene (n = 345), 0.05% DMSO (Control(+), n = 347) and 355 were the Control(-). Blastocyst yield was recorded on days 6, 7, 8 and 9. Day 7 blastocysts from each experiment and group were snap frozen and stored for mRNA extraction. Quantification of transcripts for mRNA of genes related to metabolism (AKR1B1, PTGS2, GADPH, SLC2A5, G6PD); oxidation (GPX1); and implantation (PLAC8) was carried out by real time quantitative RT-PCR. Data for embryo development and on transcript abundance were analysed by χ(2) and anova respectively. In the first experiment no differences were found between groups in terms of cleavage rate (Control(-): 74.20%; Control(+): 74.58%; 0.01 mM: 71.61%; 0.1 mM: 71.63%) or day 9 blastocyst yield (Control(-): 28.26%; Control(+): 25.80%; 0.01 mM: 25.86%; 0.1 mM: 25.25%). In the second experiment, cleavage rate tended to be higher in 0.01 mM group than in Control(-) (77.87% vs 71.41% respectively, p = 0.07). No other differences were detected in cleavage rate (Control(+): 71.32%; 0.1mM: 72.75%) or in the overall blastocyst yield on day 9 (Control(-): 25.50%; Control(+): 26.71%; 0.01 mm: 29.58%; 0.1 mM: 25.75%). In both experiments the relative abundance of genes studied varied between groups but these differences were not statistically significant. Our results imply that oxidation has minimal effect on the in vitro embryo production. Guaiazulene, a compound possessing no biological properties other than those of a strong antioxidant, while it increased cleavage rate, it failed to improve either the blastocyst formation rate, or the quality of the produced embryos under 5% O(2) .

Variation of gynogenic ability in passion fruit (Passiflora edulis Sims.) accessions

With 3 figures and 3 tables Abstract Haploid induction in passion fruit via gynogenesis provides inbred lines that can be used in production of hybrid cultivars. In the present study, gynogenesis was induced in 11 hybrid lines of passion fruit from Southern Brazil. The overall goal of this experiment was to characterize the gynogenic responsiveness of 10 hybrids and a particular accession (II-10), derived from line CI-6, a highly responsive accession. The results from the gynogenic induction of 3226 ovules cultured showed significant differences among genotypes. The accession II-10 was highly responsive, showing a mean yield of 21 haploid embryos per 274 cultured ovules (7.67%). The high embryo yield of this genotype was determined by its parental particular genotype. However, the induction rate of gynogenic embryos obtained from the hybrid plant (I-20), which is derived from the responsive plant (CI-6) also produced gynogenic embryos (5.02%) and several calli lines. The choice of genotypes as donor plants and inheritance for successful gynogenesis are discussed. The haploidy (2n = 1x = 9) of gynogenic plants was confirmed by cytological analysis.

Effect of different FSH/LH ratios on superovulatory response and embryo yield in goats

Previous studies carried out in sheep (Chupin et al., 1985) and in goats (Nowshari et al., 1995; Martemucci et al., 1996) have underlined the importance of both FSH and LH to induce a good superovulatory response, but the results on the LH amount necessary to give a higher ovulatory response and embryo production are rather contradictory. The aim of this study was to evaluate, in goats, the effect of 2 different FSH/LH ratios (1:1 vs 2:1) kept constant during the treatment, on ovulatory response and embryo production. Moreover, according to the FSH/LH ratio=2:1,.........

The parthenogenetic development of buffalo (Bubalus bubalis) oocytes after chemical stimulation

In buffalo the overall in vitro embryo production efficiency is lower than cattle, mainly due to the lower cleavage rate (Gasparrini, 2002). In fact in our experience, comparable embryo yields are obtained in the two species only in relation to the cleaved oocytes. It is thought that a major limiting factor is the quality of the frozen semen. It has been demonstrated that freezing results in acrosomal damage, leakage of enzymes, alterations in ionic strength and pH, complete withdrawal of the hydration shell of protein in the solution and loss of motility (Meur et al., 1988). On the other hand we cannot role out that the lower cleavage rate is related to the failure in the accomplishment of oocyte competence after maturation. In fact although nuclear maturation is successfully achieved in our current system

A high throughput Brassica napus microspore culture system: influence of percoll gradient separation and bud selection on embryogenesis

Microspore culture for the purpose of developing doubled haploid plants is routine for numerous plant species; however, the embryo yield is still very low compared with the total available microspore population. The ability to select and isolate highly embryogenic microspores would be desirable for high embryo yield in microspore culture. To maximize the efficiency of canola microspore culture, a combination of bud size selection and microspore fractionation using a Percoll gradient was followed. This approach has consistently given high embryo yields and uniform embryo development. Microspores isolated from buds 1.5 to 4.4 mm in length of Brassica napus genotypes Topas 4079, DH12075, Westar and 0025 formed embryos at different frequencies. The most embryogenic bud size range varied with each cultivar: Topas 4079 3.5–3.9 mm, DH12075 2.0–2.4 mm, and Westar and 0025 2.5–2.9 mm. When the microspores from 2.0 to 2.4 mm buds of DH12075 were carefully layered on top of a discontinuous Percoll gradient of 10, 20 and 40%, and subsequently spun through the Percoll layers by centrifugation, bands were formed containing populations of microspores of uniform developmental stage. The middle layer of the gradient contained the late uninucleate and early binucleate microspores that were the most embryogenic. In addition, the relationship between the bud size, developmental stage of isolated microspores, Percoll gradient concentration and the embryogenic frequency of each cultivar were studied. Optimization of these factors is required for each genotype evaluated.

Improvement of microspore culture method for multiple samples in Brassica

Microspore culture is an important method for production of haploids and doubled haploids. Although the routine protocol of isolated microspore culture of Brassica species has been established, the protocol for a large number of genotypes is laborious. In this paper, we report an improvement of the microspore culture method for dealing with multiple samples. The improved method showed the same results as the conventional one in the number of isolated microspores per bud and embryo yield per dish. The improved protocol is easier and can deal with two to four times more genotypes than the conventional one during the same period. This method provides some advantages to plant breeders and/or geneticists.

고추의 소포자 배양 시 전처리 후 배지의 교환, 배지의 첨가 및 2층배양 시 하층고체 배지의 양이 배의 생산에 미치는 영향

The effect of the addition of the fresh medium, volume of under solid medium in double layer culture as well as the medium change after pretreating microspores on the production of embryos in microspore culture of hot pepper (Capsicum annuum L.) has been studied. When cultured after heat pre-treatment, changing pretreatment media with fresh culture media proved to be more effective for embryo production rather than supplementing additional culture media. Heat-pretreating for 3 days turned out more effective for embryo production than pretreating for 1 or 2 days. In the case of anther pretreatment, the addition of fresh medium after culture was not effective for embryo production. In pretreating microspores, however, supplementing additional fresh culture media greatly improved embryo yield and quality. The best time point of media addition was 4 days after culture commenced, and the most effective number of times of media addition was one time addition. Moreover, the effective volume of added medium in double layer culture for embryo production was 1.5 ml. The addition of media more than 1.5 ml reduced both embryo yield and quality. Double layer medium was more effective for embryo development than liquid medium. When the volume of under solid medium increased ranging from 3 ml to 7 ml, more cotyledonary embryos were produced in either 5 ml or 7 ml compared to 3 ml, even though the total number of embryos were highest in 3 ml. These results can be used as an important data for establishing an efficient microspore culture system for producing high frequency of normal embryos in hot pepper.

Effects of Season and Superovulatory Treatment on Embryo Yields in Fine‐Wool Merinos Maintained Under Field Conditions

The aim of the study was to assess the effects of superovulatory treatment (multiple FSH-dose vs single-shot FSH treatment) and seasonality on embryo yields in fine-wool Merino ewes. Treatment based on multiple FSH-dose consisted of 200 mg of FSH (Folltropin(®)) administered in seven decreasing doses. Single-shot treatment consisted of a single dose of 70 mg of FSH + eCG. In ewes treated with multiple FSH doses, number of recovered embryos was higher (6.0 ± 0.5 vs 3.5 ± 1.0), while non-fertilization rate was lower (12.8 ± 3.9 vs 40.3 ± 9.5) during the breeding season when compared to the non-breeding season (p < 0.05); although similar values of recovered Grades 1-2 embryos were observed between seasons. During the breeding season, proportion of responding ewes (98.1 vs 57.1%), ovulation rate (13.9 ± 0.8 vs 3.2 ± 1.2), recovered structures (7.9 ± 0.6 vs 1.7 ± 0.7), total recovered embryos (6.0 ± 0.5 vs 1.2 ± 0.6) and good-quality embryos (5.1 ± 0.5 vs 0.9 ± 0.6) were higher for the multiple FSH-dose treatment than for the single-shot protocol. In a similar way, in the non-breeding season, ovulation rate (11.3 ± 1.8 vs 6.0 ± 1.1) and recovered structures (6.6 ± 1.2 vs 2.7 ± 0.6) were higher for the multiple FSH injections protocol than those for the single-shot treatment, resulting in higher recovered Grades 1-2 embryos (3.2 ± 0.9 vs 1.4 ± 0.5). Current results indicate that seasonal anestrus affected embryo yields when applying multiple FSH-dose superovulatory treatment in Merino ewes, by decreasing the number of recovered embryos although the number of recovered good-quality embryos was not affected. During both seasons, multiple FSH injections produced higher ovarian response and number of viable embryos than the single-shot treatment.

127 EFFECT OF DIETARY FATS ON EMBRYO QUALITY AND YIELD IN HEAT-STRESSED DAIRY CATTLE

The thermal environment is a major factor that can negatively affect production and reproduction of dairy cattle (Kadzere et al. 2002 Livest. Prod. Sci. 77, 59–91). Heat stress (HS) at and immediately after the time of breeding may result in a lower conception rate (Epperson and Zalesky 2007; http://wrac.sdstate.edu/pubs/ansci/ExEx2018.pdf). High temperatures compromise endometrial function and alter its secretory activity, which may lead to termination of pregnancy (Wolfenson et al. 2000 Anim. Reprod. Sci. 60–61, 535–547). One of the strategies to reduce thermal environment stress is improved nutritional management schemes like increasing dietary fat (Umphrey et al. 2001 J. Dairy Sci. 84, 2680–2685). Our objective was to determine (define) effects of protected fats on ovulation rate, embryo yield, and quality in heat-stressed Holstein cattle in Turkey. Twenty Holstein cows were assigned to control and treatment groups (86.50 ± 8.0 and 83.17 ± 10.8 DIM, respectively). Intravaginal devices (PRID) were placed in all cows (Day 0) and were superstimulated with twice-daily treatments with FSH (5, 4, 3, and 1 mL, respectively) on days 9 to 12. Also cows were treated with PgF2α (Dynolytic, 2 mL) twice on day 11. On the night of day 12, PRID were removed and animals were artificially inseminated on days 13 and 14. On day 20, inseminated cows were palpated per rectum and flushed transcervically. Following uterine flushings, each cows received PgF2α (Dynolytic, 2 mL) to induce luteolysis of developing corpora lutea. Embryos were scored and quantified, and good-quality embryos were fixed to determine embryonic cell counts. Two-twelve cell embryo and blastocyst counts were statistically different between seasons (P &lt; 0.01 and P &lt; 0.05, respectively); only 2-12 cell embryos were different between groups (P &lt; 0.01) and only 2-12 cell embryos were different (P &lt; 0.05) for season × treatment interaction. In the cool season experiment, morula and expanded blastocyst embryos were higher; and in the hot season experiment, unfertilized egg and morula embryos were higher for treatment group than those of controls. Also in the hot season experiment, total embryo yield was higher than the control group. However, dietary fats reduced rectal temperatures in these experiments. According to these results, we can assume that high rectal temperatures caused changes in blood parameters and very early embryonic deaths occurred in the control group. Consequently, dietary fats added to dairy cattle rations in hot seasons can positively affect embryo quality and embryo yield in dairy cattle.

230 OVUM PICKUP - IN VITRO PRODUCTION EFFICIENCY FOR CONSERVATION OF THE FLAMENGA CATTLE BREED

The introduction of more specialised and productive dairy breeds into commercial herds in recent decades has caused a gradual loss of interest in dual-purpose cattle breeds, such as the Flamenga. Because of its importance to biodiversity, the high risk of loss of this small Brazilian herd, located in one geographic region in Southern Brazil, justifies the need for and efforts in genetic conservation of the Flamenga breed. The ovum pickup (OPU) procedure is a potential tool for such conservation purposes. Thus, the aim of this study was to evaluate the oocyte and embryo yield potential of the Flamenga breed, as compared with Holstein control counterparts, after in vivo oocyte retrieval followed by in vitro embryo production (IVP). Four Flamenga and 4 Holstein multiparous milking cows from the same herd were subjected to weekly ultrasound-guided OPU sections for 10 consecutive weeks. The number of visible follicles per ovary per female was recorded before the aspiration of ≥3-mm follicles. Retrieved cumulus–oocyte complexes (COC) were morphologically graded according to the method of (Leibfried and First 1979 J. Anim. Sci. 48, 76–86), and viable COC (Grades I, II, and III) from both breeds and from slaughterhouse ovaries (IVP controls) were used for the IVP of embryos, based on our established procedures (Vieira et al. 2002 Cryobiology 45, 91–94). Frozen semen from the same Flamenga breed bull was used in all groups. Quantitative (visible and aspirated follicles, collected COC), and qualitative (COC recovery efficiency, cleavage and blastocyst rates) data were compared between breeds and IVP controls (when pertinent) by Student’s t-test and chi-square test, respectively, for P &lt; 0.05. Results are presented in Table 1. The pools of visible and aspirated follicles were similar between breeds. However, the COC recovery efficiency was higher for Flamenga than for Holstein females. In vitro embryo development was also different between breeds, with cleavage rate being significantly lower for Holsteins than for Flamenga and IVP controls. Blastocyst rates also differed between treatments. Nonetheless, development to the blastocyst stage, when based on cleavage, was similar between Flamenga and Holstein embryos, but with both breeds being lower than the IVP controls. Oocyte quality was likely the cause of such differences in embryo developmental potential between groups, with Flamenga females yielding more oocytes and IVP embryos than Holstein cows. In summary, the use of OPU-IVP procedures was demonstrated to be suitable for the preservation of the Flamenga breed in Southern Brazil. Table 1.Follicular pool and oocyte and embryo yield between Flamenga and Holstein cows following ovum pickup–in vitro production (IVP) procedures This study was supported by CAPES and CNPq/Brazil.

125 EFFECT OF GLYCERALDEHYDE-3-PHOSPHATE DURING BOVINE IN VITRO EMBRYO CULTURE

Most systems for producing mammalian embryos in vitro use glucose as an energy source in the media despite putative toxic effects (Schini and Bavister 1988 Biol. Reprod. 39, 1183–1192; Takahashi and First 1992 Theriogenology 37, 963–978). Currently there is a tendency to identify other suitable energy sources in an attempt to replace glucose from culture media. Glyceraldehyde-3-phosphate (G3P), a glucose-derived high-energy compound, is the end product of the energy-consuming phase of glycolysis that enters the pay-off phase of the pathway characterised by a net gain of energy. The aim of this study was to determine whether G3P is a valid energy source for supporting in vitro embryo development in cattle. Abattoir-derived oocytes (n = 832, over 4 replicates) were matured in vitro in TCM-199 with 15% bovine serum (BS), 0.5 μg mL–1 FSH, 5 μg mL–1 LH, 0.8 mM L-glutamine, and 50 mg mL–1 gentamicin. Mature COC were fertilized in Tyrode’s modified medium, with 30 mg mL–1 heparin, 30 mM penicillamine, 15 mM hypotaurine, 0.15 mM epinephrine, and 1% BS. Both IVM and IVF were carried out at 39°C and 5% CO2 in air. After 20 to 22 h of gamete co-incubation, presumptive zygotes were denuded and cultured in SOF containing either 1.5 mM glucose (control group) or G3P at 3 different concentrations (0.125, 0.5, and 1.5 mM). It is worth specifying that in the 3 G3P-supplemented groups small amounts of glucose were left (0.15 mM) because it is known that a complete removal would affect embryo development by interfering with ribose synthesis through the pentose–phosphate pathway. In vitro culture was carried out at 39°C under humidified air with 5% CO2, 7% O2, and 88% N2 in air for 7 days, when the percentages of tight morulae-blastocysts (TMBL) and superior quality blastocysts (BL) were recorded. Differences in embryo yields among groups were analysed by chi-square test. Supplementation of IVC medium with 1.5 mM G3P reduced (P &lt; 0.01) TMBL (5.0%) and BL (5.0%) rates compared with all other groups, indicating a toxic effect. However, when G3P was added at lower concentrations, no differences in TMBL (37.3 and 26.1, respectively, with 0.125 and 0.5 mM G3P) and in BL rates (35.3 and 25.5%, respectively, with 0.125 and 0.5 mM G3P) were observed compared with the control (32.7% TMBL and 31.4% BL, respectively). Within G3P-treated groups, the higher embryo yields were recorded with 0.125 mM compared with 0.5 mM (P &lt; 0.05) and 1.5 mM (P &lt; 0.01). Interestingly, embryos produced with G3P at the lower concentrations (0.125 and 0.5 mM) seemed to show a faster development compared with the control. In conclusion, these results demonstrated that G3P is a valid energy source for bovine preimplantation embryos and, hence, that G3P supplementation of IVC medium may be a suitable option for reducing glucose concentration in the media. However, further studies are needed to investigate lower concentrations of G3P and to better evaluate embryo viability.

318 EFFICIENCY OF PROTOCOL P-36, ASSOCIATED WITH EQUINE CHORIONIC GONADOTROPIN OR LUTEINIZING HORMONE ADMINISTRATION, IN THE LAST DAY OF SUPERESTIMULATORY TREATMENT, IN NELLORE COWS

The objective of this study was to evaluate the use of pLH in replacement of eCG on the last day of P-36 superstimulatory treatment in Nellore donors. Recent studies demonstrated improvement in embryo production when the last 2 doses of FSH were replaced by eCG. However, consecutive use of eCG in bovine superstimulatory protocols may induce antibody against eCG, decreasing embryo production. Twenty-five Nellore cows were randomly allocated in 4 groups: P-36 (control), P-36/eCG, P-36/LH2, and P-36/LH4. All animals underwent 4 treatments in a cross-over design. Donors received an intravaginal device containing 1.0 g of progesterone (IVD, Primer®, Tecnopec, Sao Paulo, Brazil) and oestradiol benzoate (2.0 mg, IM; Estrogin®, Farmavet, Sao Paulo, Brazil) at a random stage of the oestrous cycle (D0). Cows from the control group were superestimulated with decreasing doses of pFSH (133 mg, IM; Folltropin-V®, Bioniche, Ontario, Canada; D5-8). In the P-36/eCG group, the last 2 doses of pFSH were replaced by 2 doses of eCG (200 IU each dose, IM; Folligon®, Intervet, Boxmeer, the Netherlands). In the P-36/LH2 and P-36/LH4 groups, the last 2 doses of pFSH were replaced by 2 doses of 1 and 2 mg of pLH, respectively (IM; Lutropin®, Bioniche). All animals were treated with prostaglandin F2α (150 μg d-cloprostenol, IM; Prolise®, Tecnopec) on D7, and the IVD was removed 36 h after. Ovulation was induced with 12.5 mg of pLH (IM) on D9, and all animals received fixed-time artificial insemination 12 and 24 h after pLH. Embryo flushing was performed on D16. Data were analysed by ANOVA (Proc Mixed, SAS). There was a significant difference in the number of corpus luteum in the eCG group (19.2 ± 2.4) when compared with the LH2 (12.7 ± 2.0) and LH4 groups (12.3 ± 1.5; P &lt; 0.05). In addition, there was a tendency of lower ovulation rate in the LH2 group as compared with the eCG group (50.6 and 67.8%, respectively; P = 0.06). However, there was no difference in viable embryo yield among groups P-36 (3.3 ± 0.7), P-36/eCG (4.5 ± 0.5), P-36/LH2 (3.7 ± 0.8), and P-36/LH4 (4.2 ± 1.0); P &gt; 0.05. In conclusion, eCG can be replaced by pLH (4.0 mg), in the last day of P-36 protocol, without affecting the production of viable embryos in Nellore cows. The authors acknowledge FAPESP (Sao Paulo, Brazil) for funding and fellowships for A. C. S. Oliveira, M. C. C. Mattos, and M. R. Bastos.

Influence of climatic conditions on in vitro production of bovine embryos

Temperature and rainfall were analyzed daily during six years to evaluate their influence on in vitro production of bovine embryos. Weekly replications (n=480) were performed on 14,778 ovaries collected at slaughterhouses. Cumulus oocyte complexes (n=19,180) were fertilized with a pool of Bos taurus taurus semen in one incubator with 5% CO2. Presumable zygotes were cultured in gasified plastic bags with 5% CO2, 5% O2, and 90% N2. In the first year, cleavage and embryo yield were 60.3% and 15.6%, respectively, being lower (P&lt;0.05) than in the following years. Average cleavage rates were always lower in winter (P&lt;0.0001), thus producing less embryos. Winter climatic conditions had a negative influence on in vitro production, when cleavage and embryo yield declined, possibly because of reduced availability and growth of native pasture.

Day 2 embryo transfer (ET) and day 3 ET afford similar reproductive outcomes in the poor responder

A retrospective review of 237 initial, fresh nondonor IVF cycles in which all embryos generated during the cycle were transferred on either day 2 (n = 109) or day 3 (n = 128) were evaluated with regards to reproductive outcomes. Patients who underwent a day 2 ET had similar conception (18% vs. 16%; relative risk [RR], 1.1; 95% confidence interval [CI], 0.64-1.95), clinical pregnancy (13% vs. 16%; RR, 0.8; 95% CI, 0.44-1.55), implantation (6% vs. 7%; RR, 0.9; 95% CI, 0.50-1.68), and live-birth (10% vs. 16%; RR, 0.7; 95% CI, 0.32-1.29) rates as those who underwent a day 3 ET.