Goat Oocytes Research Articles

Evaluation of cholesterol- treated dromedary camel sperm function by heterologous IVF and AI

Cholesterol (cholesterol-loaded cyclodextrins: CLC) treatment of dromedary camel sperm prior to freezing enhances cryosurvival. The present study first validated the efficacy of a heterologous zona-free goat oocyte assay (n=115 oocytes) to evaluate camel sperm function in vitro (Experiment 1: n=6 bulls), then examined the effects of CLC treatment (1.5mg/mL CLC; CLC+) versus no treatment (0 CLC) of fresh (Experiment 2: n=4 bulls) and frozen-thawed (Experiment 3: n=5 bulls) camel sperm to penetrate, de-condense and form pro-nuclei in in vitro-matured goat oocytes. Finally, the ability of fresh 0 CLC and CLC+ sperm to fertilize in vivo was studied by artificially inseminating super-ovulated females (n=7–9 per treatment) and examining embryo production (Experiment 4: n=4–5 bulls/treatment). Camel spermatozoa penetrated (60%) and formed pro-nuclei (33%) in goat oocytes demonstrating the utility of this heterologous system for assessing sperm function in vitro. For fresh spermatozoa, 0 CLC-treated sperm performed better than their CLC+ counterparts for all parameters measured (P<0.05). In contrast, cryopreservation resulted in a sharp decline in sperm-oocyte interaction in 0 CLC aliquots but remained unaltered in CLC+ aliquots demonstrating a protective effect of cholesterol treatment. There was no difference between treatments in the in vitro fertilizing ability of frozen-thawed sperm or in the numbers of embryos retrieved following AI with fresh 0 CLC or CLC+ sperm. We conclude that although CLC treatment of dromedary camel sperm improves sperm motility it fails to confer an advantage to them in terms of improved in vitro sperm-oocyte interaction or in vivo fertilization under the conditions tested.

Variability in in vitro fertilization outcomes of prepubertal goat oocytes explained by basic semen analyses.

This work analyses the changes that caprine spermatozoa undergo during in vitro fertilization (IVF) of in vitro matured prepubertal goat oocytes and their relationship with IVF outcome, in order to obtain an effective model that allows prediction of in vitro fertility on the basis of semen assessment. The evolution of several sperm parameters (motility, viability and acrosomal integrity) during IVF and their relationship with three IVF outcome criteria (total penetration, normal penetration and cleavage rates) were studied in a total of 56 IVF replicates. Moderate correlation coefficients between some sperm parameters and IVF outcome were observed. In addition, stepwise multiple regression analyses were conducted that considered three grouping of sperm parameters as potential explanatory variables of the three IVF outcome criteria. The proportion of IVF outcome variation that can be explained by the fitted models ranged from 0.62 to 0.86, depending upon the trait analysed and the variables considered. Seven out of 32 sperm parameters were selected as partial covariates in at least one of the nine multiple regression models. Among these, progressive sperm motility assessed immediately after swim-up, the percentage of dead sperm with intact acrosome and the incidence of acrosome reaction both determined just before the gamete co-culture, and finally the proportion of viable spermatozoa at 17 h post-insemination were the most frequently selected sperm parameters. Nevertheless, the predictive ability of these models must be confirmed in a larger sample size experiment.

Influence of follicular fluid and gonadotropin supplementation on the expression of germ cell marker genes during in-vitro maturation of caprine (Capra hircus) oocytes

The present study aimed to investigate whether follicular fluid, gonadotropins (FSH, LH and estradiol-17β), and combination of both types of supplementation (follicular fluid+gonadotropins) in maturation medium influence the transcript abundance of germ cell marker genes (MATER, ZAR1, GDF9 and BMP15) in goat oocytes. Grade I and II oocytes were collected from the goat ovaries and matured further under in-vitro conditions using four different maturation regimens viz, group B containing basal media (TCM 199+BSA+serum), group C (basal media+10% follicular fluid), group D (basal media+FSH+LH+estradiol-17β) and group E (basal media+10% follicular fluid+FSH+LH+estradiol-17β). Group A consisted of immature oocytes. Expression profile of MATER, ZAR1, GDF9, and BMP15 was analyzed in matured oocytes of different groups as well as immature oocytes using real-time PCR. The relative abundance of MATER gene in immature oocytes (group A) was found to be significantly higher (p<0.05) compared to experimental groups. The relative expression of ZAR1 was found to be significant different (P<0.05) between group D and E. The relative expression of GDF9 and BMP15 was significantly higher (P<0.05) in group E as compared to other experimental groups. These results indicated that the expression of MATER and ZAR1 transcript were down regulated after maturation; however BMP15 and GDF9 transcripts were upregulated after maturation. For better in vitro embryo production (IVEP) out comes goat oocytes may be matured in vitro in TCM-199 supplemented with follicular fluid and gonadotropins.

Intrinsic quality of goat oocytes already found denuded at collection for in vitro embryo production

Although cumulus cells are essential for efficient oocyte maturation, the establishment of protocols that support IVD of embryos obtained from denuded oocytes (DOCs) is important for optimizing the use of reproductive biotechnologies. Thus, this study aimed to establish a protocol for IVD of goat DOC using different strategies of IVM and methods of oocyte activation. Four experiments were performed. Similar developmental competence of slaughterhouse DOC was obtained, regardless of maturation media (complex, semidefined or simplified). However, the ability to reach the blastocyst stage was affected by the activation method. Denuded oocytes subjected to parthenogenetic activation had greater (P < 0.05) development capacity, compared with those undergoing IVF with average cleavage rate of 83% and 75%, blastocyst rate of 49% and 28%, and blastocysts in relation to the cleaved embryos of 59% and 38, respectively. In addition, the quality of embryos evaluated after vitrification/warming was similar between parthenogenetic activation and IVF. Finally, we demonstrated that the coculture of cumulus-oocyte complex (COC) with DOC increased the competence of DOC at a ratio of 1:1 and 1:9 (DOC:COC). We believe that presence of cumulus cells (CCs) is not essential to the meiotic maturation, if at the time of removal of the oocyte from follicular environment, they already acquired competence to development. However, when the oocytes still need to acquire competence, the presence of CC may significantly contribute in their developmental capacity acquisition during IVM. Thus, regardless of the source, these oocytes will require longer time in IVM, contrary to what happens in the absence of CC. In conclusion, although DOC had a lower developmental potential, especially after IVF, they were able to produce blastocysts and the coculture of DOC with COC increased this developmental capacity.

Incidence of apoptosis after retinoids and insulin-like growth factor-I (IGF-I) supplementation during goat in vitro embryo production.

The addition of growth factors and vitamins enhances goat embryonic development in vitro. However, few attempts have been reported trying to identify supplementation regimens for oocyte maturation or embryo culture with additive properties. The present report was aimed to evaluate if retinoids [0.3 μM retinyl acetate (RAc) and 0.5 μM 9-cis-retinoic acid (RA)] supplementation during goat oocyte maturation and retinoids and/or 50 ng mL-1 IGF-I during embryo culture synergically enhanced embryonic development while diminishing the incidence of apoptosis. All combinations of RAc and RA treatment produced blastocysts with similar efficiencies, while IGF-I enhanced embryos yields irrespectively of retinoid addition. Moreover, retinoids and IGF-I supplementation showed similar caspase activity or DNA fragmentation indexes in blastocysts. In conclusion, supplementation with retinoids and IGF-I during goat embryo culture enhances blastocysts development without synergic reduction of apoptosis.

Using cysteine/cystine to overcome oxidative stress in goat oocytes and embryos cultured in vitro

Assisted reproductive techniques expose gametes to excessive concentrations of reactive oxygen species. The present study aimed to evaluate the effects of oxidative stress on apoptosis in goat oocytes and embryonic development. The results demonstrated that the addition of 100µM hydrogen peroxide (H2O2) into media produces an oxidative environment during oocyte maturation. The number of cumulus cells positive for terminal deoxynucleotidyl transferase UTP nick end labeling, and the activity of caspase 3 in mature oocytes were increased, compared with the control group (P<0.05). In addition, the expression levels of mitochondrial regulators, including peroxisome proliferator‑activated receptorγ coactivator-1α (PGC-1α) and nuclear respiratory factor‑1 (NRF‑1) were increased in the oxidative oocytes, compared with those in the control group (P<0.05). The ratio of the proapoptotic gene, Bcell lymphoma (Bcl-2)-associatedXprotein(BAX), to the anti‑apoptotic gene, BCL‑2, was higher in the H2O2 group, compared with the control group (P<0.05). To overcome oxidative stress in oocytes and embryos cultured invitro, 200µM cysteine and 200µM cystine were added to the media, thereby increasing the concentration of intracellular glutathione (GSH) and assisting in maintaining the redox state of the cells. In conclusion, cysteine and cystine reduced the oxygen tension caused by H2O2, thereby providing a novel strategy for optimizing invitro embryonic development systems.

Linolenic acid improves oocyte developmental competence and decreases apoptosis of in vitro-produced blastocysts in goat.

The effects of α-linolenic acid (ALA) on developmental competence of oocytes in goats were evaluated in this study. Initially, the level of ALA in small and large antral follicles was determined to be in a range of 0.018-0.028 mg/ml (64.6-100.6 μM, respectively). In vitro maturation was performed in the presence of various concentrations (10, 50, 100, or 200 μM) of ALA. Cumulus expansion, meiotic maturation, levels of intracellular glutathione (GSH), embryonic cleavage, blastocyst formation following parthenogenetic activation (PA) and in vitro fertilization (IVF), number of total and apoptotic cells in blastocyst, and expression of Bax, Bcl-2, and p53 genes in blastocyst cells were determined. Compared with the control, no improvement was observed in cumulus expansion in ALA-treated groups. At 50 μM concentration, ALA increased meiotic maturation rate but had no effect on GSH level. When oocytes treated with 50 μM ALA were subsequently used for PA or IVF, a higher rate of blastocyst formation was observed, and these embryos had a higher total cell number and a lower apoptotic cell number. Expression analyses of genes in blastocysts revealed lesser transcript abundances for Bax gene, and higher transcript abundances for Bcl-2 gene in 50 μM ALA group. Expression of p53 gene was also less observed in ALA-treated blastocysts. Our results show that ALA treatment at 50 μM during in vitro maturation (IVM) had a beneficial effect on maturation of goat oocytes and this, in turn, stimulated embryonic development and regulated apoptotic gene expression.

Effect of C-Type Natriuretic Peptide on Maturation and Developmental Competence of Goat Oocytes Matured In Vitro

The developmental competence of oocytes during in vitro maturation (IVM) is compromised due to asynchronous nuclear and cytoplasmic maturation. To improve IVM efficiency, a pre-maturation culture or two-step maturation strategy has been established, involving meiosis arrest induced by pharmacological agents to provide oocytes with sufficient time to synchronize the maturation of the nucleus and cytoplasm. C-type natriuretic peptide (CNP), which has been demonstrated to function as an oocyte maturation inhibitor (OMI) in many species, provides a new alternative to improve the developmental capacity of oocytes matured in vitro. However, the effect of CNP on meiosis arrest and the maturation of goat oocytes remains unclear. In the present study, CNP was shown to function as an OMI in goat oocytes. CNP could temporarily maintain the meiotic arrest of goat oocytes cultured in vitro for 4 hours. This transient effect was partly due to the reduction of natriuretic peptide receptor 2 (Npr2). Estradiol could delay the decrease in Npr2 expression and prolong the duration of meiosis arrest up to 6 hours. Based on the above results, a two-step method was established for goat oocyte maturation, in which the oocyte maturation rate was significantly increased. After parthenogenetic activation, the cleavage rate, blastocyst rate and total cell number of blastocysts were significantly improved. Our results suggested that CNP can be used to delay meiotic resumption and enhance the developmental competence of goat oocytes matured in vitro.

Chemically assisted somatic cell nuclear transfer without micromanipulator in the goat: effects of demecolcine, cytochalasin-B, and MG-132 on the efficiency of a manual method of oocyte enucleation using a pulled Pasteur pipette

The present study aimed to facilitate widespread application of a previously described manual method of somatic cell nuclear transfer (SCNT) by investigating the effects of demecolcine (a microtubule-depolymerizing chemical), cytochalasin-B (a microfilament-depolymerizing chemical: 2.5μg/ml for 15min) and MG-132 (a proteasome inhibitor chemical) on the (i) incidence of cytoplasmic protrusion of MII chromosomes, (ii) improvement of manual oocyte enucleation, and (iii) in vitro and in vivo developmental competence of SCNT embryos in the goat. Following in vitro maturation, around 65% of goat oocytes contained a characteristic cytoplasmic protrusion of MII-chromosomes. Treatment with demecolcine (0.4μg/ml for 30min) significantly increased this rate to 92.2±4.5%. Treatment with MG-132 (2μM for 30min) could not improve this rate when used alone (61.4±11.5%), but when combined with demecolcine (86.4±8.1%). Treatment with cytochalasin-B completely suppressed this rate whenever used, either alone (7.7±5.1%) or in combination with demecolcine (3.9±1.3%). In a direct comparison, there was no significant difference in quantity and quality of embryos propagated by the manual vs. micromanipulation-based methods of SCNT (cleavage: 85.3±4.5 vs. 89.5±8.9%, blastocyst: 19.5±4.3 vs. 24.3±4.4%, grade 1 and 2 blastocyst: 33.8±7.1 vs. 29.5±6.3%, total cell count: 125±11.1 vs. 122±10.5, respectively). Furthermore, development to live kids at term was not significant between the two SCNT methods. From both technical and economical points of view, the overall in vitro and in vivo efficiency of this manual method of SCNT proved it a simple, fast and efficient alternative for large scale production of cloned goats.

305 ROYAL JELLY TREATMENT DURING OOCYTE MATURATION IMPROVES IN VITRO MEIOTIC COMPETENCE OF GOAT OOCYTES BY INFLUENCING INTRACELLULAR GLUTATHIONE SYNTHESIS AND APOPTOSIS GENE EXPRESSION

Royal jelly (RJ) is a secretion product from the cephalic glands of nurse bees that has extraordinary properties and remarkable health effects. Over the years, antioxidative and antiapoptotic properties of RJ have been experimentally investigated. Here we hypothesised that supplementary RJ in in vitro maturation (IVM) medium would (i) improve cumulus expansion (ii) oocyte nuclear maturation, (iii) glutathione (GSH) content, and (iv) mitochondrial activity, and (v) also affect the mRNA abundance of the (Bax, Bcl-2, and p53) transcripts involved in oocyte apoptosis. To test these hypotheses, goat ovaries were collected from a local abattoir and transported to the laboratory. Cumulus-oocyte complexes (COC) with multilayered compact cumulus investment and evenly granulated cytoplasm were selected and randomly allocated to the experiments. To evaluate the effects of RJ on meiotic competence after maturation in vitro, IVM medium was supplemented with concentration of 0.0 (RJ-0), 2.5 (RJ-2.5), 5.0 (RJ-5), and 10.0 (RJ-10) mg mL–1 of RJ. After IVM, oocytes of each group were evaluated for cumulus expansion (visual assessment), stage of nuclear maturation (Hoechst staining), intracellular level of GSH (Cell Tracker blue staining), mitochondrial activity (MitoTracker Deep Red staining), and relative expression of Bax, Bcl-2, and p53 genes (qRT-PCR assay). Differences were analysed for significance by one-way ANOVA using SAS version 8.0 (SAS Institute Inc., Cary, NC, USA), considering P &lt; 0.05 to be significant. Supplementation of the maturation media with RJ did not appear to affect cumulus expansion (P &gt; 0.05). Our results revealed that maturation rate was higher (88.0%) in the RJ-10 group when compared with the RJ-2.5 (71.5%) and control (RJ-0) groups (60.0%; P &lt; 0.05), but similar with the RJ-5 group (81%; P &gt; 0.05). A higher (P &lt; 0.05) GSH content was detected when comparisons were made between each concentration of RJ-treated (i.e. RJ-2.5, RJ-5, and RJ-10) oocytes and the control (RJ-0) oocytes; however the differences were not significant when RJ groups were compared. No difference (P &gt; 0.05) was observed among RJ-treated and untreated oocytes regarding their mitochondrial activity after IVM. Based on these results, the concentration of 10 mg mL–1 (RJ-10) was selected for evaluation of Bax, Bcl-2, and p53 transcripts abundance. Our results revealed that the expression of Bax mRNA was decreased (P &lt; 0.05) in RJ-10 group when compared with control (RJ-0) group. Furthermore, there was an increased (P &lt; 0.05) expression of Bcl-2 transcripts in RJ-10 group when compared to the control (RJ-0) group. The p53 transcript also tended to be higher in RJ-10 group than in the control (RJ-0) group, although this difference was not statistically significant (P &gt; 0.05). In conclusion, results of this study showed that adding RJ to maturation medium at optimum concentration increased the nuclear maturation and GSH synthesis, but not activity of the mitochondria; this improvement was associated with expression of apoptosis-related genes in goat oocytes.

253 EFFECTS OF ADDITION OF ALPHA-LINOLENIC ACID INTO MATURATION MEDIUM ON IN VITRO DEVELOPMENT AND EXPRESSION OF APOPTOSIS-RELATED GENES OF GOAT EMBRYOS

Fatty acids are important sources of energy for oocytes and embryos. In bovine, an increased level of rumen-inert fatty acids in the diet improved the developmental competence of oocytes to the blastocyst stage and also embryo quality. As described in our previous report, providing appropriate levels of α-linolenic acid (ALA) in maturation medium had beneficial effects on nuclear maturation and embryonic development in the goat. Considering these beneficial effects, here we have conducted experiments to evaluate whether addition of ALA to goat oocyte maturation medium can affect the quality of blastocyst and the transcription of apoptosis-related Bax, Bcl-2, and p53 genes. Ovaries were collected from goats, and cumulus-oocyte complexes (COC) were recovered by the slicing method. The COC were placed in maturation medium supplemented with 50 µM ALA. Oocytes in the control group were incubated in the same maturation medium without ALA. In vitro maturation (IVM) was performed in a humidified atmosphere containing 5% CO2, 5% O2, and 90% N2 at 38.5°C for 24 h. After IVM, oocytes from both the treatment (n = 113) and control (n = 104) groups were subjected to IVF followed by culture in CR1aa medium for 8 days under the conditions stated above. The cleavage and blastocyst rates were recorded at Days 3 and 8 of culture, respectively. To examine the effect of ALA on total cell number and apoptosis of the blastocyst cells, the blastocysts from 50 μM ALA-treated and control oocytes were stained with 4′,6-diamidino-2-phenylindole to count total cell number, and apoptotic cells in these blastocysts were detected with TUNEL assay. Blastocysts derived either from 50 μM ALA-treated oocytes or control oocytes were also evaluated for the expression of Bax, Bcl-2, and p53 genes. The cleavage and blastocyst rates were compared by chi-square analysis. Differentially expressed genes were analysed by 1-way ANOVA. A P-value of less than 0.05 was considered significant. Although cleavage rates after IVF were similar (P &gt; 0.05) between 50 μM ALA-treated (68.1%) and control (55.8%) groups, 50 μM ALA-treated oocytes produced more (25.7%) blastocysts than the control group (13.5%; P &lt; 0.05). Blastocysts derived from oocytes supplemented with 50 μM ALA not only had a greater (P &lt; 0.05) total cell number (115.2), but also a lower (P &lt; 0.05) number of apoptotic cells (3.1) compared with the control blastocysts (110.8 and 4.2, respectively). The relative transcript abundance of Bax and p53 was decreased (P &lt; 0.05) in blastocysts that originated from the 50 μM ALA group compared with control blastocysts. Furthermore, there was an increased (P &lt; 0.05) expression of Bcl-2 transcripts in blastocysts derived from the 50 μM ALA-treated oocytes compared with the control. In conclusion, our findings revealed that ALA-treated medium led to an improvement in blastocyst rate and quality as determined by greater total cell number, lower number of apoptotic cells, and altered expression of apoptosis-related genes.

307 MATURATION RATE AND GENE EXPRESSION ANALYSIS OF GOAT OOCYTES SELECTED BY FOLLICLE SIZE AND BRILLIANT CRESYL BLUE STAINING

Oocyte quality plays a critical role in determining the success of embryo development. Studies on cattle and goats indicate that oocytes derived from large follicles (LFO) have greater developmental competence than those derived from small follicles (SFO). Brilliant cresyl blue (BCB) staining determines the activity of glucose-6-phosphate dehydrogenase and is a commonly used noninvasive marker of oocyte competence. Studies in pigs, goats, cows, mice, and dogs showed that the maturation and blastocyst developmental rate of BCB+ oocytes is significantly higher than BCB– oocytes. The aim of this study was to evaluate the maturation rate of goat oocytes selected based on follicular size and BCB staining and compare their relative patterns of gene expression. Maturation rate and gene expression profile were expected to be different in these oocyte groups. Cumulus-oocyte complexes were recovered from abattoir-derived ovaries using a slicing technique. Eleven rounds of oocyte maturation and 4 rounds of BCB staining were carried out. During each replicate, oocytes from large (≥3 mm) and small (&lt;3 mm) follicles were collected separately from the same group of ovaries. Oocyte maturation rates were 54.3 ± 5.4% for LFO (n = 378) and only 33.5 ± 3.7% for SFO (n = 981; P &lt; 0.01). The BCB+ (n = 223) oocytes yielded a significantly higher maturation rate than the BCB– (n = 194) oocytes (56.1 ± 1.8 v. 20.6 ± 3.8%, respectively; P &lt; 0.001). Gene expression analysis was conducted on individual MII oocytes (21 oocytes per group). Specific target amplification was performed on a single oocyte directly by using the CellsDirect One-Step qRT–PCR Kit (Invitrogen). Quantitative real-time PCR was then performed using the 48.48 BioMark platform from Fluidigm. Forty two genes were selected from the following categories: growth factors, transcription factors, metabolism, pluripotency, cell cycle, apoptosis, and oocyte-specific genes. Relative expression values were calculated using the ΔΔCT (fold change) method and analysed by ANOVA. The significance was assigned at P &lt; 0.05. The relative expression of CCNA2, CDK2, CCNB1, POU5F1, SOX2, EGF, FGF2, GDF9, ZP3, BCL2, GJA1, DDR1, PFKFB3, IGF2R, and GRB10 was significantly greater (P &lt; 0.05) in both LFO and BCB+ oocytes compared to SFO and BCB– oocytes, respectively. The proapoptotic gene BAX, the ACSL3 gene involved in fatty acid oxidation, and the growth factor IGF1 were expressed significantly higher (P &lt; 0.05) in SFO compared to LFO. By investigating these differentially expressed transcripts, we will better understand pathways involved in oocyte developmental competence and potentially use them as markers of oocyte quality. We expect that the ability to select oocytes of better quality based on BCB staining will improve outcomes of IVF and SCNT.

254 IN VITRO BLASTOCYST PRODUCTION FROM PREPUBERTAL GOAT OOCYTES ACCORDING TO SEASON

In our laboratory, we have been working for more than 20 years on in vitro embryo production from prepubertal goat oocytes. We have observed significant differences on blastocyst production according to the season of year. The present study is aimed to asses these differences in summer, autumn, winter, and spring. Oocytes with more than 3 compact cumulus layers (cumulus-oocyte complex, COC) and homogeneous cytoplasm were obtained after slicing prepubertal goat ovaries (45-day-old Murciano-Granadina rearing animals) recovered from slaughterhouse. Groups of 30 COC were matured in 100-µL drops of TCM199 medium (10% FBS) for 24 h with 5% CO2 and humidified atmosphere. Fresh buck semen (pool of 3 males) was selected by swim-up and capacitated with 0.05 mg mL–1 of heparin for 45 min. The IVF was performed by transferring 20 oocytes to 100-μL drops of Tyrode's medium with 4 × 106 capacitated sperm with 5% CO2 and humidity. After 17 h of IVF, a sample of oocytes was fixed in ethanol:acetic (3 : 1) for staining (1% lacmoid) to assess nuclear stage (Table 1). At 20 h post-insemination (pi), groups of 10 presumptive zygotes were transferred to 10-μL drops of SOF medium (10% FBS) under paraffin oil in a humidified atmosphere with 90% N2 for 8 days. Cleavage rate was evaluated at 48 h pi, and blastocyst rate was recorded at Day 8 pi (Table 1). Table 1 shows a decrease in normal pronuclear formation during winter (43%) and spring (47%). However, winter (16%) and spring (16%) produced significantly more blastocysts than summer (10%) and autumn (5%). Similar results were found by Sousa et al. (2014 Theriogenology 82, 1149–1162) in adult goats producing significantly higher blastocysts in the anoestrus season. We hypothesise that prepubertal goat oocytes in winter and spring may have a better cytoplasmic quality, producing more blastocyst even though the penetration rate is significantly lower than in autumn. In conclusion, under in vitro conditions and using fresh semen, the rate of penetration, cleavage, and blastocysts in prepubertal goats is influenced by season. Further studies in oocyte and sperm quality should be done to clarify this result. Table 1.Nuclear stage of prepubertal goat oocytes according to season (3 replicates; top) and in vitro blastocyst production of prepubertal goat oocytes according to season at Day 8 postinsemination (7 replicates; bottom)1

Pengukuran PFRET Menggunakan Metode Sensitized Emission dalam Pengapatan Perubahan Kalsium Pada Oosit

Fluorescence Resonance Energy Transfer (FRET) is a process of non-radiative energy transfer from donor molecule to an acceptor. We expect to know the quantitative FRET measurements in immature and mature goat oocyte with the presence of calcium. FRET was detected using a laser scanning confocal microscope. We assume that calcium as a donor and fluo-3 as an acceptor. The methods of sensitized emission have been used for quantitative FRET measurement. We also describe the precision FRET (PFRET) data analysis. The value of PFRET describe the presence of calsium in immature and mature oocytes. For these immature and mature oocytes, the results of PFRET are 622,5 and 754,57 respectively. Mature oocyte has more calsium than immature oocyte.

Expression of CD44 Gene in Goat’s Oocytes and Embryos

The CD44 family belongs to a larger group of hyaluronic acid-binding proteins and plays important roles in oocyte maturation, fertilization and preimplantational embryo development. We analyzed the CD44 receptor in goat oocytes and embryos. Immature oocytes (N = 90) were obtained from a local abattoir; mature oocytes (N = 40) and embryos (N = 25) were obtained by laparotomy from adult hair ewes submitted to ovarian stimulation treatment. The CD44 mRNA was detected by PCR, after reverse transcription, while proteins were located by indirect immunofluorescence, using anti-human CD44 monoclonal antibody. Human lymphocytes and immature bovine oocytes were used as positive and negative controls respectively. Assessment of the oocyte nuclear stages as well as classification of the embryonic Growth phase were made with Hoechst 33342 staining. Indirect immunofluorescence detected CD44 expression on the surface of mature oocytes and embryos; immature oocytes did not take up the stain. These findings were supported by the RT-PCR data, which showed no mRNA templates for CD44, in material from immature oocytes and cumulus cells. The CD44 amplicons were detected after a second hemi- nested PCR in mature oocytes and embryos. The finding of CD44 in mature oocytes and preimplantational embryos could reflect the expression profile of hyaluronic acid during terminal folliculogenesis and preimplantational embryo development in goats.

All‐trans retinoic acid improves goat oocyte nuclear maturation and reduces apoptotic cumulus cells during in vitro maturation

All-trans retinoic acid (t-RA) is a natural component and representative physiologically active metabolite of vitamin A, having multiple physiologic functions. The objective of this study was to evaluate the effect of t-RA on goat oocyte maturation and cumulus cell apoptosis during in vitro maturation (IVM). Immature goat cumulus-oocyte complexes (COCs) were matured in vitro in the absence or presence of t-RA at concentrations of 10 nmol/L, 100 nmol/L and 1000 nmol/L. Oocyte maturation and embryo development were assessed by polar body formation and parthenogenetic activation, respectively. Oocyte survival was checked by Trypan blue staining. Apoptosis of cumulus cells was analyzed by terminal deoxynucleotidyl transferase nick end labeling staining and quantitative real-time PCR. In comparison with the control group, 100 nmol/L and 10 nmol/L t-RA significantly improved goat nuclear oocyte maturation and survival (P < 0.05). Addition of 1000 nmol/L t-RA improved nuclear maturation (P < 0.05), but had no effect on survival of goat oocytes. t-RA had no positive effect on goat parthenogenetic embryonic cleavage, blastocyst formation or total cell numbers. However, t-RA inhibits the apoptosis of cumulus cells (P < 0.01). t-RA treatment up-regulated the expression of B-cell lymphoma 2 (BCL-2), catalase (CAT) (P < 0.05) and down-regulated the expression of Caspase-8 (P < 0.05). In conclusion, t-RA has positive effects on goat oocyte nuclear maturation and reduces apoptotic cumulus cells during IVM.

Goat oocyte quality and competence to undergo IVM and embryo development after parthenogenetic activation from goats fed with different levels of cashew nut bran as source of dietary lipids

Lipid-rich and energy-dense diets can have significant effects on the reproductive physiology, including the ovarian function and fertility. The aim of this study was to assess the effect of cashew nut bran supplementation as a lipid source on follicle development, plasma and intrafollicular concentrations of cholesterol, and developmental competence of in vitro–matured goat oocytes. The inclusion of cashew nut bran as 24% of the goats' diet for 28 days increased the percentage and number of degenerated oocytes compared with the control (P < 0.05), and also the plasma cholesterol levels and the proportion of grade IV oocytes compared with all other treatments (P < 0.05). Moreover, a significant reduction was observed in the proportion of viable oocytes compared with the control and in the percentage of grade II oocytes compared with all other treatments (P < 0.05). Oocyte maturation, cleavage, and blastocyst rates after parthenogenetic activation of viable oocytes were not affected by the type of diet. In conclusion, the inclusion of cashew nut bran as 24% of the diet of adult goats for 28 days changed plasma cholesterol levels and reduced the proportion of viable immature oocytes; however, the 12% and 24% diet supplementations with cashew nut bran did not interfere with competence of resulting viable oocytes to reach the metaphase II stage after IVM, and to develop after parthenogenetic activation.

Dose dependent effect of pregnant mare serum gonadotropin and human chorionic gonadotropin on in vitro maturation of goat oocytes

This study was aimed to evaluate the dose dependent effect of pregnant mare serum gonadotropin and human chorionic gonadotropin on in vitro maturation goat oocytes. The study was planned in 2 experiments. In the first experiment effect of pregnant mare serum gonadotropin supplementation on nuclear maturation of goat oocytes using 5 different concentrations of PMSG, was studied. The treatment was divided into group 1 (control medium containing TCM-199, sodium pyruvate (0.25mM), gentamycin (50µg/ml), L-glutamine (100µg/ml), estradiol 17-ß (1µg/ml), BSA (3mg/ml), supplemented with 10% FBS); group 2 (control medium + 10 IU/ml PMSG); group 3 (control medium + 20 IU/ml PMSG); group 4 (control medium + 30 IU/ml PMSG); group 5 (control medium + 40 IU/ml PMSG); and group 6 (control medium + 50 IU/ml PMSG). The oocytes of these treatment groups were matured in maturation media for 27 h in humidified atmosphere of 5% CO2 at 38.5°C in a CO2 incubator. The maturation rates achieved in groups 1–6 were 20.0, 48.51, 74.63, 74.15, 75.0 and 76.33%, respectively. In experiment 2, the effect of hCG supplementation on nuclear maturation of goat oocytes using 3 different concentrations of PMSG, was studied. Second experiment was divided into group 1 (control medium + 20 IU/ml PMSG +10IU/ml hCG), group 2 (control medium + 20 IU/ml PMSG +20IU/ml hCG) and group 3 (control medium + 20 IU/ml PMSG +30IU/ml hCG). The maturation rates achieved in groups 1–3 were 77.0, 85.19 and 84.22%, respectively. It can be concluded that supplementation of 20 IU/mL PMSG and 20IU hCG in maturation media significantly increased the maturation rate.

Effect of vitrification and slow freezing on in-vitro matured prepubertal goat oocytes

The present study was conducted to study the post thaw recovery rate of morphologically normal matured oocytes subjected to vitrification and slow freezing method. Oocytes were matured in TCM-199, which comprises sodium pyruvate (0.25 mM), L- glutamine (1 mg/10 ml), gentamycin (50 μg/ml) and supplemented with 10% fetal bovine serum at 38.5°C in CO2 incubator under humidified air. After 27 h of in-vitro maturation oocytes were assigned for freezing, viz. vitrification, slow cooling and control cooling. The percentage of oocytes recovered after thawing was significantly higher for control than those for vitrification (69.67%) and slow freezing (85.00%), respectively. Moreover, the cleavage rate in vitrification, slow freezing and control were 1.14, 3.92 and 13.37%, respectively. Thus, the cleavage rate of oocytes in vitrification and slow freezing method did not differ significantly. However, there was a significant difference in cleavage rates of freezing groups and the control groups.

In vitro embryo production in goats: Slaughterhouse and laparoscopic ovum pick up–derived oocytes have different kinetics and requirements regarding maturation media

A total of 3427 goat oocytes were used in this study to identify possible differences during in vitro embryo production from slaughterhouse or laparoscopic ovum pick up (LOPU) oocytes. In experiment 1, one complex, one semi-defined, and one simplified IVM media were compared using slaughterhouse oocytes. In experiment 2, we checked the effect of oocyte origin (slaughterhouse or LOPU) on the kinetics of maturation (18 vs. 22 vs. 26 hours) when submitted to semi-defined or simplified media. In experiment 3, we determined the differences in embryo development between slaughterhouse and LOPU oocytes when submitted to both media and then to IVF or parthenogenetic activation (PA). Embryos from all groups were vitrified, and their viability evaluated in vitro after thawing. In experiment 1, no difference (P > 0.05) was detected among treatments for maturation rate (metaphase II [MII]; 88% on average), cleavage (72%), blastocyst from the initial number of cumulus oocyte complexes (46%) or from the cleaved ones (63%), hatching rate (69%), and the total number of blastomeres (187). In experiment 2, there was no difference of MII rate between slaughterhouse oocytes cultured for 18 or 22 hours, whereas the MII rate increased significantly (P < 0.05) between 18 and 22 hours for LOPU oocytes in the simplified medium. Moreover, slaughterhouse oocytes cultured in simplified medium matured significantly faster than LOPU oocytes at 18 and 22 hours (P < 0.05). In experiment 3, cleavage rate was significantly greater (P < 0.001) in all four groups of embryos produced by PA than IVF. Interestingly, PA reached similar rates for slaughterhouse oocytes cultured in both media, but improved (P < 0.05) the cleavage rate of LOPU oocytes. Slaughterhouse oocytes had acceptable cleavage rate after IVF (∼67%), whereas LOPU oocytes displayed a lower one (∼38%), in contrast to cleavage after PA. The percentage of blastocysts in relation to cleaved embryos was not affected by the origin of the oocytes (P > 0.05). Therefore, slaughterhouse oocytes developed a greater proportion of blastocysts than LOPU ones, expressed as the percentage of total cumulus oocyte complexes entering to IVM. Vitrified-thawed blastocysts presented similar survival and hatching rates between the oocyte origin, media, or method of activation. In conclusion, slaughterhouse and LOPU derived oocytes may have different IVM kinetics and require different IVM and IVF conditions. Although the IVM and IVF systems still need improvements to enhance embryo yield, the in vitro development step is able to generate good quality embryos from LOPU-derived oocytes.