Number Of Viable Oocytes Research Articles

Effect of various antioxidants on ovarian tissue vitrification success in sheep

BACKGROUND: Vitrification is a technique of cryopreservation that has been proposed as a promising alternative method for the preservation of oocytes, embryos and gonadal tissue. OBJECTIVE: To determine the effect of different antioxidants on post-thaw viability, morphology of retrieved oocytes and histology of vitrified ovarian tissue.MATERIALS AND METHODS: Four different antioxidants [i.e., resveratrol (20 μM), ZnSO4 (500 μM), curcumin (25 μM) and quercetin (1 μM)] were evaluated after their addition to the vitrification and warming media for their effects on the viability and morphology of retrieved oocytes and the histology of vitrified ovarian tissue. RESULTS: The number of oocytes retrieved from ovarian tissue from the above mentioned antioxidants and vitrified control were 34, 41, 26, 31 and 46 respectively. Among these the number of viable oocytes were found to be 24 (70.6%), 30 (73.1%), 20 (76.9%), 26 (83.9%) and 33 (71.7%) and the number of oocytes found morphologically normal were 24 (70.6%), 26 (63.4%), 18 (69.2%), 21 (67.7%) and 34 (73.9%) for the above mentioned different antioxidants and vitrified control, respectively. Nonsignificant (P>0.05) differences were found between different treatment groups. Histomorphological evaluation of the ovarian cortical tissue showed that the percentage of intact follicles was significantly (P≤0.05) higher in the fresh control (84.19±3.9) than in other groups. Non-significant differences were found between resveratrol (50.2±5.5), curcumin (48.7±5.7), quercetin (51.6±4.8) and the vitrified control (42.7±6.1) groups; however, the ZnSO4 supplemented group (23.1±8.54) differed significantly (P≤0.05) from other antioxidant groups but was non-significant (P>0.05) with the vitrified control group (42.7±6.1). CONCLUSION: The addition of antioxidants resveratrol, curcumin and quercetin at these concentrations tended to non-significantly improve the follicular integrity after vitrification.

Inheritance of genomic regions and genes associated with number of oocytes and embryos in Gir cattle through daughter design

Over the past decades, daughter designs, including genotyped sires and their genotyped daughters, have been used as an approach to identify quantitative trait loci (QTL) related with economic traits. The aim of this study was to identify genomic regions inherited by Gir sire families and genes associated with number of viable oocytes (VO), total number of oocytes (TO) and number of embryos (EMBR) based on a daughter design approach. In total, 15 Gir sire families were selected. The number of daughters per family ranged from 26 to 395, which were genotyped with different SNP panels and imputed to the Illumina BovineHD BeadChip (777K) and had phenotypes for oocyte and embryo production. Daughters had phenotypic data for VO, TO, and EMBR. The search for QTL was performed through Genome-Wide Association Study (GWAS) based on genomic best linear unbiased prediction (gBLUP). QTL were found for each trait among and within families based on the top 10 genomic windows with the greatest genetic variance. For EMBR, genomic windows identified among families were located on BTA4, BTA5, BTA6, BTA7, BTA8, BTA13, BTA16 and BTA17, and they were most frequent on BTA7 within families. For VO, genomic windows were located on BTA2, BTA4, BTA5, BTA7, BTA17, BTA21, BTA22, BTA23 and BTA27 among families, being most frequent on BTA8 within families. For TO, top 10 genomic windows were identified on BTA2, BTA4, BTA5, BTA7, BTA17, BTA21, BTA22, BTA26 and BTA27, being most frequent on BTA7 and BTA8 within families. Considering all results, the greatest number of genomic windows was found on BTA7, where VCAN, XRCC4, TRNAC-ACA, HAPLN1 and EDIL3 genes were identified in the common regions. In conclusion, 15 Gir sire families with 26 to 395 daughters per family with phenotypes for oocyte and embryo production helped to identify the inheritance of several genomic regions, especially on BTA7, where EDIL3, HAPLN1 and VCAN candidate genes were associated with number of oocytes and embryos in Gir cattle families.

Parent-of-origin effects for the number of oocytes and embryos in Gir cattle

Imprinting is a phenomenon that alters the expression of genes according to the parental origin of their alleles. A quantitative form to evaluate the imprinting effect is known as parent-of-origin effect. Our aim with this work is to identify parent-of-origin effects that influence the number of oocytes and embryos in Gir dairy cattle. A dataset with 17,526 Ovum Pick Up observations from 1641 Gir donors was used to estimate parent-of-origin effects for the traits number of total oocytes (TO), number of viable oocytes (VO) and number of embryos (EM). To identify parent-of-origin effects, dam and sire gametic effects were included, individually or together, in an animal model for TO, VO and EM traits. For TO, inclusion of paternal origin effects in the model was significant (P < 0.05), and explained 6 % of the total phenotypic variance. For VO and EM no significant parent-of-origin effects were found for either parental line. In conclusion, paternal effects appear to influence the total oocyte production in the Gir cattle breed.

Effects of Heat-Stress on Oocyte Number and Quality and In Vitro Embryo Production in Holstein Heifers

Background: This study aimed to determine the effects of environmental temperature on the number and quality of oocytes and embryo production rates obtained by performing ovum pick up (OPU). Heat stress leads to long-term, short-term, visible, and invisible effects in dairy cows. Its effects on reproduction are evident in all stages, from oocyte development to birth. Disturbance in ovarian follicle development, follicular dominance deficiency, anoestrus, polyspermia, embryoniclosses, decreased fetal growth, and abortion are some examples of responses to these effects. The aim of the present study was aimed to determine the effects of ambient temperature on oocyte quality and number and embryo production rates.Materials, Methods &amp; Results: The animal material used in this study comprised 10 Holstein heifers. At the beginning of the study, the heifers were 13-15 months old. OPU was performed at different times of the year, and weather conditions were recorded. Grouping according to ambient temperature was done as &lt; 10°C (group 1), 10-25°C (group 2), and &gt; 25°C (group 3). The veterinary ultrasonography device and a set of compatible intravaginal OPU probe, catheter, and aspiration device were used for OPU application. All antral follicles with diameters of 2-8 mm in the ovaries were aspirated. The aspirated follicle fluids were examined under a stereo microscope, and the cumulus-oocyte complexes (COC) were collected and classified according to their structure. A, B, and C-quality oocytes were included in the in vitro embryo production process. After performing 69 OPUs on random days of the cycle, the number of oocytes per OPU was found to be 8.72, 6.32, and 6.85 in groups 1, 2, and 3, respectively (P &lt; 0.05). The number of viable oocytes per OPU was 6.83, 4.64, and 4.65 in groups 1, 2, and 3, respectively (P &lt; 0.05). The statistical difference between the first group and the other groups was significant for cleavage and blastocyst counts (P &lt; 0.05).Discussion: All the negative effects of heat stress on animals resulted from the increased body temperature. Reproductive performance is adversely affected by high temperatures and humidity during periods of high ambient temperatures. Metabolic heat is released, and the heat load increases due to the metabolism of nutrients in cattle. Internal body temperature is regulated via the dissipation of metabolic heat to the environment. The amount of heat dissipated via conduction andconvection depends on the unit body weight, surface area, skin and coat color, difference in temperature gradient of the animal and ambient temperature, and humidity. In the present study, it was determined that the blastocyst development rates of the oocytes obtained in the warm season (&gt;25°C [group 3]) were lower than those of the other groups. It was concluded that this may be because the oocytes developed under chronic heat stress in the animals, and several cycles were required to enhance oocyte quality and developmental potential. Additional studies are needed to investigate the response of oocytes obtained with OPU to heat stress during embryonic developmental stages and to determine the sensitivity and effects of embryonic tissue damage according to developmental stages. Based on the results of the present study, it was concludedthat performing OPU and in vitro embryo production (IVEP) when the ambient temperature is close to the thermoneutral limits may increase the blastocyst development rates. Keywords: blastocyst, heat stress, heifer, in vitro embryo production, oocyte quality, ovum pick-up.

Repeatability and random regression models to estimate genetic parameters for oocyte and embryo production in the Gir breed

Context Greater production of oocytes and embryos from Gir donors contributes to greater fertility and genetic progress. Aims This study aimed to obtain genetic parameters for oocyte and embryo production in the Gir breed. Methods Repeatability and random regression models were applied to data consisting of 17 526 Ovum Pick Up observations from 1641 Gir donors from five different herds. Single and multi-trait analyses were carried out with the application of both models for the traits: number of viable oocytes, number of total oocytes and number of embryos, using the BLUPF90 family programs. Legendre polynomials of second order were used in the random regression model. Key results Considering the repeatability model, additive genetic variance ranged from 0.06 to 0.13 and permanent environment variance ranged from 0.05 to 0.08 for all evaluated traits. Residual variance ranged from 0.30 to 0.45. Heritability estimates were 0.10 for number of embryos, 0.24 for total oocytes, and 0.25 for viable oocytes. Repeatability estimates were moderate, ranging from 0.20 to 0.40, and genetic correlation estimates were always above 0.80. Phenotypic correlation was high only between viable and total oocytes (0.95), and moderate in the other cases. Random regression model results were consistent with those from the repeatability model. The heritability values remained similar throughout the donors’ ages, with moderate values for viable and total oocytes, and low values for number of embryos. Genetic correlations among ages for each trait were moderate to high. Also, the genetic correlations between traits within each age were high, with values always above 0.7. Conclusions Selection of Gir donors for total oocyte production at any time, between 1 and 16 years of age, might lead to an increase in the number of viable oocytes and embryos obtained, but it’s preferable at younger ages to hasten genetic progress. Repeatability models could be the best method, as they require less computational effort when compared to the random regression models and the parameter estimates do not vary substantially throughout different ages of the donor. Implications The use of repeatability models to estimate genetic parameters of oocytes and embryos resulted in similar results compared to random regression models.

Improvement in early antral follicle development and gene expression modulation prior to follicle aspiration in bovine cumulus-oocyte complexes by equine chorionic gonadotropin

We aimed to evaluate the morphological ovarian response to equine chorionic gonadotropin (eCG) prior to ovum pick-up (OPU) and its effects on the molecular phenotype of immature cumulus-oocyte complexes (COCs) from Nelore cow (Bos indicus) donors. To this end, 20 Nelore cows were distributed randomly into the synchronized-OPU (Sync-OPU) and synchronized plus stimulated-OPU (Sync + eCG-OPU) groups using a cross-over experimental design, as each cow was used in both treatments. On a random day of the estrus cycle (Day 0), all cows received an intravaginal implant with 1.0 g of progesterone and 2 mg IM of estradiol benzoate. On the morning of Day 3, only the Sync + eCG-OPU group received 400 IU of eCG IM. On the morning of Day 5, the P4 device was removed and OPU was conducted in both groups. Before OPU management, ultrasonography was used to identify and measure the follicles. The aspirated COCs were morphologically classified based on their cumulus cells (CC) layers and the texture of the ooplasm. The COCs classified as Grade 1, Grade 2, and Grade 3 were considered viable and used for the assessment of quality markers. Oocytes and CC were mechanically separated from pools of 25 immature COCs of the Sync-OPU and Sync + eCG-OPU groups immediately after the follicular aspiration and stored at −80 °C until RNA extraction. Relative quantification of several markers for oocyte quality was assessed by RT-qPCR. The eCG treatment increased the number of follicles sized 3.0–5.0 mm and >5.0 mm compared to that in Sync-OPU group. Moreover, the protocol with eCG improved the total number of oocytes and the number of viable oocytes, which is related to a high number of oocytes in Grade 3. Regarding the impact on transcriptional regulation in immature oocytes, the mRNA encoding BMP15, SMAD1, SMAD2, SMAD3, ACACA, and CPT1A was upregulated in Sync + eCG-OPU compared with the Sync-OPU group. Moreover, the relative mRNA abundance of CTSZ, a member of the cathepsins family functionally related to reduced oocyte competence, was lower in the Sync + eCG-OPU group than in the Sync-OPU group. In addition, CC CTSB, CTSS, and CTSK mRNA abundances were lower in the Sync + eCG-OPU group than in the Sync-OPU group. However, the relative abundance of AREG and EREG mRNA was higher in CC recovered from cows stimulated with eCG. In conclusion, the eCG approach addressing follicular stimulation in Nelore cows had a positive impact on early antral follicle development, followed by a positive morphological and molecular phenotype in bovine COCs.

Synchronization with estradiol benzoate in the presence of the corpus luteum in Wagyu cows increases the number of medium follicles but does not interfere with in vitro production of embryos

Reproductive biotechnologies are emerging as an important element for livestock; however, some strategies must be modified to adapt to different breeding systems, such as the use of follicular synchronization protocols. This study aimed to evaluate follicular synchronization using estradiol benzoate (EB), in the presence of the corpus luteum (CL) from Wagyu oocyte donors in in vitro embryo production (IVEP). Rounds of IVEP were performed in heifers and cows (n=19) that were classified into three groups: G1/CL - animals with CL, G2/WCL - animals without CL, and G3/CL + EB - animals with CL that were subjected to follicular synchronization with EB at D0. The groups G1/CL and G2/WCL were considered the control and undertook the natural process of follicular dynamics. The results showed that the synchronization of the follicular wave with the application of EB in the presence of CL, presented a smaller number of small (6.05 ± 0.55) and large follicles (0.45 ± 0.15), but increased (P &lt; 0.05) the number of medium-sized follicles (16.20 ± 0.90). However, the results of ovum pick up showed that regardless of whether or not EB was applied, and regardless of the presence or absence of CL in the Wagyu donor, there was no difference among the groups (P &gt; 0.05) concerning the number of viable oocytes and the viability rate. It was concluded that follicular synchronization using EB in Wagyu oocyte donors that presented a CL, increased the number of medium-sized follicles. However, there was no improvement in the efficiency of ovum pick up, in vitro embryo production, and pregnancy rate.

Influence of antral follicle count on in vitro embryo production, sexual precocity and conception rate of Senepol cattle

Context There are contradictory results concerning the relationship between antral follicle count (AFC) and reproductive traits for different breeds of cattle. Understanding this relationship could be useful to selecting animals for breeding. Aims We evaluated the effects of AFC on parameters related to in vitro embryo production (IVP), and the influence of AFC on sexual precocity and conception rate at first fixed timed artificial insemination (FTAI) of Senepol females. Methods Ovarian ultrasound was performed to count antral follicles, and females were classified as low AFC (≤22 follicles), intermediate AFC (23–36 follicles) or high AFC (&gt;36 follicles). Expt 1 evaluated the influence of AFC on IVP, with data from 146 adult cows that were submitted to follicular aspiration. Expt 2 evaluated the influence of AFC on heifer sexual precocity, determined by the presence of a corpus luteum (n = 608), and on conception rate at first FTAI (n = 143). Key results High AFC cows had a greater (P &lt; 0.0001) number of viable oocytes and cleaved embryos and a higher average number of embryos than low AFC cows (38.28 vs 9.92, 38.28 vs 7.75 and 9.34 vs 2.41, respectively). However, the conversion rate (total viable embryos per oocytes) was not affected (P = 0.46) by AFC category. High AFC heifers tended (P = 0.07) to be more sexually precocious than low AFC heifers (16.73% vs 9.47%). An influence of AFC or of AFC category on conception rate at first FTAI was not detected (P = 0.35 and 0.19, respectively). Conclusions Despite greater IVP in high AFC cows, the conversion rate of viable oocytes to IVP was not affected by AFC category. High AFC heifers tended to be more sexually precocious, although AFC did not influence the conception rate at first FTAI. Implications Oocyte quality is probably not influenced by AFC, but selection of animals according to AFC may improve the sexual precocity of the herd.

In vitro performance of Zebu (Bos indicus) and Taurus (Bos taurus) donor cow embryos

ABSTRACT In this study, the in vitro production of bovine embryos from zebu and taurine donors was compared. Cumulus-oocyte complexes (COCs) were obtained from 167 Bos taurus and 161 Bos indicus donors by ovum pick-up. COCs were classified based on their morphological quality, matured in incubators for 22 to 24 h in maturation medium, and then fertilized for 18 to 22 h. The zygotes were transferred to the culture medium for seven days. The embryos were classified as morula (OM), initial blastocyst (BI), blastocyst (BL), and expanded blastocyst (BX), before being transferred to synchronized recipient cows. Pregnancy was diagnosed 30-45 days post-transfer. The Bos indicus donors had a higher oocyte yield (n = 2556) than Bos taurus donors (n = 1903) (P = 0.008). The COCs from zebu donors had a better morphological quality than those from taurine donors (n = 689 vs. 444 for grade 1 COC, P &lt; 0.0001; n = 681 vs. 509 for grade 2 COC, P = 0.010, for zebu and taurine donors, respectively). There were differences in embryo production percentages obtained from OM (0.44% from zebu and 6.42% from taurine, P = 0.017), BL (14.18% from zebu and 3.74% from taurine, P &lt; 0.0001), and BX (81.43% from zebu and 75.13% from taurine, P &lt; 0.0001). No significant difference was observed for embryo production from BI and pregnancy rate (P &gt; 0.05). The Bos indicus cows showed greater oocyte recovery, number of viable oocytes, and production of viable embryos than the Bos taurus cows.

176 Synchronisation of follicle wave emergence prior to superstimulation with purified FSH for ovum pickup affects blastocyst rate in pregnant Holstein heifers

The timing of initiation of superstimulatory treatments relative to follicle wave emergence has been shown to affect ovulatory response and invivo embryo production. The significant increase of invitro embryo production (IVP) and the possibility of using pregnant animals as oocyte donors has created the need to optimise superstimulatory treatments for IVP in pregnant cattle. Thus, the objective of the present study was to evaluate the effect of synchronisation of follicle wave emergence before superstimulation for ovum pickup (OPU) and IVP in pregnant heifers. Pregnant (47-62 days of gestation) Holstein heifers (n=28) 19.5±0.3 months of age were assigned in a completely randomised design to one of two groups: synchronisation of follicular wave emergence by dominant follicle removal (DFR; all follicles &amp;gt;6mm) or untreated control (no DFR). Superstimulatory treatments were initiated 36h after DFR or at random stages of the follicular wave in the no-DFR group and consisted of the administration of 160mg of purified FSH (Folltropin-V, Vetoquinol) over four injections 12h apart as follows: 48.0, 42.7, 37.3, and 32.0mg. Ovum pickup was performed in all heifers 40h after the last purified FSH injection. Heifers were subjected to OPU for oocyte recovery, and the number of follicles was determined. Recovered oocytes were processed in groups by treatment, and IVP was performed. Differences between treatment groups were evaluated using generalised linear mixed models. Results are presented in Table 1 and are expressed as means±s.e.m. for data collected at the time of OPU or as proportions for embryo production results. The number of small follicles (&amp;lt;6mm) at the time of OPU was greater in the no-DFR group than in the DFR group (P=0.04). Conversely, there were no differences between treatments in the number of medium follicles (6-10 mm; P=0.17), large follicles (&amp;gt;10 mm; P=0.11), total follicles (P=0.93), total number of recovered oocytes (P=0.4), or number of viable oocytes (P=0.53). The mean oocyte percentage recovery rate was not different between heifers in the DFR (53.6±4.7%) and no-DFR (56.5±4.7%) groups (P=0.52). Both cleavage and blastocyst rate were greater (P&amp;lt;0.008) in the DFR group than in the no-DFR group; as a result, the number of transferable embryos per animal was 5.6 in the DFR group and 2.8 in the no-DFR group. In summary, initiation of superstimulatory treatments at the time of follicle wave emergence improves cleavage and blastocyst rates, thus leading to greater embryo production. Table 1.Ovarian response and embryo production in pregnant heifers superstimulated with or without synchronisation of follicle wave emergence Variable DFR No DFR Small follicles, n 8.1±1.2A 12.1±1.8B Medium follicles, n 18.3±1.3 13.7±2.0 Large follicles, n 2.4±0.6 1.4±0.4 Total follicles, n 28.8±1.4 27.2±2.2 Total oocytes, n 15.4±1.5 16.0±1.9 Viable oocytes, n 13.7±1.5 13.4±1.8 Cleavage rate,% (n) 77.1 (192)A 64.4 (188)B Blastocyst rate,% (n) 40.6 (192)A 20.7 (188)B A,BMeans within a row with different superscripts differ (P&amp;lt;0.05).

132 Effect of treatment with follicle-stimulating hormone on in vitro embryo production of Gyr (Bos indicus) calves, pubertal heifers and adult cows

The aim of this study was to evaluate the effect of FSH treatment on the in vitro embryo production of Gyr (Bos indicus) calves (3-10 months), pubertal heifers (16-21 months) and adult cows. Thirty females were used: 10 calves, 10 pubertal heifers (puberty was determined by the presence of a corpus luteum) and 10 cows. The animals were distributed as follows: calves: control (C-C, n=5) and with FSH (FSH-C, n=5); heifers: control (C-H, n=5) and with FSH (FSH-H, n=5); and adult animals: control (C-A; n=5) and with FSH (FSH-A; n=5). All animals received an intravaginal progesterone device (calves-Primer PR®, Agener União, Brazil; heifers and cows-Prociclar®, Ceva Saúde Animal, Brazil) and oestradiol benzoate (calves and heifers: 1mg, cows: 2 mg; Fertilcare Sincronização®, MSD Saúde Animal, Brazil) on Day 0. The control animals of each category received no additional treatment. The FSH-C received 80mg IM of FSH (Folltropin®, Vetoquinol, Mairipora, Brazil), performed in 4 injections twice a day in decreasing doses [25mg (Day 4, p.m.), 25mg (Day 5, a.m.), 15mg (Day 5, p.m.), and 15mg (Day 6, a.m.); coasting period: 24 h]. The FSH-H received 100mg IM of FSH, performed in 4 injections twice a day in decreasing doses [30mg (Day 4, p.m.), 30mg (Day 5, a.m.), 20mg (Day 5, p.m.), and 20mg (Day 6, a.m.); coasting period: 24 h]. The FSH-A received 140mg IM of FSH, performed in 4 injections twice a day in decreasing doses [40mg (Day 4, a.m.), 40mg (Day 4, p.m.), 30mg (Day 5, a.m.), and 30mg (Day 5, p.m.); coasting period: 48 h]. On Day 7 the intravaginal devices were removed and all animals were submitted to epidural anaesthesia (2% lidocaine) followed by ovum pickup guided by transvaginal ultrasound (guide EC9-5 Heifer, WTA, Sao Paulo, Brazil; ultrasound S8®, SonoScape, Shenzhen, China). The recovered oocytes were sent to a commercial laboratory for the in vitro embryo production. The obtained data were analysed by the GLIMIX procedure of SAS® (SAS Institute Inc., Cary, NC, USA; means are presented as standard error of the mean. The oocyte recovery rate was lower (P=0.01) for calves and heifers treated with FSH (FSH-C: 42.8%; FSH-H: 55.2%) when compared with control calves and heifers (C-C: 65.1%; C-H: 81.1%); however, no difference was observed for cows (C-A: 63.7%; FSH-A: 63.1%). The number of viable oocytes differed according to the category (P=0.001); calves and cows had higher numbers of viable oocytes (calves: 11.6±0.7; cows: 11.7±0.9) when compared with heifers (23.4±0.6). The number of cleaved oocytes increased (P=0.03) when calves (FSH-C: 8.0; C-C: 5.8) and cows (FSH-A: 8.8, C-A: 6.0) were treated with FSH. Cleavage rate was higher (P=0.05) when animals of all categories were treated with FSH (calves=52.7%; heifers=68.0%; cows=67.9%), when compared with nontreated animals (45.8%; 60.1%; 51.8%, respectively). The number of blastocysts per ovum pickup increased (P=0.04) when calves (FSH-C: 3.2; C-C: 1.5) and cows (FSH-A: 5.8; C-A: 2.8) were treated with FSH. The number of vitrified embryos (percentage of blastocysts surviving vitrification) increased (P=0.02) when calves (FSH-C: 2.6; C-C: 1.0) and cows (FSH-A: 5.0; C-A: 2.4) were treated with FSH. These results show that treatment with FSH increases the efficacy of in vitro embryo production in Gyr calves and cows.

Nutritional impact on gene expression and competence of oocytes used to support embryo development and livebirth by cloning procedures in goats

Changes in the nutritional plan have been shown to affect oocyte quality, crucial to oocyte donors animals used in cloning. This study aimed to evaluate the impact of diets with increasing nutritional levels (maintenance diet=M; 1.3M; 1.6M; 1.9M) fed to goats for four weeks on follicular fluid composition, gene expression and oocyte competence used to cloning in goats. Donor females were superovulated for the retrieval of matured oocytes and physical measurements reported. After four weeks, groups receiving diets above maintenance increased thickness of subcutaneous adipose tissue and body weight, with higher values in 1.9M Group (P<0.05). Treatments did not affect follicular density, number of aspirated follicles, retrieved and matured oocytes. Animals from 1.3M group had lower (P<0.05) maturation rate (44.0%) and number of viable oocytes (65.3%) than M (68.8%) and 1.9M (76.0%). Follicular fluid glucose concentrations increased with nutritional levels (P=0.010), with a difference (P<0.05) between groups 1.9M (11.4±2.6mg/dL) and M (2.6±0.5mg/dL). The diet did not affect the expression of GDF9, BMP15, and BAX genes in oocytes, but BCL2 and apoptotic index were significantly higher (P<0.05) in the 1.3M and 1.6M groups than the other groups. Following the transfer of cloned embryos, one fetus was born live of a twin pregnancy in the 1.9M Group. The association between energy intake and oocyte quality suggests better nutritional use by oocytes when the maximum flow was used (1.9M), but the optimal feeding level in cloning still needs refinement.

Count Bayesian models for genetic analysis of in vitro embryo production traits in Guzerá cattle

Four models for in vitro embryo production traits in Guzerá cattle were compared: Gaussian (untransformed variable – LIN and transformed in logarithmic scale – LOG), Poisson (POI) and zero-inflated Poisson (ZIP). Data consisted of 5716 ovum pick-up and in vitro fertilization records performed in 1205 cows from distinct regions of Brazil. Analyzed count traits were the number of viable oocytes (NOV), number of grade I oocytes (NGI), number of degenerated oocytes (NDG), number of cleaved embryos (NCLV) and number of viable produced embryos (NEMB). Heritability varied from 0.17 (LIN) to 0.25 (POI) for NOV; 0.08 (LOG) to 0.18 (ZIP) for NGI; 0.12 (LIN) to 0.20 (POI) for NDG; 0.13 (LIN) to 0.19 (POI) for NCLV; 0.10 (LIN) to 0.20 (POI) for NEMB depending on the considered model. The estimated repeatability varied from 0.53 (LOG) to 0.63 (POI) for NOV; 0.22 (LOG) to 0.39 (ZIP) for NGI; 0.29 (LIN) to 0.42 (ZIP) for NDG; 0.42 (LIN) to 0.59 (POI) for NCLV; 0.36 (LIN) to 0.51 (POI) for NEMB. The goodness of fit, measured by deviance information criterion and mean squared residuals, suggested superiority of POI and ZIP over Gaussian models. Estimated breeding values (EBV) obtained by different models were highly correlated, varying from 0.92 for NOV (between LIN-POI) and 0.99 for NGI (between POI-ZIP). The number of coincident animals on the 10% top EBV showed lower similarities. We recommend POI and ZIP models as the most adequate for genetic analysis of in vitro embryo production traits in Guzerá cattle.

Use of FSH in two different regimens for ovarian superstimulation prior to ovum pick up and in vitro embryo production in Holstein cows

We aimed with the present study to evaluate the effects of FSH treatment (200 mg) split in four or six administrations on ovarian follicle stimulation and in vitro oocyte competence for embryo production in dairy cows with synchronized follicular wave emergence. On random days of the estrous cycle (Day 0), non-lactating Holstein cows received a progesterone (P4)-releasing intravaginal device and 2 mg estradiol benzoate IM. On Day 3, they received 0.530 mg sodium cloprostenol (PGF2α) IM. Control cows (n = 35) received no further treatments, whereas FSH-treated cows received 200 mg FSH split in four (FSH4 group; n = 33) or six (FSH6 group; n = 33) administration regimens. Starting on Day 4, cows in FSH4 group received 200 mg FSH split in four equivalent doses of 50 mg 12 h apart. Cows in FSH6 group received the same total FSH dose split in six equivalent doses of 33.3 mg 12 h apart, but treatments started on Day 3. On Day 7 AM (36 h of “coasting” period for FSH-treated groups), the P4 devices were removed and cows were subjected to ovum pick up (OPU). Viable oocytes were in vitro fertilized using sexed-sorted semen. Although FSH treatment did not (P > 0.1) increase the total number of follicles (Control, 53.2 ± 4.5 vs. FSH-treated, 51.4 ± 3.1), the two hormonal stimulation regimens, FSH4 and FSH6, increased the number of medium follicles (6–10 mm; 5.2 ± 0.5 vs. 18.1 ± 1.4; P < 0.0001) and reduced the number of small follicles (2–5.9 mm; 46.3 ± 5.1 vs. 31.0 ± 2.4 P < 0.0001). Also, FSH treatment or regimen did not increase (P > 0.1) the number of viable oocytes (Control, 12.6 ± 1.26 vs. FSH-treated, 12.70 ± 1.03), recovery rate (Control, 36.5% vs. FSH-treated, 36%) and the number of in vitro produced blastocyst (Control, 4.1 ± 0.52 vs. FSH-treated 4.3 ± 0.5). We concluded that FSH stimulation protocol proposed herein is effective to stimulate the growth of small antral follicle population prior to OPU, but it was ineffective to improve in vitro oocyte competence for embryo production in non-lactating Holstein cows with synchronized follicular wave emergence.

Parâmetros da produção in vitro de embriões da raça Sindi

Resumo: O objetivo deste trabalho foi avaliar os efeitos da doadora, estação do ano, do touro e do tipo de sêmen, na produção in vitro de embriões da raça Sindi (Bos indicus). Para isso, avaliaram-se 434 sessões de aspiração folicular in vivo (OPU), realizadas em 152 doadoras, com a utilização de sêmen sexado e não sexado de 22 touros. Analisaram-se as seguintes variáveis: doadora; idade da doadora; estação do ano; touro; tipo de sêmen; oócitos recuperados; viáveis e degenerados; e as taxas de clivagem e de blastocistos. Observaram-se efeitos da doadora e do touro sobre os oócitos recuperados, viáveis e degenerados e sobre a taxa de blastocisto. O ano foi dividido em estações chuvosa, de outubro a março, e seca, de abril a setembro. Na estação chuvosa, a proporção de oócitos viáveis aumentou, enquanto a de degenerados diminuiu. As doadoras com menos de seis anos tiveram maior proporção de oócitos viáveis e menor, de degenerados. O sêmen não sexado obteve as melhores taxas de clivagem e de blastocistos. Doadoras de até seis anos apresentam a maior proporção de oócitos viáveis, e o maior número de oócitos viáveis é produzido na estação chuvosa. O sêmen não sexado resulta em melhores taxas de clivagem e de blastocistos.

Development of insulin resistance in dairy cows by 150 days of lactation does not alter oocyte quality in smaller follicles

The objective of this study was to test the hypothesis that high-producing dairy cows become increasingly resistant to insulin throughout lactation and that, consequently, oocyte quality is compromised. We used Holstein cows at 50 (51.5±3.7; n=30), 100 (102.3±9.4; n=30), and 150 (154.5±18.9; n=30) days in milk (DIM). We measured circulating insulin and glucose and performed a glucose tolerance test (GTT) after 5h of fasting. To evaluate oocyte quality, we performed ovum pickup on the day before the GTT (581 oocytes). We performed statistical analyses using the MIXED procedure of SAS. The model included the fixed effects of DIM, period, time, parity, and an interaction between DIM and time. We observed no difference in the GTT between groups for any variable related to circulating glucose (for example, glucose peak=203.3±7.2, 208.8±6.3, and 194.3±5.9mg/dL). However, various measures of circulating insulin were different in cows at 150 DIM compared with 50 or 100 DIM: higher basal insulin (8.8±0.9, 8.8±0.8, and 11.9±0.8 µIU/mL), peak insulin (61.9±6.2, 69.1±5.7, and 89.0±6.1 µIU/mL), delta maximum insulin (51.1±5.5, 59.4±5.0, and 73.5±5.4 µIU/mL), and area under the curve 5-60 (1,874.8±171.0, 2,189.5±157.8, and 2,610.5±174.0 µIU/mL × min). Nevertheless, we observed no difference among groups in the number of viable oocytes (3.2±0.7, 3.9±0.7, and 3.6±0.7 per cow per ovum pickup) or percentage of viable oocytes (49.3, 52.2, and 51.8%). Increased circulating insulin before and throughout the GTT in cows at 150 DIM indicates that cows develop increasing insulin resistance with increasing DIM; however, increased insulin resistance was not associated with a detectable alteration in the quality of oocytes aspirated from small and medium-sized follicles.

Genetic analysis of oocyte and embryo production traits in Guzerá breed donors and their associations with age at first calving.

The objective of this study was to estimate variance components for oocyte and embryo production traits in Guzerá breed female donors, and investigate their associations with age at first calving (AFC). The traits analyzed were the number of viable oocytes (NOV), the number of grade I oocytes (NGI), the number of cleaved embryos (NCLV), and viable embryos produced (NEMB), and the percentages of viable oocytes (POV), grade I oocytes (PGI), cleaved embryos (PCLV), and viable embryos (PEMB). Data were obtained from 5173 ovary puncture and in vitro fertilization (IVF) sessions using 1080 Guzerá female donors of different ages, occurred from March 2005 to July 2013. Variables were log-transformed (logeX+1) prior to analysis. (Co)variance components were estimated by restricted maximum likelihood (REML), using one- and two-trait animal models. Permanent environment and IVF sire (father of the embryos) random effects were included. Estimated heritabilities for NOV, NGI, NCLV, NEMB, POV, PGI, PCLV, and PEMB were 0.19, 0.08, 0.16, 0.14, 0.04, 0.03, 0.01, and 0.07, respectively. Repeatabilities for count traits (NOV, NGI, NCLV, and NEMB) varied from 0.14 and 0.32, higher than estimated for percentage traits (POV, PGI, PCLV, and PEMB), which varied from 0.01 to 0.08. Selection for NOV may be more appropriate in breeding programs than selection for NEMB, because of its strong genetic correlation (0.68) with NEMB and its greater time- and cost-effectiveness. AFC was only weakly associated with the oocyte and embryo production traits, which indicates that there would be no effect on AFC when selecting for these traits.

176 SERUM ANTIMULLERIAN HORMONE CONCENTRATIONS AND IN VITRO EMBRYO PRODUCTION IN BEEF CATTLE

The collection of high numbers of oocytes for in vitro embryo production in the cow depends on the number of antral follicles available for aspirations. The number of antral follicles varies significantly among cows and can be estimated by ovarian ultrasonography or measurement of serum concentrations of antimullerian hormone (AMH). The AMH is secreted by the granulosa cells of small antral follicles and serum concentrations are independent of the stage of the oestrous cycle. Therefore, it may be useful as a tool to select oocyte donors for in vitro embryo production. An experiment was designed to evaluate the effect of breed of cattle and the correlation of serum AMH concentrations on the number of viable oocytes and embryos produced in vitro. Cumulus-oocyte complex were collected during 44 ultrasound-guided follicle aspiration sessions performed at random stages of the oestrous cycle without superstimulation in 14 Bonsmara, 11 Braford, and 19 Brangus cows that were used for commercial in vitro embryo production using frozen-thawed semen from bulls of the same breed. Blood samples were collected by jugular venipuncture at the time of OPU, centrifuged at 3000 × g for 30 min for serum separation which was frozen at –20°C until AMH analysis. Serum AMH was evaluated using an ELISA (AnshLabs, Webster, TX, USA). The sensitivity of the AMH assay was 0.011 ng mL–1 and intra-assay CV were &lt;5%. The number of viable oocytes collected and embryos produced in each breed were compared by ANOVA and means were compared by the protected l.s.d. test when ANOVA revealed statistically significant differences. Regression analysis and Pearson correlation were used to determine the relationship between the number of viable oocytes aspirated and the number of embryos produced. The mean (± SEM) number of viable oocytes collected was greater (P &lt; 0.05) from Brangus (23.3 ± 4.3) and Braford (22.5 ± 3.6) than from Bonsmara donors (12.2 ± 2.5). Although not statistically different, the numbers of embryos produced followed the same pattern (Brangus, 4.4 ± 0.9; Braford, 3.4 ± 0.6; Bonsmara, 2.9 ± 0.5). Serum AMH concentration was positively correlated with the number of viable oocytes collected in all breeds (R2 = 0.63 for Bonsmara, 0.50 for Braford, 0.32 for Brangus; P &lt; 0.01). Serum AMH concentrations were also correlated with the number of embryos produced in all breeds (R2 = 0.32 for Bonsmara, 0.16 for Braford, 0.39 for Brangus). However, AMH concentrations were not significantly correlated with the proportion of viable oocytes collected and embryos produced. Results demonstrate a breed effect on the number of oocytes collected per session and that circulating AMH concentrations are correlated with the number of viable oocytes collected per session in commercial in vitro embryo production programs.

169 OVUM PICK-UP, IN VITRO EMBRYO PRODUCTION, AND EMBRYO TRANSFER IN CATTLE IN TROPICAL AND SUBTROPICAL MEXICO

Biotechnology continues to evolve rapidly, allowing the development of artificial reproductive techniques (ART) to increase reproductive efficiency and contribute to the genetic improvement of domestic animals. The present study examines the results obtained after 30 months of starting a commercial practice for ovum pickup (OPU) in vitro embryo production (IVP) and embryo transfer (ET) in cattle in tropical and subtropical Mexico. This research was conducted from 2013–2015 in beef and dairy cattle kept under different environmental and management conditions in 6 states (Chiapas, Oaxaca, Tabasco, Tamaulipas, Veracruz, and Yucatan). Oocytes were collected by OPU, without hormonal treatment, from 10 donor cows: 2 Bos taurus (Bt), 5 Bos indicus (Bi), and 3 Bt × Bi. A total of 98 oocyte recovery sessions were performed on 756 cows and produced 12 524 viable oocytes (1349 GI, 3383 GII, 7792 GIII), which were sent to a central laboratory for IVP. Both conventional and sexed semen were used for IVF, from 9 breeds: 2 Bt, 5 Bi and 2 Bt × Bi. The overall cleavage rate was 69% (8587/12 524). The embryo production efficiency rate was 31% (3905/12 524). Fresh sexed and conventional embryos were transferred to recipients synchronised with the following protocol: Day 0, application of an intravaginal device (ID) with progesterone and 2 mg of oestradiol benzoate IM; Day 8, removal of the ID, 400 IU eCG IM, 0.5 mg cloprostenol sodium IM, and 0.5 mg oestradiol cipionate IM. Day 10 was considered the day of oestrus. Pregnancy rate after ET was 38% (945/3905). The average number of viable oocytes per donor cow was 16.57; the average number of transferred embryos per donor cow was 5.17, and the average number of pregnancies per donor cow was 1.25. The OPU-IVP were successful in producing pregnancies even under several adverse conditions, such as a tropical environment, many donors being prepuberal females and the majority of the adult cows having a previous non-productive history due to hormonal mishandling in superovulation programs or because of overfeeding for show purposes. The consequences of these factors can be observed in the lower overall cleavage rate obtained (69%), compared to the expected (75%). Some variables require further analysis (e.g. different OPU and ET technicians, time of year, cow age, cow breed, and use of conventional and sexed semen). A successful IVP practice has to face and overcome multiple problems that may arise in some geographic locations but, perhaps, not in others. It is interesting to note the use of sexed versus conventional embryos, of the 98 ET sessions, 25 involved sexed embryos versus 73 for conventional embryos. The use of sexed semen for OPU-IVP and ET in this region of Mexico continues to expand. We thank Genemex Internacional and the ranch owners that were involved with this research.

233 HIGH NUMBERS OF ANTRAL FOLLICLES INFLUENCE THE IN VITRO EMBRYO PRODUCTION, BUT NOT THE CONCEPTION RATE OF FIXED-TIME ARTIFICIAL INSEMINATION IN NELORE CATTLE

The aim of this study was to compare the conception rates to fixed-time artificial insemination (FTAI) and in vitro embryo production between Nelore cows with high or low antral follicle counts (AFC). First, multiparous Nelore cows (Bos indicus, n = 547, 40–60 days postpartum) were subjected to synchronization of ovulation. Randomly during their oestrous cycle (Day 0), cows received an intravaginal device containing 1.9 g of P4 (CIDR®) and 2 mg of oestradiol benzoate (Estrogin®), intramuscularly. At device removal (Day 8), cows received 500 µg of PGF2α (Ciosin®), 300 IU of eCG (Novormon®), and 1 mg of oestradiol cipionate (ECP®), intramusculary. All cows were inseminated 48 h after P4 device removal. Antral follicles = 3 mm were counted using an intravaginal microconvex transducer (Day 0), and cows were assigned to groups of high (G-High, = 25 follicles, n = 183), intermediate (G-Intermediate, 16–20 follicles, n = 183), or low AFC (G-Low, = 10 follicles, n = 181). In another study to compared the in vitro embryo production, Nelore cows (n = 66, 72–96 months) were subjected to ultrasound-guided follicular aspiration using an intravaginal microconvex array transducer (7.5 MHz). The COC were selected and cows were assigned to groups according to the oocyte production: G-High (n = 22, = 40 oocytes), G-Intermediate (n = 25, 18–25 oocytes), or G-Low (n = 19, = 7 oocytes). Previously tested semen from a single bull was used for IVF using a previously described protocol (Silva-Santos et al. 2014 Reprod. Domest. Anim. 49, 228–232). The oocyte and embryo production (viable embryo: grade I, II, III; vitrifiable embryo: grade I, II) were evaluated. The number of follicles was evaluated by Kruskal-Wallis, and the chi-square test was used for data on oocyte and embryo production (P = 0.05). The average follicular population was 30.7 ± 5.7 (G-High), 18.6 ± 1.64 (G-Intermediate), and 7.8 ± 2.4 follicles (G-Low; P &lt; 0.05), but there were no differences in the conception rates among groups (51.9 v. 48.6 v. 58.6%, respectively; P &gt; 0.05). The total number of oocytes recovered were 1109 (G-High), 534 (G-Intermediate), and 101 (G-Low; P &lt; 0.05). The mean number of viable oocytes was 40.4 ± 10.6 (G-High), 14.8 ± 3.0 (G-Intermediate), and 3.8 ± 1.1 (G-Low; P &lt; 0.05) and the percentage of viable oocytes was 80% (G-High), 69% (371/534, G-Intermediate), and 71% (G-Low; P &lt; 0.05). Cleavage rate was 79% (G-High), 74% (348/472, G-Intermediate), and 71% (G-Low; P &lt; 0.05), and blastocyst rate was 42% (G-High), 32% (153/472, G-Intermediate), and 13% (G-Low; P &lt; 0.05). The number of viable embryos was 18.4 ± 6.7 (G-High), 6.1 ± 3.6 (G-Intermediate), and 0.6 ± 0.7 (G-Low; P &lt; 0.05) and the percentage of vitrifiable embryos was 81% (G-High), 77% (118/153, G-Intermediate), and 58% (G-Low; P &lt; 0.05). Therefore, Nelore cows with high oocyte production had ~10-fold higher oocyte production and produced ~30-fold more embryos compared with the low AFC group. In conclusion, AFC had no influence on the conception rates to FTAI; however, Nelore cows with high oocyte production exhibited higher in vitro embryo production.