Development Of Bovine Oocytes Research Articles

Supplementation of media with gamma-oryzanol as a novel antioxidant to overcome redox imbalance during bovine oocyte maturation invitro.

This study evaluated the effect of supplementing IVM media with γ-oryzanol (ORY), a nutraceutical derived from rice bran oil, on the development of bovine oocytes and hindering the compromising effect of redox imbalance. An invitro model of the bovine cumulus-oocyte complex was used for the evaluation of nuclear maturation and development. Antioxidant activity was investigated by assessing the level of ROS (Reactive Oxygen Species) and GSH (glutathione) in oocytes and quantitative changes in gene expression in matured oocytes and their respective cumulus cells. ORY supplementation increased the proportion of MII oocytes, cleaved embryos, and total blastocysts (p < .05) and was linked to higher and lower levels of intracellular GSH and ROS, respectively (p < .05). The treated oocytes and their respective cumulus-granulosa cells showed a modulation in the expression of genes related to apoptosis (downregulation of BAX and CHOP) and oxidative stress (upregulation of NRF2, CAT, and SOD). Also, relative upregulation of OCT-4 and IGF2R in treated oocytes was concomitant with higher subsequent development in terms of cleavage and total blastocyst rates (p < .05). Based on our findings, it appears that ORY supplementation can improve the nuclear maturation and development of bovine oocytes into blastocysts and augment their enzymatic and non-enzymatic antioxidant systems, maintaining the Redox balance and high enzymatic activity against ROS generation.

Review: The putative role of Progesterone Receptor membrane Component 1 in bovine oocyte development and competence.

Acquisition of developmental competence is a complex process in which many cell types cooperate to support oocyte maturation, fertilisation, and preimplantation embryonic development. In recent years, compelling evidence has shown that Progesterone Receptor Membra Component 1 (PGRMC1) is expressed in many cell types of the mammalian reproductive system where it exerts diverse functions. In the ovary, PGRMC1 affects follicular growth by controlling cell viability and proliferation of granulosa cells. PGRMC1 has also a direct role in promoting a proper completion of bovine oocyte maturation, as altering its function leads to defective chromosome segregation and polar body extrusion. Strikingly, the mechanism by which PGRMC1 controls mitotic and meiotic cell division seems to be conserved, involving an association with the spindle apparatus and the chromosomal passenger complex through Aurora kinase B. Conclusive data on a possible role of PGRMC1 in the preimplantation embryo are lacking and further research is needed to test whether the mechanisms that are set in place in mitotic cells also govern blastomere cleavage and subsequent differentiation. Finally, PGRMC1 is also expressed in oviductal cells and, as such, it might also impact fertilisation and early embryonic development, although this issue is completely unexplored. However, the study of PGRMC1 function in the mammalian reproductive system remains a complex matter, due to its pleiotropic function.

LncRNA-mediated effects of vitrification temperatures and cryoprotectant concentrations on bovine oocyte development following vitrification at the GV stage

We evaluated the effects of different vitrification temperatures (VTs) and cryoprotective agent concentrations (CPAs) on the viability and expressions of long non-coding RNA (lncRNA) in bovine oocytes following vitrification at the germinal vesicle (GV) stage. Our findings provide a theoretical support for improvement of the cryopreservation technology of bovine immature oocytes (BIOs). Bovine cumulus oocyte complexes (COCs) were collected and randomized into five groups: fresh oocytes (control), oocytes vitrified in liquid helium (LHe; −269 °C) with 5.6 M CPAs (LHe 5.6 M), oocytes vitrified in LHe with 6.6 M CPAs (LHe 6.6 M), oocytes vitrified in liquid nitrogen (LN; −196 °C) with 5.6 M CPAs (LN 5.6 M), and oocytes vitrified in LN with 6.6 M CPAs (LN 6.6 M). Of the four vitrification groups, the LHe 5.6 M group exhibited the highest blastocyst rate (13.22%), followed by the LHe 6.6 M group (10.19%) and LN 6.6 M group (9.77%), while the LN 5.6 M group had the lowest blastocyst rate (1.87%). Then, lncRNA expressions in the five groups were profiled. A total of 18,271 lncRNAs were identified, of which 2,158 were differentially expressed lncRNAs (DELs) in the vitrified groups, compared to the fresh group (P < 0.05; fold-change > 2). Co-location (cis) and co-expression (trans) prediction revealed 14 differentially expressed target genes (DETGs), which corresponded to 17 DELs. Based on grouping data and expression profiles of the DELs, we demonstrated that different VTs (−269 °C vs. −196 °C) can affect the expressions of MSTRG.12295.5, MSTRG.37123.1, MSTRG.37930.2, MSTRG.40464.9, MSTRG.8869.3 and MSTRG.26680.6. Expressions of these lncRNAs were affected by CPAs only in the condition of vitrification with LHe (−269 °C). Expressions of MSTRG.35129.6 were associated with exposures to both VTs and CPAs; while expressions of MSTRG.3578.3, MSTRG.40576.3, MSTRG.6723.5, MSTRG.32862.4, MSTRG.1184.4, MSTRG.33110.3, MSTRG.40454.2, MSTRG.41073.2, MSTRG.44732.4 and MSTRG.6729.3 might be related to vitrification. Co-expression analysis showed that MSTRG.12295.5, MSTRG.37930.2, MSTRG.40454.2, MSTRG.8869.3 and MSTRG.6723.5 expressions affect oocyte development after vitrification by regulating target gene expressions. Taken together, improvement of the developmental ability of BIOs after LHe vitrification maybe attributed to changes in expressions of some lncRNAs. Our findings elucidate on the molecular mechanisms underlying the development of BIOs under different VTs and CPAs.

BAPTA-AM dramatically improves maturation and development of bovine oocytes from grade-3 cumulus-oocyte complexes.

Intracellular free calcium ([Ca2+ ]i ) is essential for oocyte maturation and early embryonic development. Here, we investigated the role of [Ca2+ ]i in oocytes from cumulus-oocyte complexes (COCs) with respect to maturation and early embryonic development, using the calcium-buffering agent BAPTA-AM (1,2-bis[2-aminophenoxy]ethane-N,N,N',N'-tetraacetic acid tetrakis [acetoxymethyl ester]). COCs were graded based on compactness of the cumulus mass and appearance of the cytoplasm, with Grade 1 indicating higher quality and developmental potential than Grade 3. Results showed that: (i) [Ca2+ ]i in metaphase-II (MII) oocytes from Grade-3 COCs was significantly higher than those from Grade-1 COCs, and was significantly reduced by BAPTA-AM; (ii) nuclear maturation of oocytes from Grade-3 COCs treated with BAPTA-AM was enhanced compared to untreated COCs; (iii) protein abundance of Cyclin B and oocyte-specific Histone 1 (H1FOO) was improved in MII oocytes from Grade-3 COCs treated with BAPTA-AM; (iv) Ca2+ transients were triggered in each group upon fertilization, and the amplitude of [Ca2+ ]i oscillations increased in the Grade-3 group upon treatment with BAPTA-AM, with the magnitude approaching that of the Grade-1 group; and (v) cleavage rates and blastocyst-formation rates were improved in the Grade-3 group treated with BAPTA-AM compared to untreated controls following in vitro fertilization and parthenogenetic activation. Therefore, BAPTA-AM dramatically improved oocyte maturation, oocyte quality, and embryonic development of oocytes from Grade-3 COCs.

162 EFFECTS OF PROLACTIN AND DITHIOTHREITOL ON THE QUALITY AND DEVELOPMENTAL CAPACITY OF IN VITRO MATURED BOVINE OOCYTES

In vitro maturation (IVM) and IVF of domestic animal oocytes is widely used for commercial and research purposes. The oocyte quality and capacity for further development acquired during in vitro maturation and reduced during the subsequent aging are the main limitative factors affecting the embryo production (Miao et al. 2009 Hum. Reprod. Update 15, 573–585). Our objective was to evaluate effects of prolactin (PRL) and dithiothreitol (DTT) on apoptosis and the embryo development of bovine oocytes matured in vitro using 2 different systems. A total of 1437 slaughterhouse-derived cumulus-oocyte complexes (COC) were matured for 24 h in TCM-199 supplemented with 10% FCS, 0.2 mM sodium pyruvate, 10 μg mL–1 porcine FSH, and 5 μg mL–1 ovine LH. In system 1, 251 COC from a total of IVM oocytes were transferred to the aging medium (TCM-199 supplemented with 10% FCS) and cultured for 24 h in the absence (control) and the presence of either PRL (20 and 50 ng mL–1) or DTT (2.5, 5, and 10 μM). At the end of culture, oocyte apoptosis was detected using the TUNEL method. In system 2, another part of IVM oocytes (1186 COC) was co-incubated for 18 h with sperm in Fert-TALP medium modified by addition of 10 μg mL–1 heparin, PHE (20 μM penicillamine, 10 μM hypotaurine, 1 μM epinephrine), and 0.1% modified Eagle’s medium (MEM) nonessential amino acids. In this case, PRL and DTT (at the above listed concentrations) were added directly to the fertilization medium. After IVF, oocytes were cultured in CR1aa medium for assessment of the cleavage and blastocyst rates on Days 2 and 8, respectively. The nuclear status of blastocysts was evaluated by the cytogenetic method. The data from 3–7 replicates were analysed by ANOVA. Culture of matured COC in the aging medium (system 1) increased the rate of apoptotic oocytes from 8.1 ± 4.7% (0 h) to 48.6 ± 5.8% (24 h) (P &lt; 0.01). This rate was reduced (P &lt; 0.05) up to 22.5 ± 3.1% and 17.8 ± 5.1% in the presence of PRL (20 and 50 ng mL–1) and up to 15.0 ± 6.9% and 19.5 ± 3.7% in the presence of DTT (2.5 and 5 μM). The direct addition of PRL at a concentration of 20 ng mL–1 to the IVF medium raised the blastocyst rate from 21.6 ± 2.2% to 29.8 ± 2.4% (P &lt; 0.05) but did not affect the cleavage rate (72.1 ± 2.2% v. 74.3 ± 2.1%). By contrast, 50 ng mL–1 PRL did not increase the yield of blastocysts and decreased the cleavage rate (from 74.3 ± 2.1% to 62.9 ± 2.4%, P &lt; 0.05). When added to the IVF medium, DTT raised the blastocyst rate only at a concentration of 2.5 μM (P &lt; 0.05). No effects of PRL and DTT on the number of cells in embryos at the blastocyst stage were found. Our findings indicated that PRL and DTT supplements during in vitro fertilization of bovine oocytes may improve their capacity for the subsequent embryo development. This effect was probably due to the inhibitory influence of PRL and DTT on apoptosis of matured oocytes. The study was supported by the Federal Agency for Scientific Organizations and RFBR (project No. 14–48–03681).

Influence of Maternal Nutritional Factors on Ovarian Folliculogenesis in Cattle

Assisted reproductive technologies, such as artificial insemination and embryo transfer, have produced significant outcomes in cattle. These technologies are useful not only for improving fertility but also for increasing the rate of genetic change in beef and dairy live stock. Today, embryo transfer in cattle is both practical and commercially viable. However, bovine embryo transplantation still remains suboptimal under on-farm conditions, one of the main reasons for which may be inefficient nutritional management. The role of nutri tion in modulating reproduction and particularly in ovarian function remains unclear. Considerable effort has been made to elucidate the nutritional factors regulating the development of follicles in either a direct or an indirect manner. Moreover, evidence indicates that bovine folliculogenesis is modulated by gonadotropins as well as by metabolic hormones, including growth hormone, leptin, insulin and insulin-like growth factor-1. In addition, metabolites – such as glucose, urea and non-esterified fatty acids – are considered to play a role in regulating ovarian follicular development. Nutrition affects folliculogenesis indirectly by altering the activity of reproductive hormones. Furthermore, nutrition has local impact on the development of follicles at the ovarian level. As a result, nutrition is possibly decisive to the fate of bovine oocyte development, since oocyte quality influences reproductive outcomes such as fertilization and pregnancy rates. Although numerous reviews in the published literature describe nutritional roles in reproduction, the primary focus of this paper is the effect of nutritional factors on folliculogenesis in cattle. A greater understanding of this influence may facilitate nutritional manipulation in order to enhance fertility in cattle in a cost-effective yet very efficient way.

The effects of PHE mixture, theophylline, and sperm concentrations on fertilization and development of bovine oocytes in vitro

The effects of PHE mixture, theophylline, and sperm concentrations on fertilization and development of bovine oocytes in vitro

MicroRNA expression profile in bovine cumulus–oocyte complexes: Possible role of let-7 and miR-106a in the development of bovine oocytes

The objectives of this study included: (1) identify the expression of miRNAs specific to bovine cumulus–oocyte complexes (COCs) during late oogenesis, (2) characterize the expression of candidate miRNAs as well as some miRNA processing genes, and (3) computationally identify and characterize the expression of target mRNAs for candidate miRNAs. Small RNAs in the 16–27bp range were isolated from pooled COCs aspirated from 1- to 10-mm follicles of beef cattle ovaries and used to construct a cDNA library. A total 1798 putative miRNA sequences from the cDNA library of small RNA were compared to known miRNAs. Sixty-four miRNA clusters matched previously reported sequences in the miRBase database and 5 miRNA clusters had not been reported. TaqMan miRNA assays were used to confirm the expression of let-7b, let-7i, and miR-106a from independent collections of COCs. Real-time PCR assays were used to characterize expression of miRNA processing genes and target mRNAs (MYC and WEE1A) for the candidate miRNAs from independent collections of COCs. Expression data were analyzed using general linear model procedures for analysis of variance. The expression of let-7b and let-7i were not different between the cellular populations from various sized follicles. However, miR-106a expression was greater (P<0.01) in oocytes compared with COCs and granulosa cells. Furthermore, all the miRNA processing genes have greater expression (P<0.001) in oocytes compared with COCs and granulosa cells. The expression of potential target mRNAs for let-7 and let-7i (i.e., MYC), and miR-106a (i.e., WEE1A) were decreased (P<0.05) in oocytes compared with COCs and granulosa cells. These results demonstrate specific miRNAs within bovine COCs during late oogenesis and provide some evidence that miRNAs may play a role regulating maternal mRNAs in bovine oocytes.

Histone Deacetylase 1 Down-Regulation on Developmental Capability and Histone Acetylation in Bovine Oocytes and Parthenogenetic Embryos

Histone deacetylase 1 (HDAC1) is one of the most conserved enzymes present in the nuclei of cells, including bovine oocytes and pre-implantation embryos. However, the biological functions of HDAC1 in supporting the growth and development of bovine oocytes and embryos are still not fully elucidates. In this study, three siRNAs (si299, si672, and si1272) targeting to HDAC1 mRNA sequence were designed. After transfection into bovine fibroblast cells, si299, the most effective one in HDAC1 knock-down, was selected. The selected siRNA was microinjected into bovine germinal vesicle (GV) stage oocytes to determine the functions of HDAC1 in the maturation of bovine oocytes. Finally, the siRNA was microinjected into mature oocytes, which were then parthenogenetically activated and cultured in vitro until the blastocyst stage. The rates of cleavage, blastocyst development and acetylation of lysine 14 of H3 (H3K14) state were checked. The results suggest that HDAC1 knock-down in oocytes did not influence the rates of maturation or cleavage of parthenogenetic embryos. However, the rates of blastocyst decreased after siRNA microinjection. Furthermore, histone H3K14 acetylation level increased after siRNA microinjection into parthenogenetic embryos.

214 EFFECT OF LONG-DISTANCE TRANSPORTATION OF OVARIES ON THE DEVELOPMENT OF IN VITRO-MATURED - IN VITRO-FERTILIZED - IN VITRO-CULTURED BOVINE EMBRYOS

Keeping ovaries for a long time before oocyte recovery affects the maturation rate and blastocyst rate (BL rate), but a change in the temperature of the ovary at the time of transportation does not affect the BL rate (Ribeiro et al. 2008 Anim. Reprod. Sci. 108, 171–179). The objective of this study was to investigate the effect of long-distance transportation of ovaries on the in vitro development of bovine oocytes into blastocysts. Ovaries in the transportation group (Group T) were collected from a slaughterhouse, placed inside a Thermos flask, and transported to the laboratory within 17 to 21 h. The Thermos flask was covered with a freezer pack in a foam polystyrene box (Nakatate et al. 2006 Reprod. Fertil. Dev. 18, 193). The temperature of the Thermos flask was measured with a temperature recorder. Cumulus–oocyte complexes (COC) were collected by aspirating 2- to 6-mm follicles from the ovaries. The COC were matured in TCM-199 supplemented with 5% calf serum and 0.02 AU mL–1 of FSH. Groups of 20 COC were incubated in 100-μL drops of IVM media at 38.5°C under an atmosphere of 5% CO2 in air for 20 h. After 18 h of gamete co-culture (3 × 106 sperm mL–1), the presumptive zygotes were cultured in CR1aa medium supplemented with 5% calf serum for 9 days at 38.5°C under an atmosphere of 5% CO2, 5% O2, and 90% N2. Embryonic development was evaluated at 48 h after IVF (total cleavage rates, CL rate) and on Days 7 to 9 (BL rate). The ovaries in the control group (Group C) were collected from a slaughterhouse different from that of the Group T ovaries and transported to the laboratory in saline (23°C) in a Thermos flask within 3 h. These ovaries were then used for the collection of COC in the same way as for Group T. Data were analysed by chi-square test. A total of 25 experiments were performed using the ovaries from Groups C and T (n = 3945 v. n = 7218). The CL rate and the BL rate for Group T were significantly lower than those for Group C (41.8 v. 62.0% and 16.1 v. 29.7%; P &lt; 0.01). In Group T, the duration of ovary transportation did not affect the CL rate (within 19 h, between 19 and 20 h, and more than 20 h), but the BL rate was significantly decreased with an increase in transport time (18.7 v. 16.1 v. 14.8%). The temperature change in the Thermos flask during transportation ranged from 12.0 to 25.1°C; the temperature was maintained virtually constant or decreased. The CL rates of the groups that underwent a temperature (t) change (t ≤ 5.0 and 5.0 &lt; t ≤ 10.0) were not significantly different, but the BL rate in the group with 5.0 &lt; t ≤ 10.0 was significantly lower than that of the other group (16.7 v. 13.5%; P &lt; 0.01). These results suggest that the long-distance transportation of ovaries affects in vitro embryo development. Moreover, a decrease in temperature during transportation may have an adverse effect on blastocyst formation.

The effects of methyl-β-cyclodextrin on in vitro fertilization and the subsequent development of bovine oocytes

In this study, we investigated the effects of methyl-β-cyclodextrin (MBCD) on in vitro fertilization and the subsequent development of bovine oocytes. Bovine oocytes matured in serum-free medium were inseminated with frozen-thawed sperm pre-incubated in protein-free modified Brackett and Oliphant medium (BO) containing various concentrations of MBCD for various periods. MBCD decreased the frequency of live sperm, however enhanced the capacitation and acrosome reaction of the live sperm. Pre-incubation of sperm with 0.5, 1.0 and 1.5 mM MBCD for 2 and 4 h increased the frequency of normal fertilization. Embryos derived from oocytes fertilized with spermatozoa pre-incubated with MBCD developed normally to the blastocyst stage and term. There were individual differences and similar tendencies in four different sires in terms of the effects of MBCD upon fertilization. These results indicate that the pre-incubation of bovine sperm with MBCD affects viability and capacitation status of the sperm and promotes fertilization in vitro. Embryos derived from oocytes fertilized with sperm pre-incubated with MBCD developed normally to the blastocyst stage and term.

Roles of BMP15 and GDF9 on the In Vitro Maturation, Fertilization and Following Development of Bovine Oocytes.

Roles of BMP15 and GDF9 on the In Vitro Maturation, Fertilization and Following Development of Bovine Oocytes.

Effect of Ovary Storage on Development of Bovine Oocytes after Intracytoplasmic Sperm Injection, Parthenogenetic Activation, or Somatic Cell Nuclear Transfer

ABSTRACT The purpose of this study was to examine the effect of ovary storage on the development of bovine oocytes after intracytoplasmic sperm injection (ICSI), parthenogenetic activation, or somatic cell nuclear transfer (SCNT). Oocytes were obtained from ovaries stored in PBS for 2 to 6 h (control group) or 26 to 30 h (stored group) at 15°C. The maturation rate of the oocytes was significantly lower in the stored group (67%) than in the control group (78%). The degeneration rate of the oocytes was significantly higher in the stored group (24%) than in the control group (2%). ICSI and parthenogenetic oocytes from stored ovaries had a significantly decreased development to the blastocyst stage compared with the control (ICSI 8% vs. 24%, parthenogenetic activation 15% vs. 31%). However, the development rate to blastocysts of SCNT embryos derived from cumulus cells was not different between the two groups (38% vs. 38%). Also, the storage period of ovaries did not decrease the pregnancy rate of SCNT embryos...

Oocyte activation and preimplantation development of bovine embryos obtained by specific inhibition of cyclin-dependent kinases

The efficiency of bohemine and roscovitine in combination with ionomycin on parthenogenetic activation and initial embryonic development of bovine oocytes was studied. Two experiments were performed: in the first, different concentrations (0, 50, 75 or 100µM) and different exposure periods (2, 4 or 6 hours) to bohemine or roscovitine were tested for activation rates of in vitro matured (IVM) bovine oocytes, which were pre-exposed to ionomycin. The best treatments, 75µM bohemine and 50µM roscovitine, both for 6h, were used in the second experiment, in which IVM bovine oocytes were exposed to ionomycin, followed or not by bohemine or roscovitine treatment, and evaluated for nuclear status, activation rate and blastocyst development were assessed. The combined treatments (ionomycin + cyclin-dependent kinases inhibitors - CDKIs) showed better results for activation rates (77.3%) and initial embryonic development (35.2%) than the single ionomycin treatment (69.4% for activation and 21.9% for development); and also lead to a more uniform activation (nearly 90% single pronucleus development). The results showed that CDKIs improve the effects of ionomycin on parthenogenetic activation and blastocyst development in bovine oocytes and could help to achieve more efficient activation protocols, increasing the developmental competence of embryos obtained by reproductive biotechniques.

The In Vitro Development of Bovine Oocytes after Maturation in Glucose and β‐Hydroxybutyrate Concentrations Associated with Negative Energy Balance in Dairy Cows

Negative energy balance (NEB) in high yielding dairy cows early postpartum may affect oocyte quality. Therefore, we tested the effect of two different beta-hydroxybutyrate (BHB) and glucose concentrations, which are associated with subclinical or clinical ketosis, during in vitro maturation (IVM) on the developmental competence of bovine oocytes. In Expt 1, subclinical ketosis conditions were imitated. Oocytes were matured in four different serum-free media with two glucose concentrations (g1=2.75 mm or G1=5.5 mm glucose) and with or without BHB (BHB1=1.8 mm BHB). Following maturation groups were used: g1, G1, g1:BHB1 and G1:BHB1. In Expt 2, clinical ketosis conditions were mimicked by using the concentrations: g2=1.375 mm or G2=3.1 mm glucose and BHB2=4.0 mm BHB. The combinations used were: g2, G2, g2:BHB2 and G2:BHB2. After IVM and in vitro fertilization (IVF), presumptive zygotes were routinely cultured for 7 days in synthetic oviduct fluid [SOF; 5% fetal calf serum (FCS)]. At 48 h and 8 days pi, cleavage rate and number of blastocysts were recorded respectively. The results demonstrated that the maturation conditions mimicking subclinical (g1:BHB1) and clinical ketosis (g2 : BHB2) resulted in an impaired developmental competence of the oocyte after maturation. Especially the moderately low (g1) or extremely low glucose (g2) concentrations were responsible for this detrimental effect that was associated with a blocked cumulus expansion. Only in moderately low glucose conditions (g1:BHB1), BHB exerted an additive toxic effect during oocyte maturation resulting in a reduced blastocyst rate. Conclusively, our results may suggest that subclinical and clinical ketosis can affect the oocyte's developmental competence most likely through a directly adverse effect of the low glucose concentrations on oocyte maturation. Only in subclinical conditions this harmful effect may be aggravated by BHB.

373 OOCYTE-ACTIVATING CAPACITY OF BOVINE SPERMATOGENIC CELLS AND ACTIVATION PROTOCOLS FOR INTRACYTOPLASMIC INJECTION OF BOVINE ROUND SPERMATIDS

In vitro spermatogenesis can be applied to generate spermatids or spermatozoa and produce a genetically modified male germ line. Intracytoplasmic injection of the spermatids or spermatozoa is an important technique for effective production of offspring. The objective of this study is to evaluate oocyte-activation capacity of bovine spermatids or spermatozoa and to determine the effective activation treatment for in vitro development of bovine oocytes injected with round spermatids. Cryopreserved testicular spermatogenic cells and cauda epididymal spermatozoa obtained from a 1-year-old Japanese bull were used. In the first experiment, we injected bovine round (ROS) and elongated (ELS) spermatids, or testicular (TES) and cauda epididymal (CES) spermatozoa into mouse oocytes to examine their oocyte-activating capacity. The presence of pronuclei within whole-mounted oocytes was observed 4 h after injection. In the second experiment, we injected similar spermatids and spermatozoa into bovine oocytes without additional activation, and examined cleavage and blastocyst development. In the third experiment, bovine oocytes injected with ROS were activated with 7% ethanol or 5 �M ionomycin for 5 min (1 � Et or 1 � Iono) immediately after injection; some were further activated repeatedly at 3 h after injection (2 � Et or 2 � Iono), and some of these were subjected to 1.9 mM 6-dimethylaminopurine (DMAP) for 3 h after the second activation (2 � Et + DMAP or 2 � Iono + DMAP). Data were analyzed by the chi-square test in all experiments. The vast majority of bovine ROS failed to activate mouse oocytes (activation rate 10%). Activation rates of mouse oocytes injected with bovine ELS, TES, and CES were 61%, 75%, and 91%, respectively. The results suggest that oocyte-activation capacity is acquired during transformation from ROS to ELS. Cleavage and blastocyst rates of bovine oocytes injected with CES (59% and 19%, respectively) were significantly higher (P &amp;lt; 0.05) than the rates obtained with injections of TES (37% and 9%) and ROS (5% and 0%) without additional activation. However, cleavage and blastocyst rates of bovine oocytes injected with ROS in the groups of 2 � Et + DMAP (80% and 19%) and 2 � Iono + DMAP (76% and 19%) were significantly higher (P &amp;lt; 0.05) than those in the groups of 1 � and 2 � Et (37% and 2%, 59% and 4%), 1 � and 2 � Iono (10% and 7%, 22% and 4%), or those receiving a sham injection and activated with 2 � Iono + DMAP (43% and 4%). These results demonstrate that intracytoplasmic injection of ROS with repeated Et or Iono activation followed by DMAP treatment is more efficient than single or double Et or Iono activation.

275 THE EFFECTS OF MATURATION CULTURE PERIOD ON THE SEX RATIO OF BOVINE IVF EMBRYOS

The maturational stage of oocytes at the time of insemination has been suggested as influencing the sex ratio of resulting embryos (Dominko and First, 1997 Theriogenology 47, 1041–1050). However, there are very few reports concerning the relation between the maturation culture period of oocytes and the sex ratio of resulting embryos. The objective of this study was to investigate the effects of maturation culture period of oocytes on the sex ratio of bovine IVF blastocysts, using a novel technique of loop-mediated isothermal amplification (LAMP). Cumulus-oocyte complexes (COCs) were collected from ovaries of slaughtered cows. The COCs were cultured for various times (16, 22, 28 and 34 h) in maturation medium (TCM 199 supplemented with 5% fetal cow serum (FCS), 0.02 mg mL−1 of follicle-stimulating hormone and 50 μg mL−1 of gentamicin). After maturation culture for each period, the oocytes were inseminated with frozen-thawed spermatozoa (4 × 106 spermatozoa mL−1). After incubation with spermatozoa for 5 h, oocytes were transferred into culture medium (TCM199 supplemented with 5% FCS, and 5 μg mL−1 of insulin) and cultured for 7 days at 38.5°C in a humidified atmosphere of 5% CO2 in air. The zona pellucida of blastocysts collected on Day 7 after insemination (Day 0) was removed by brief exposure to 0.2% pronase, and the sex determination of embryos was conducted by the LAMP method, using a bovine embryo sexing kit (Eiken Chemical Co., Ltd., Tochigi, Japan). The rates of blastocyst formation were analyzed by ANOVA, and the sex ratios of embryos were compared by chi-square analysis. As shown in Table 1, the rate of blastocyst formation after insemination was significantly higher (P &lt; 0.05) in the oocytes matured for 22 h than in the oocytes matured for 16, 28, and 34 h. The proportion of male blastocysts derived from oocytes matured for 34 h was significantly higher (P &lt; 0.05) than from oocytes matured for 16 and 22 h. Moreover, the proportions of male blastocysts increased with delaying insemination. These results indicate that skewing of the sex ratio of IVF blastocysts is apparently influenced by the maturation culture period of oocytes. Table 1. Effect of maturation culture period on the development of bovine oocytes after IVF and the sex ratio of blastocysts

Activation with ethanol improves embryo development of ICSI-derived oocytes by regulation of kinetics of MPF activity.

Developmental potential of bovine embryos that are not artificially activated after intracytoplasmic sperm injection (ICSI) is generally very low. In this study, we investigated effects of artificial activation with ethanol on kinetics of maturation promoting factor (MPF) activity (p34(cdc2) kinase activity) and development of bovine oocytes following ICSI. Treatment of oocytes with ethanol at 4 h after ICSI improved their first cleavage and further preimplantation development (51% vs. 13%, 14% vs. 4%: treatment with vs. without ethanol, respectively). MPF activity of oocytes was lowered until at least 2 h after ICSI. In oocytes without activation after ICSI, MPF activity temporarily elevated at 6 h after ICSI, whereas this phenomena was not observed in the oocytes treated with ethanol. Furthermore, MPF activity was elevated 20 h after ICSI in oocytes activated with ethanol, whereas this elevation of MPF activity was not shown in oocytes without activation. These results indicate that the stimulus of sperm was sufficient to lower MPF activity of oocytes following ICSI, and moreover the activation treatment of bovine oocytes with ethanol after ICSI served to maintain the low levels of MPF activity until the next cell cycle started.

Different activation treatments for successful development of bovine oocytes following intracytoplasmic sperm injection.

In this study, the developmental capacity and cytogenetic composition of different oocyte activation protocols was evaluated following intracytoplasmic sperm injection (ICSI) of in vitro matured bovine oocytes. Motile spermatozoa selected by Percoll density gradient were treated with 5 mM dithiothreitol (DTT) and analysed for ultrastructural changes of the head using transmission electron microscopy (TEM). The alterations in sperm morphology after DTT treatment for different times (15, 30 and 60 min) were 10%, 45-55% and 70-85%, respectively. Further, a partial decondensation of sperm heads was observed after DTT treatment for 30 min. Oocytes were injected with sperm treated with DTT for 30 min. In group 1, sperm injection was performed without any activation stimulus to the oocytes. In group 2, sham injection without sperm was performed without activating the oocytes. Oocytes injected with sperm exposed to 5 microM ionomycin for 5 min (group 3), 5 microM ionomycin + 1.9 mM dimethylaminopurine (DMAP) for 3 h (group 4) and 5 microM ionomycin + 3 h culture in M199 + 1.9 mM DMAP (group 5) were also evaluated for cleavage, development and chromosomal abnormality. Cleavage and development rates in groups 1, 2 and 3 were significantly (p < 0.05) lower than those in groups 4 and 5. The incidence of chromosomal abnormality in the embryos treated directly with DMAP after ionomycin (group 4) was higher than in group 5. We conclude that immediate DMAP treatment after ionomycin exposure of oocytes results in arrest of release of the second polar body, and thus leads to changes in chromosomal pattern. Therefore, the time interval between ionomycin and DMAP plays a crucial role in bovine ICSI.

Effect of the Absence or Presence of Various Protein Supplements on Further Development of Bovine Oocytes During In Vitro Maturation1

The evaluation of culture medium for bovine oocytes has progressed toward more defined conditions during the last few years. The main objective of this study was to evaluate different sources of albumin as a protein supplement during in vitro maturation (IVM) of bovine oocytes in synthetic oviduct fluid medium (SOF). The replacement of protein with polyvinyl pyrrolidone (PVP) or polyvinyl alcohol (PVA) was also evaluated. The effect of recombinant human FSH on cumulus expansion and nuclear maturation in SOF containing BSA (BSA-V) or PVP-40 was also studied. Addition of BSA-V during IVM retarded nuclear maturation when compared with addition of PVP-40 or use of SOF alone. The inclusion of different concentrations of BSA-V, fetal calf serum (FCS), or PVA during IVM had no positive effect on the developmental capacity of the oocytes compared with the use of SOF alone with no supplement but significantly decreased the percentage of embryos reaching the morula and blastocyst stages. However, when BSA-V was replaced with purified BSA, BSA that was essentially free of fatty acids, or chicken egg albumin, embryonic development rates were restored. The presence of PVP-40 but not PVP-360 during IVM significantly increased morula and blastocyst production. These results indicate that although SOF alone can support bovine oocyte maturation, a high proportion of morulae and blastocysts can be produced from IVM oocytes cultured in medium containing PVP-40. These studies are the first to show that the effect of FSH on nuclear maturation and cumulus expansion is dependent on substrates present in IVM medium.