In Vitro Embryo Production Efficiency Research Articles

PSXIII-14 Effect of melatonin supplementation during in vitro embryo culture on cleavage rates following in vitro maturation and fertilization of bovine oocytes

Abstract In vitro embryo production (IVEP) is an important advanced reproductive technology and has great potential in enhancing livestock production. However, in vitro culture (IVC) of embryos exposes them to stress resulting in compromising quality and efficiency of embryo production. Melatonin has been reported in several studies to reduce oxidative stress and improve the efficiency of IVEP. Our objective was to evaluate the effect of melatonin supplementation in IVC media on cleavage rates of bovine embryos. Bovine ovaries (n = 22) were obtained from local abattoir with ovarian weight of 7.29 ± 3.31 g, and size of 3.65 ± 0.69 cm. Cumulus-oocyte complexes (COCs) were recovered from 2 to 8 mm size follicles by aspiration using an 18-gauge needle attached with a 10 mL syringe. COCs were matured, fertilized, and cultured in vitro using IVF Bioscience bovine media and their protocols. Oocytes were in vitro matured for 23 h at 38.8°C, 5.5% CO2 in a humidified atmosphere and assessed for maturation by assessing COC expansion. Fully expanded and partially expanded COCs were fertilized for 20 h in a 4-well dish with frozen/thawed bull semen. Following IVF, the cumulus cells were removed by vertexing, and the presumptive zygotes were cultured in vitro with and without 1 µM melatonin in 4-well dishes in triplicates under the oil for embryo development at 38.8°C and 5.5% CO2. Cleavage rates were determined on d 3 of culture. The percentage of cleaved embryos was calculated as (number of zygotes cleaved/total number of fertilized oocytes) x100. Data were analyzed by student’s unpaired t- test with Welch’s correction using GraphPad Prism software. The cleavage rate was greater in melatonin supplemented group (54.17 ± 11.24%) than the control (49.83 ± 7.36%) but the difference was not significant (P > 0.05). In conclusion, under our experimental conditions, melatonin supplementation during embryo culture, following IVM/IVF of the bovine oocytes, did not significantly improve cleavage rates.

Outstanding Gir oocyte donors: How does individual factor affect in vitro embryo production efficiency?

The oocyte donor plays a pivotal role in bovine in vitro embryo production (IVP) success. The individual factor affects blastocyst/oocyte ratio and determine the existence of outstanding performing animals. The aim of this study was to assess the extent of individual factor effect to IVP efficiency, in a population of Gir oocyte donors. Extreme (high or low IVP efficiency based on blastocyst/oocyte ratio) animals were selected out of a population of 250 oocyte donors (1,734 observations) to form high (>0.48, n= 40), average (0.17-0.48, n= 168), and low (<0.17, n= 42) efficiency donor groups. Cumulus-oocyte complex indicators (total number, IVF-grade number, and IVF-grade/total ratio) were lower (p< 0.05) in high efficiency donors. The number of blastocysts per OPU was analyzed for highest performing bull, and an increase (p< 0.05) in high efficiency donors and a decrease (p< 0.05) in low efficiency donors were noticed, compared to average efficiency donors. The number of pregnancies obtained per OPU was affected (p= 0.017) by donor's efficiency (low: , average: , high: ), being 4.3-fold higher in high than in low efficiency donors. We conclude that producing embryos from high efficiency blastocyst/oocyte ratio donors increases blastocyst and pregnancy numbers by OPU, being an important indicator for donor selection in IVP programs.

Detection of Brucella abortus during in vitro bovine embryo production

The in vitro embryo production (IVP) technology has emerged as a potential biotechnological approach to multiply genetically high yielding dairy cows. Its commercial application is increasing in many developed and developing countries over the years. Bangladesh livestock Research Institute (BLRI) adopted in vitro embryo production protocol from bovine ovaries of slaughterhouse. However, the risks of transmission of contagious diseases like Brucella abortus with embryos are not evaluated so far. Considering these facts, the present experiments were conducted to evaluate the efficiency of in vitro embryo production protocol with slaughterhouse ovaries as well as risk of contamination of produced embryos with Brucella abortus. To identify sources of contamination of embryos with Brucella abortus (if any), the laboratory water, different media used in the IVP process, semen, and follicular fluids were evaluated for confirmation of the organisms. In addition, vaginal swabs were collected from 2 buffaloes aborted due to suspected Brucella abortus infection. Molecular test were used to detect Brucella abortus contamination. Brucella abortus specific PCR product was not detected on agarose gel electrophoresis. The efficiency of IVP measured by cleavage and blastocyst development rates were 75.5±2.7% and 16.6±3.9%, respectively. The present study inferred that the in vitro produce embryos are free from Brucella abortus infection. Bang. J. Livs. Res. Vol. 27 (1&amp;2), 2020: P. 105-112

Increased antral follicle population and in vitro embryo production in pregnant Holstein

This study used Holstein cattle to evaluate the following: I) the productive and reproductive characteristics of cows with low, intermediate and high antral follicle counts (AFCs) that were subjected to artificial insemination (AI) and comparison of AFC variability at the time of AI (nonpregnant) and at two time points during pregnancy (30 and 60 days), and II) whether the pregnancy status and different pregnancy periods in heifers improves the efficiency at in vitro embryo production (IVEP). In study I, 75 high-milk production cows with body condition scores (BCSs) of 2.75 to 4.75 and ages of 23 to 99 months were selected. On the day of estrus, AFCs (follicles ≥ 3 mm), BCS, body weight, and diameters of the dominant follicle, ovaries and the corpus luteum were evaluated. Posteriorly, AFC was monitored in pregnant cows at 30 days (n = 35) and at 60 days for comparison of AFC before pregnancy versus different periods. In study II, heifers (n = 9) with BCS of 2.5 to 3.5 and ages of 10 to 16 months were selected for ovum pick-up and an IVEP program before AI (nonpregnant) as well as at 0 to 30, 31 to 60, and &gt; 60 days of pregnancy. Statistical analysis was performed using PROC GLM and binary logistic regression model (P ≤ 0.05). In study I, AFC was not associated with variations in productive and reproductive parameters. However, AFCs in the low group increased following pregnancy (AI: 14.82 ± 1.36, 30 days: 23.45 ± 2.31 and 60 days: 35.18 ± 3.17 follicles). AFCs increased from AI to 60 days in the intermediate group but did not vary among time points in the high group. AFCs varied among AFC groups in AI and at 30 days of pregnancy but not at 60 days. In study II, the mean of total oocytes (17.43 ± 4.52 vs. 41.67 ± 3.79) and viable oocytes (13.77 ± 3.63 vs. 30.56 ± 3.45) increased from AI (nonpregnant) to the first 30 days of pregnancy. The mean number of embryos produced increased during pregnancy and during the first 30 days of pregnancy (4.55 ± 0.75) compared to the nonpregnant period (1.39 ± 0.85). In conclusion, pregnancy positively influences AFC, and the greatest effect is noted in low count cows. In addition, the OPU at the time of pregnancy increased the efficiency of IVEP in heifers.

Cattle breed affects in vitro embryo production in a large-scale commercial program on dairy farms

Sexed cryopreserved semen is widely used in vitro embryo production (IVEP) for cattle reproduction in Brazil. However, differences between donor breeds regarding variables related to the IVEP process have been reported. This study aimed to evaluate factors affecting the efficiency of IVEP using donors of different genotypes and sexed sperm from Gir and Holstein bulls in a commercial program of IVEP. Higher oocyte and blastocyst means per OPU session were observed in ½ HolsteinxGir donors. Gir donors showed higher percentage of blastocyst than Holstein donors, with blastocyst production of crossbreed donors being similar to the other breeds. Moreover, embryos in the stage of expanded blastocyst and hatching blastocyst had a higher capacity of resulting in pregnancy than embryos in the blastocyst stage. In relation to the crosses of the donor and the bull used, no effect was found on the percentage of blastocyst and pregnancy. However, donors with greater number of oocytes recovered had a higher number of blastocysts and pregnancies per OPU session. Finally, OPU sessions performed with 21-day intervals for two years did not affect the pe rformance of Gir donors on IVEP. Thus, the conclusion is that donor breed influences the rate of oocyte retrieval. Moreover, embryo production is affected by donor breed and the number of viable oocytes recovered. Only embryo developmental stage influences the pregnancy rate. Finally, bull breed does not affect IVEP results and there was no effect of repeated OPU sessions on Gir donors in these variables. These results provide new insights related to IVEP in large scale.

Beneficial Effects of Melatonin in the Ovarian Transport Medium on In Vitro Embryo Production of Iberian Red Deer (Cervus elaphus hispanicus)

Simple SummaryThe development of in vitro embryo production (IVP) in wild species, such as Iberian red deer, can become a daunting challenge since prolonged ovary transport times to the laboratory are often unavoidable. This may have detrimental effects on the quality and developmental capacity of oocytes. We evaluated the effect of supplementing the ovary transport medium with the antioxidant melatonin and observed an increased level of oocyte intracellular reduced glutathione content. Moreover, melatonin enhanced cleavage and blastocyst rates and had a positive effect on embryo quality in terms of the expression of essential embryo development-related genes. In conclusion, the addition of melatonin to the ovary storage medium could mitigate the negative impacts that long transport times may have on oocyte developmental competence and quality of the resulting blastocysts in Iberian red deer.A major limiting factor for the development of in vitro embryo production (IVP) in wild species, such as Iberian red deer, compared to livestock animals is the poor availability and limited access to biological material. Thus, the use of post-mortem ovaries from slaughtered animals represent a source of oocytes for the large scale production of embryos needed for research and to improve the efficiency of IVP. However, these oocytes are not as developmentally competent as their in vivo counterparts. Moreover, oocytes are usually obtained from ovaries that have been transported for long distances, which may also affect their quality. In order to overcome the issues associated with prolonged storage times of post-mortem material, in this study we examined the effect of melatonin supplementation to the ovary transport medium on oocyte quality, embryo yield, and blastocyst quality in Iberian red deer. When necessary, sheep was used as an experimental model due to the large number of samples required for analysis of oocyte quality parameters. Oocytes were in vitro matured and assessed for early apoptosis; DNA fragmentation; reactive oxygen species (ROS); reduced glutathione (GSH) content, mitochondrial membrane potential, and distribution; and relative abundance of mRNA transcript levels. After in vitro fertilization, embryo rates and blastocyst quality were also investigated. The results revealed that melatonin treatment significantly increased intracellular level of GSH in sheep oocytes. Moreover, the percentage of cleavage and blastocyst yield in red deer was greater compared to the Control group and there was lower abundance of oxidative stress- and apoptosis-related SHC1, TP53, and AKR1B1 mRNA transcripts in blastocysts for the Melatonin group. In conclusion, the supplementation of melatonin to the ovary storage medium had a positive effect on the developmental competence and quality of resulting blastocysts in Iberian red deer.

Likelihood of pregnancy after the transfer of embryos derived from follicle aspiration and in vitro embryo production sessions with different relative efficiencies

The aim of the present study was to evaluate the likelihood of pregnancy of in vitro-produced (IVP) embryos from batches with distinct relative efficiencies. Data were retrospectively analyzed from 605 transvaginal ultrasonic-guided follicle aspiration sessions (OPU) followed by in vitro embryo production (IVEP) and 2456 fresh embryo transfers (ET), performed between 2008 and 2012 in individuals of the Gir (dairy Bos indicus) breed. The OPU and IVEP were performed using standard procedures by a single group of technicians at the same laboratory facility. Records were stratified into quartiles (I to IV) according to the total of cumulus-oocytes complexes (COC) produced per donor, or in percentile ranges (0%–25%, 26%–50%, 51%–75%, and 76%–100%) for endpoints related to COC quality or efficiency of embryo production. Pregnancy per embryo transfer (P/ET) was compared among quartiles or ranges using the chi-squared test. Donors producing a greater number of total COC (quartile I) also had more viable and grade I COC, and a greater number of embryos than donors ranked in quartiles II, III or IV, respectively (P < 0.0001). Nevertheless, P/ET did not differ (P > 0.05) among embryos produced by donors ranked in Quartiles I to IV. Similarly, there was no difference (P > 0.05) in P/ET for embryos derived from OPU sessions with a relatively greater or lesser percentage of viable or Grade I COC. Cleavage and blastocyst rates within each IVEP batch had no effect (P > 0.05) on P/ET. In conclusion, data suggest that there is no relationship among oocyte yield after OPU, or efficiency of IVEP, and the likelihood of pregnancy after ET of fresh IVP embryos.

101 Effects of Active Immunization Against GnRH in Oocyte Donors with Cystic Ovarian Disease

Cows intensively used as oocyte donors for in vitro embryo production (IVEP) are usually kept nonpregnant for prolonged intervals, exposed to successive hormonal treatments, and frequently become overweight. These are all risk factors for the development of endocrine unbalance and, consequently, cystic ovarian disease (COD). The aim of this study was to evaluate the effect of active immunization against gonadotropin-releasing hormone (GnRH) on (1) ovarian follicular population, and (2) development potential of oocytes used for IVEP. Nelore (Bos indicus) cows (n = 14), previously diagnosed with chronic COD (Faria et al. 2017 Anim. Reprod., in press), weighing 620.0 ± 12.8 kg and with body condition score of 4.1 ± 0.2, were assigned to control (n = 6) or treatment (n = 8) group. Cows in the treatment group received 2 SC injections of 1.0 mL of anti-GnRH vaccine (Bopriva, Zoetis, Brazil), 28 days apart (weeks 0 and 4), whereas cows in the control group received placebo on the same schedule. Transrectal ultrasonography was performed weekly from week 0 to evaluate the number and distribution of follicles among size classes, endometrial thickness, and clinical presence of mucometra. Immunization was considered effective (E-IM) when no follicles ≥5.0 mm were observed on the ovaries during a given examination. Cows having E-IM were then used as oocyte donors for IVEP. Cumulus-oocyte complexes (COC) were collected in 5 consecutive ovum pick-up weekly sessions. As a control for IVEP, oocytes from a slaughterhouse were used, with similar procedures performed on the same days and using the same semen batch. The MIXED procedure of SAS (SAS Institute Inc., Cary, NC, USA) with repeated-measures statement was used to evaluate the effects of treatment, time, and interactions on ovarian endpoints; and the GLM procedure was used to analyse embryo production data. Results are shown as mean ± SEM. There were time and time × treatment effects on ovarian parameters. Treated cows had a decrease (P &lt; 0.05) in the average diameter of the largest follicle and in the number of follicles ≥8 mm, and an increase (P &lt; 0.05) in follicular population after week 6. Nonetheless, individual response to treatment was variable: only 50% of the cows (4 of 8) were E-IM at week 8, whereas 25% (2 of 8) still had COD (largest follicle ≥18.0 mm) at this timepoint. Overall, a negative correlation was detected between follicular population and the diameter of the largest follicle (r = –0.60, P &lt; 0.0001) or the number of follicles ≥8 mm (r = –0.47, P &lt; 0.0001). There was no effect (P &gt; 0.05) of treatment on endometrial thickness or mucometra score. Cows with E-IM produced 22.2 ± 3.6 total and 12.9 ± 2.3 viable COC. Cleavage rate did not differ between E-IM and control (slaughterhouse) oocytes (70.8 ± 7.0 v. 75.1 ± 3.0%, respectively; P &gt; 0.05); however, blastocyst rate was greater in the E-IM group compared with controls (39.7 ± 5.5 v. 20.5 ± 4.7%, respectively; P &lt; 0.02). In summary, our results suggest that active immunization against GnRH leads to variable results in the distribution of the follicular population in cows with COD, but it does not negatively affect IVEP efficiency. This research was supported by Zoetis, CNPq, and CAPES.

Beef donor cows with high number of retrieved COC produce more in vitro embryos compared with cows with low number of COC after repeated ovum pick-up sessions

The aim of the present study was to evaluate whether repeated ovum pick-up (OPU) procedures may affect the efficiency of OPU programs for in vitro embryo production (IVEP) in Bos indicus (Nelore) donors. In addition, the repeatability (r) efficiency of IVEP was also assessed. Data available were from 432 OPU-IVEP sessions that were performed at random stages of the estrous cycle in 36 cycling, nonlactating Nelore donors. Semen from three Nelore bulls was used for the IVF. Donors were submitted to 12 consecutive OPU procedures, with an interval of approximately 30 days between sessions. Data were analyzed as repeated measures using the GLIMMIX procedure of SAS 9.3. Cows yielding ≥15 COCs were defined as “high” COCs and cows with less than 15 COCs were defined as “low” COCs donors. The number of COCs retrieved decreased over time in donors classified with high COCs and remained fairly steady in cows with low COCs at the beginning of the program (Psession*COC retrieved groups = 0.02). Moreover, the number of COCs retrieved (P < 0.0001), and number of blastocysts produced per OPU (P = 0.001) was greater for the high COCs donors compared with the low COCs category, and these results were consistent across OPU sessions. Interestingly, there was no effect of COCs category on the rate of blastocyst development (P = 0.83). In addition, number of blastocysts produced were not affected by repeated OPU (P = 0.37) and interactions between time and COC category (P = 0.72). Similarly, blastocyst rate was not affected by repeated OPU (P = 0.21) and interactions between time and COC category (P = 0.58). Despite of COC category of the donor cows, repeatability was high for the number of COCs retrieved (r = 0.81), number of blastocysts produced per OPU (r = 0.79), and blastocyst rate (0.69). In conclusion, overall numbers of COCs decreased over time in donors classified as having high COCs. However, cumulative amounts of produced blastocysts were greater in donors with high COCs. More importantly, high repeatability was observed in terms of IVEP efficiency. Therefore, IVEP programs can be significantly improved with more aggressive selection toward donors with greater numbers of COCs.

241 EFFECT OF PRODUCTION EFFICIENCY IN THE LIKELIHOOD OF PREGNANCY OF IN VITRO-DERIVED BOVINE EMBRYOS

Improving in vitro culture systems to optimize embryo yield has been a major research goal. The relationship between the efficiency of embryo production systems and the pregnancy outcomes, however, remain controversial. The aim of the present study was to evaluate the likelihood of pregnancy of in vitro-produced embryos derived from batches with different relative efficiency indexes. Data of 702 ovum pick-up (OPU) and in vitro embryo production (IVEP) sessions, and of 2456 embryo transfers, recorded from 2008 to 2012, were evaluated. All donors were from the same herd, and were of the same breed (Gir, Bos indicus), as well as the semen used for IVF. The cumulus-oocycte complex (COC) recovery and IVEP were performed by the same team, in a single IVF laboratory, and using standard medium and procedures. Only data from embryos transferred as fresh were used, and records from 97 OPU/IVEP sessions in which no embryo was produced, or embryos were frozen or discharged due to lack of recipients, were discharged. The remaining 605 sessions were stratified in quartiles (I to IV, each one corresponding to 25% of total data) according to COC production of the donors, or stratified in ranges (0–25%, 26–50%, 51–75%, and 76–100%) according to COC quality (percentage of viable COC or of grade I COC) and to embryo production efficiency endpoints (cleavage rate, blastocyst rate). Pregnancy rates were compared among quartiles or ranges by the chi-square method. On average, the Gir donors produced 24.8 ± 0.6 COC per OPU, from which 14.4 ± 0.4 were classified as viable (57.8%), and 3.2 ± 0.1 as grade I (12.9%). On average 6.1 ± 0.2 embryos (morulas and blastocysts) were produced per OPU per donor, and mean pregnancy rate was 30.9%. As expected, donors with greater total COC yield (quartile I) also produced more viable oocytes (25.5 ± 0.7 v. 15.7 ± 0.3, 10.5 ± 0.2 and 5.8 ± 0.2), more COC grade I (4.8 ± 0.4 v. 3.9 ± 0.3, 2.6 ± 0.2 and 1.6 ± 0.1), and more embryos (9.0 ± 0.4 v. 6.9 ± 0.3, 5.0 ± 0.2 and 3.3 ± 0.1) than donors from quartiles II, III, or IV, respectively (P &lt; 0.0001). Nevertheless, there was no difference (P &gt; 0.05) in pregnancy rates for embryos produced from donors ranked in the different quartiles (30.9 v. 29.3, 31.5, and 30.5% for quartiles I to IV, respectively). Similarly, there was no difference (P &gt; 0.05) in the pregnancy rate of embryos derived from OPU sessions in which there was a high or low percentage of viable or grade I COC. In vitro production efficiency (cleavage and blastocyst rates) also had no effect (P &gt; 0.05) on further pregnancy rates. In conclusion, these results suggest that there is no relationship among the average number or quality of the COC recovered by OPU, the efficiency of IVEP, and the likelihood of pregnancy of in vitro-derived embryos.Research was supported by Fazendas do Basa, CNPq, and Fapemig.

Osteopontin improves sperm capacitation and in vitro fertilization efficiency in buffalo (Bubalus bubalis)

The aim of this study was to evaluate the effect of osteopontin (OPN), an ubiquitous acid glycoprotein, on in vitro sperm capacitation and on in vitro embryo production (IVEP) efficiency in buffalo. In experiment 1, after swim-up separation the sperm were incubated in Tyrode albumin lactate pyruvate medium in the absence of capacitating agents (control), with the standard concentration of heparin (0.01 mM) and three different concentrations of OPN (0.1, 1, and 10 mcg/mL), both in the presence and absence of heparin, for 2 and 4 hours. Capacitation was assessed indirectly by estimating the percentage of acrosome-reacted sperm after incubation with lysophosphatidylcholine. In order to determine the effect of OPN, in the presence of heparin, on fertilization (Experiment 2) and in vitro embryo development (experiment 3), in vitro–matured buffalo oocytes were fertilized in the presence of 0, 0.1, 1, and 10 mcg/mL of OPN. After IVF, the presumptive zygotes were dezonated, fixed, stained, and then evaluated microscopically. At Days 5 and 7 of culture, the cleavage and blastocyst rates were evaluated, respectively. Two hours of treatment with OPN at the two higher concentrations (1 and 10 mcg/mL) promoted in vitro capacitation of buffalo sperm (experiment 1). A synergic action of OPN with heparin was also done for all OPN concentrations tested. At 4 hours incubation, all treatments, including heparin (20.4%), improved (P < 0.01) capacitation compared with the control (16.2%). Interestingly, the best results were reported in all groups treated with OPN + heparin (40.8%, 38.6%, and 33.8%, respectively; P < 0.01). The addition of OPN to the IVF medium had a positive influence on total penetration, synchronous pronuclei formation (experiment 2), and IVEP efficiency (experiment 3). In particular, the two lower concentrations of OPN (0.1 and 1 mcg/mL), compared with the control, gave higher synchronous pronuclei formation (73.5%, 75.0%, and 46.5%, respectively; P < 0.01) and cleavage rates (70.3%, 71.6%, and 59.3%, respectively; P < 0.01). Interestingly, the treatments also improved blastocyst yields (29.3%, 30.3%, and 19.4%, respectively; P < 0.01). In conclusion, these results indicate that adding OPN to the IVF system improves IVEP efficiency by enhancing in vitro sperm capacitation and blastocyst yields in buffalo.

322 EFFECT OF PLASMIN ON ACROSOME REACTION OF BUFFALO (BUBALUS BUBALIS) SPERMATOZOA IN VITRO

Fertilization is a critical step of the in vitro embryo production (IVEP) technology in buffalo. It is known that proteolytic enzymes are involved in different steps of the fertilization process; among these, a critical role may be played by the plasminogen activator-plasmin system. It has been demonstrated that plasmin, the active enzyme of this system, induces acrosome reaction (AR) in bull spermatozoa (Taitzoglou IA et al. 2003 Andrologia 35, 112-116). The aim of this study was to investigate the effect of plasmin on the ability of buffalo sperm to undergo the AR. Frozen- thawed sperm from 4 buffalo bulls were treated by swim-up and incubated with 0.01 mM heparin for 4 h. At 0, 2, and 4 h, aliquots of spermatozoa were exposed for 10 min to 60 μg mL-1 of lysophosphatidylcholine (LPC), as positive control, and to 0.01 μg mL-1 of plasmin. This concentration was chosen after a preliminary dose-response trial. Another sample from each treatment was incubated with IVF medium (negative control). After 10 min, sperm motility was evaluated and sperm were fixed in 37% formaldehyde and stained with trypan blue-Giemsa for subsequent microscopic examination. The total number of sperm counted, over 3 replicates, was 1269 for the negative control, 1293 for LPC, and 1238 for plasmin, equally distributed among incubation times. Differences among groups were analyzed by chi-square test. After swim-up, acrosomal loss was observed only in 4% of the sperm. The addition of 0.01 μg mL-1 of plasmin for 10 min to buffalo spermatozoa at time 0 significantly (P &lt; 0.01) enhanced (23%) AR compared with the control (7.8%), with the same efficiency of LPC (17.1%). After 2 h of incubation with heparin, both plasmin and LPC increased the AR compared to the control (24.4, 20.1, and 14.0%, respectively; P &lt; 0.01). After 4 h, plasmin gave higher percentages of AR (27.2%) compared to both the control (21.0%; P &lt; 0.05) and LPC (19.2%; P &lt; 0.01). Another interesting result is the improved motility recorded with plasmin compared to both the control and LPC groups at 2 h of incubation (90, 75, and 75%, respectively; P &lt; 0.05) and at 4 h of incubation (75, 60, and 60%, respectively; P &lt; 0.05). Finally, no differences in sperm viability were observed between plasmin and the control, whereas a decreased viability was found when LPC was used at 0 h (96.2, 95.0, and 89.0%, respectively, for plasmin, control, and LPC; P &lt; 0.05), at 2 h (85.0, 87.5, and 77.0%, respectively, for plasmin, control, and LPC; P &lt; 0.01), and at 4 h (85.0, 93.3, and 81.1%, respectively, for plasmin, control, and LPC; P &lt; 0.01). In conclusion, we found that addition of plasmin to capacitated sperm increases the percentage of acrosome-reacted spermatozoa and improves motility. Our results suggest that plasmin may play a role in events surrounding fertilization and suggest to evaluate in further studies whether the addition of plasmin during IVF improves the IVEP efficiency in buffalo.

251 METHYLATION PATTERN OF A DIFFERENTIALLY METHYLATED REGION LOCATED IN THE LAST EXON OF THE Igf2 GENE IN OOCYTES FROM NELLORE COWS

Assisted reproductive technologies are essential in modern cattle breeding. The relevance of in vitro embryo production (IVP) has been increasing around the world, particularly in Brazil. Acknowledging the positive effect of IVP regarding the utilization of female reproductive potential, its productivity can be improved. Methylation pattern of DNA in oocytes is a key factor for the improvement of IVP efficiency because it is related to oocyte competence. The objective of this study was to evaluate the methylation pattern of a differentially methylated region (DMR) located in the exon 10 of the imprint gene IGF2 in immature and in vitro-maturated oocytes from different follicle sizes. Small follicles (1-3 mm in diameter) and large follicles (≥8.1 mm in diameter) were dissected from slaughterhouse ovaries, and the COC were removed. Immature and in vitro-maturated oocytes, presenting the first polar body, were denuded and frozen in PBS at -80°C until DNA extraction. After extraction, DNA was treated with sodium bisulfite using EZ DNA Methylation™ Kit (Zymo Research, Orange, CA, USA). Bisulfite-treated DNA was amplified in a 2-round PCR strategy (nested-PCR), the amplicons purified with Geneclean® III Kit (MP Biomedicals, Irvine, CA, USA), cloned using pGEM®-T Easy Vector System (Promega, Madison, WI, USA), and sequenced. The number of follicles and in vitro maturation data were analyzed with chi-square test, and the methylation status of 27 CpG islands was analyzed with Student (normal distribution) or Mann-Whitney tests in the Prophet software (BBN Systems and Technologies, Cambridge, MA, USA). Cumulus-oocyte complexes from ≥8.1-mm follicles presented greater percentages of viable oocytes (40.5%) and nuclear maturation (60.6%) (P ≤ 0.05). Immature oocytes from 1- to 3-mm follicles were less methylated (33.33%) than those from ≥8.1-mm follicles (83.69%). After in vitro maturation, oocytes from ≥8.1-mm follicles were less methylated (18.51%) than the immature oocytes from ≥8.1-mm follicles (83.69%) and 1- to 3-mm maturated oocytes (49.62%). The less methylated pattern in the oocytes from ≥8.1-mm maturated follicles can be associated with greater competency of those oocytes, because this was the expected pattern. The lower level of methylation in the immature oocytes group (1- to 3-mm follicles) can be associated with the incomplete establishment of imprinting reprogramming pattern. Finally, we concluded that the methylation pattern of DMR, in the last exon of the IGF2 gene, can be used as a molecular marker for epigenetic reprogramming status in oocytes, helping the development of new IVP protocols. Supported by Embrapa Genetic Resources and Biotechnology and CAPES.

Factors affecting chromatin stability of bovine spermatozoa

The structural stability of transcriptionally inert paternal chromatin is of vital importance for the fertilization process and early embryonic development. Accordingly, a series of eight experiments were conducted during a 7-month period to investigate: (1) effects of bull breed, individuality, successive ejaculations, semen quality characteristics (SQC), semen dilution rates and hypothermic storage of semen in a Tris-egg yolk extender on incidence of sperm nuclear chromatin instability (NCI), and (2) effects of the interaction between variation of NCI within a frozen ejaculate and variation of oocytes quality due to maturation time and/or season on the efficiency of in vitro embryo production (IVEP). Semen samples were collected once a week from six bulls using an AV and only ejaculates ( n = 220) of >0.30 × 10 9 sperm/ml and ≥60% motility were used. NCI was measured by: (1) detection of lysine-rich histones in sperm chromatin using aniline blue staining, (2) sperm susceptibility to acid-induced nuclear DNA denaturation in situ using acridine orange test, and (3) sperm susceptibility to nuclear chromatin decondensation (NCD). Bovine oocytes ( n = 695) were matured in vitro for 18 or 24 h, fertilized after sperm selection through a swim-up procedure and cultured for 72 h. The results showed that the 2nd ejaculates were superior to the 1st ones with respect to chromatin stability. Dilution of semen to 49.67 ± 8.56 × 10 6 sperm/ml (1:19) decreased resistance of sperm to NCD. Cooling of semen had no significant effect on chromatin stability. Cryopreservation of semen augmented sperm vulnerability to DNA denaturation. Improvement of SQC (semen volume, sperm motility, velocity, viability and morphological normalcy) was generally concomitant with increase of sperm resistance to NCI. While Blonde d’Aquitaine bulls had a resistance to NCD higher than Limousine bulls in fresh semen, the former showed a greater susceptibility to DNA denaturation than the latter in cooled semen. Individuality significantly influenced NCI. The variability of NCI within a frozen ejaculate affected efficiency of IVEP. Significant negative correlations were observed between incidence of NCI and both fertilization rate and developmental capacity of embryos after maturation of oocytes for 18 h. The significant variation in IVEP traits due to season was independent of the effect of sperm chromatin instability.

293 EFFECT OF GUAIAZULENE ON IN VITRO BOVINE EMBRYO PRODUCTION

The substrates used in in vitro embryo production (IVP) mimic the in vivo fluids in which oocytes mature, oocytes are fertilized, and the early embryos develop (follicular and oviductal fluid). It is well established that oxidative stress negatively affects in vitro culture (IVC) outcomes. Guaiazulene (G) is a component of chamomile species oil with known antioxidant properties. In the present study, all IVP media were modified by the addition of G solutions so that the former exhibited a total protection against induced lipid peroxidation (TPaLP) similar to that of the respective in vivo environment. The IVP outcomes were then compared between G-processed and control oocytes. Bovine preovulatory follicular (BF) and oviductal (BO) fluid samples were collected from 10 Holstein 4- to 5-year-old cows in estrus. TPaLP was assessed according to the samples&amp;apos; ability to inhibit rat hepatic microsomal lipid peroxidation, by determination of the 2-thiobarbituric acid reactive material. TPaLP (mean % � SEM) of the BF and BO were 70.63 � 10.03 and 16.33 � 4.33, respectively, whereas those of the IVP [in vitro-matured (IVM), in vitro-fertilized (IVF), and IVC] media were lower (17.94 � 1.66, -1.82 � 0.78, and 14.57 � 1.26, respectively). TPaLP of the 0.1 mM G-modified IVP medium increased to 67.2 � 5.85, 19.98 � 2.49, and 69.19 � 6.22, respectively. A total of 2041 class A oocytes were used. The proportion of cleavage, early embryo development (embryos with more than 4 cells), or both after IVP (18 h IVM–5% CO2 in air, and 18 h IVF, 48 h IVC–5% CO2, 10% O2, 85% N) in the presence of G (n = 1237) during each of the IVP phases or any possible combination of IVP phases was compared with the respective control (C, n = 804). Statistical analysis was performed by a chi-squared test; P &amp;lt; 0.05 was considered significant. G improved cleavage and embryo development rates when present during IVM (79.4 and 57.8% vs. 64.5 and 38.2% for C) or both IVM and IVC (78.0 and 60.7% vs. 57.8 and 36.5%, respectively). When present only during 18 h of IVF, G had no effect on embryo production. However, an increased embryo development rate resulted from the combined exposure to G during IVF and IVM (56.4 vs. 29.6%), during IVF and IVC (55.3 vs. 35.5%), or at all IVP phases (56.6 vs. 34.9%). The latter effect resembled the one obtained after G addition only to the IVC medium (62.5 vs. 39.7%, respectively). We concluded that the addition of G to IVP substrates, at concentrations that mimic the in vivo TPaLP conditions, could promote bovine IVP efficiency.

Bovine and buffalo in vitro embryo production using oocytes derived from abattoir ovaries or collected by transvaginal follicle aspiration

This study was undertaken in order to evaluate the effect of oocyte source (live animals and abattoir ovaries) on subsequent embryo development in buffalo ( Bubalus bubalis). Cow ovaries were also collected as oocyte donors for in vitro embryo production (IVEP). Three hundred thirty-eight oocytes were recovered by ovum pick up (OPU, Group A) from 8 pluriparous buffalo cows, while 1127 and 1457 oocytes were aspirated, respectively, from buffalo (Group B) and bovine (Group C) slaughterhouse ovaries. Cumulus enclosed oocytes (COCs) suitable for IVEP were in vitro matured (IVM), fertilized (IVF) and cultured (IVC) to the tight morula (Tm) and blastocyst (Bl) stage. Within buffalo species Group A had a higher Bl yield (29.7 % versus 19.9%; P<0.05) and a lower proportion of embryos arrested at Tm stage (11.1% versus 22.3%; P<0.05) than Group B. Within slaughterhouse groups cattle oocytes had a higher cleavage rate (83.9% versus 64.8%; P<0.05) and yielded 49.2% more blastocysts than buffalo. However, when data are related to the total number of cleaved oocytes, only 13.7% more blastocysts were produced in cattle than in buffalo. In conclusion, in buffalo species the source of oocytes significantly affected post-fertilization embryo development, as demonstrated by the higher Bl yields derived from OPU-derived oocytes. A higher overall IVEP efficiency, mainly related to the higher cleavage rate, was recorded in cattle compared with buffalo when ovaries from an abattoir were used as oocyte donors.

In vitro embryo production in buffalo species: state of the art

In the last several years, there has been an increasing interest in in vitro embryo production (IVEP) technologies for faster propagation of superior germplasm in buffalo because of the low efficiency of superovulation (SO) and embryo transfer (ET) programs in this species. Although the IVEP efficiency has improved, embryo yield and development to term are still very low. This paper reviews the progress made in optimizing the IVM, IVF, and IVC systems. It also highlights the importance of embryo cryopreservation, which might critically contribute to the diffusion of ET procedures in the field. The acquisition of more information on embryo physiology, metabolism, and culture requirements in this species is critical to optimize the efficiency of advanced reproductive strategies. Further studies are also needed to improve the cryopreservation of IVEP embryos. The second part of the work underlines the potential impact of ovum pick-up (OPU) technique combined with IVEP on genetic improvement of buffalos. The OPU technique is a non-invasive and repeatable procedure for recovering large numbers of meiotically competent oocytes from antral follicles of live animals. Our experience, in buffalo, has demonstrated that OPU is superior to SO because it can yield more transferable embryos (TE) per donor on a monthly basis (2 TE vs 0.6, respectively). Therefore this technology has great potential to improve the genetic progress of buffalo through the maternal lineage.