Number Of Viable Oocytes Research Articles

143 COMPARISON OF COMMERCIAL IN VITRO EMBRYO PRODUCTION AND PREGNANCY RATES OF BRAHMAN DONORS IN PANAMA VERSUS DOMINICAN REPUBLIC

It has been previously demonstrated (IETS 2011) that Panama is applying the biotechnology of in vitro embryo production (IVP) to their bovine reproduction management systems. The present work demonstrates the ability to apply the IVP technology across 2 distant country borders. Herein, we demonstrate that a country (Dominican Republic; DR) that does not have a bovine IVP laboratory can take advantage of fresh bovine IVP embryos for transfer using distant IVP facilities in another country (Panama; ~1500 km away). The objective of this study was to demonstrate that a model system for large-scale commercial in vitro bovine embryo production for beef and dairy producers, that do not have IVP technology in their home country, could be developed producing comparable results. As the same laboratory provides IVP services to the both countries, a special sanitary protocol was developed in order to legalize the exchange of biological materials (oocytes or embryos). The data obtained in DR was compared to Panamanian client data because identical conditions were utilised for IVP. Cattle production systems were similar, as Brahman (a Zebu type of cattle) is the most popular breed in both countries. Oocytes were collected from 10 different herds in Panama and 4 different herds in DR. The oocytes were transported in an oocyte transporter in both instances. However, oocytes from DR were transported in InVitro Brasil™ maturation medium from 12 to 18 h and in Panama from 6 to 12 h before they were placed in a standard CO2 incubator. In both cases, the oocytes were matured for 24 h before fertilization with conventionally frozen Brahman semen in InVitro Brasil™ fertilization medium, followed by culture for up to 7 days in InVitro Brasil™ embryo culture medium. The embryos were transferred on Day 7, either in Panama or DR. They were transported by car in Panama and via airplane back to DR. A comparison of oocyte number and quality, cleavage, embryo production, and pregnancy rate, was made using the same in vitro production system for Brahman donors from September 2012 until May 2013. The difference between sites in the relative number of viable oocytes, relative number of cleaved oocytes among viable oocytes, relative number of embryos produced among cleaved oocytes, and relative number of embryos produced among viable oocytes was tested using Fisher's exact test. Pregnancy rate was analysed with chi-squared. We realise these results represent field data; however, we believe the present work is a significant step in demonstrating the potential for wide commercial-scale dissemination of IVP technology between distant countries. The number of embryos produced in Panama was slightly, but significantly, higher than those produced in DR; this is likely due to the larger number of donors and oocytes from the Panamanian herds. However, the pregnancy rate was higher in DR, likely due to the health status of DR recipients. These data illustrate that IVP using Brahman donors could be used as a tool to improve and spread superior genetics. Furthermore, this technique can serve as a model for other Central American and Caribbean countries under similar management systems. Table 1.Panama and the Dominican Republic in vitro Brahman embryo production and pregnancy (September 2012 through May 2013)

Association of recombinant bovine somatotropin (rBST) with equine chorionic gonadotropin (eCG) on antral follicle count and oocyte production in Holstein and Tabapuã heifers

The objective of this study was to investigate whether the use of rbST and eCG prior to ultrasound-guided follicular aspiration (OPU) improves oocyte yield and quality in Tabapua and Holstein heifers. The study was conducted in two phases, 20 days apart, in a change-over design. The dominant follicle was ablated two days (D-2) before two treatments: stimulation (6 Holstein and 8 Tabapua), 500 mg of rbST (Boostin®) on D0 and 500 IU of eCG (Folligon) on D2; and control (6 Holstein and 8 Tabapua), in which heifers received injections of the excipient. Heifers were aspirated on D4. Oocytes were subjected to a well established commercial in vitro embryo production protocol (Vitrogen®) and embryos were evaluated seven days after fertilization. There was an effect from the interaction of treatment and breed, so that hormonal stimulation increased antral follicle count (2-8 mm) in Tabapua (29.9±2.6 to 41.4±2.6), but not in Holstein heifers (14.4±2.6 to 15.5±2.6). Tabapua heifers had higher mean antral follicle count than Holsteins (35.6±1.8 vs. 15.0±2.1). The number of viable oocytes was not increased by stimulation in Tabapua (from 4.7±1.0 to 5.2±1.1 in control and simulation, respectively) or in Holstein heifers (1.3±1.9 to 2.0±1.6 in control and simulation, respectively). There was no difference in the percentage of heifers with more than five viable oocytes in the group treated (33 vs 27%). The number of blastocysts was not affected by treatment (1.75 vs. 1.00 in hormonal stimulation and control, respectively). The increase in antral follicle count in the stimulated Tabapua heifers did not reflect upon oocyte yield. The differential breed response to the hormonal treatment underscores the need for additional tests, especially for the Holstein breed, in order to enhance OPU efficiency.

266 OVUM PICKUP AND IN VITRO EMBRYO PRODUCTION DURING THE SUMMER: DIFFERENCES BETWEEN HOLSTEIN HEIFERS, HIGH-PRODUCING HOLSTEIN COWS IN PEAK LACTATION, AND REPEAT-BREEDER HOLSTEIN COWS

The association of ovum pickup (OPU) and in vitro embryo production (IVP) has been widely used to improve bovine reproduction. However, previous reports have indicated the occurrence of low fertility associated with summer heat stress. In the present work, we hypothesized that different categories of Holstein cattle [heifers (H), high-producing cows in peak lactation (PL), and repeat-breeders (RB)] would respond differently to OPU and IVP during the summer, because of their different metabolisms. This experiment was conducted on 2 commercial dairy farms in southeast Brazil in summer 2009. Cattle (n = 36/category) started a protocol to synchronize follicular wave emergence: 2 mg of estradiol benzoate (Sincrodiol®, OuroFino, Minas Gerais, Brazil) + 50 mg of progesterone (OuroFino) + 150 μg of D-cloprostenol (Sincrocio®, OuroFino) i.m. + a norgestomet ear implant (Crestar®, Intervet, São Paulo, Brazil) on Day 0, implant removal and OPU on Day 5. Respiration rate (RR), rectal temperature (RT), and cutaneous temperature (CT) were recorded on Day 0. Semen from a single Holstein bull previously tested was used in IVP, and oocytes from slaughterhouse were submitted to IVP as a quality control. Statistical analyses were done using PROC GLIMMIX of SAS (SAS Institute, Cary, NC, USA). The average and maximum environmental temperature and humidity were 30 and 39.8°C and 61 and 88%. Heifers were on average 15.7 months old; PL and RB cows had 112.8 ± 5.4a v. 422.8 ± 27.6b days in milk, milk production of 32.8 ± 0.9a v. 21.7 ± 1.1b kg, and number of insemination of 0.9 ± 0.1a v. 6.8 ± 0.3b; P < 0.0001; mean ± SE). Heifers and cows had different RR (H = 11.2 ± 0.3a, PL = 18.2 ± 0.6b, RB= 18.5 ± 0.4b breaths/min; P < 0.0001), RT (H = 38.7 ± 0.1a, PL = 39.56 ± 0.2b, RB = 39.32 ± 0.1b; P < 0.0001), and CT (H = 31.3 ± 0.2a, PL = 33.2 ± 0.3b, RB = 32.9 ± 0.3b; P < 0.0001). At OPU, heifers had greater number of follicles than PL cows, but they were similar to RB cows (H= 18.5 ± 1.9a, PL = 12.4 ± 1.1b, RB = 17.2 ± 2.0a; P = 0.04). Heifers had also greater number of oocytes (H = 9.6 ± 1.6a, PL = 5.0 ± 0.9b, RB = 8.8 ± 13ab; P = 0.03) and viable oocytes (H = 7.6 ± 1.5a, PL = 3.6 ± 0.8b, RB = 6.8 ± 1.2ab; P = 0.05) recovered from OPU than PL cows and similar to RB cows. However, at IVP, heifers had greater rates than both other categories (cleavage at Day 3: H = 47.8%a, PL = 31.1%b, RB = 35.4%b, P = 0.008; blastocyst at Day 7: H = 21.0%a, PL = 4.1%b, RB = 3.8%b, P < 0.0001) and more grade I embryos (H = 1.3 ± 0.4a, PL = 0.3 ± 0.2b, RB = 0.5 ± 0.2b, P = 0.04). The quality control had 80.7% cleavage and 45.4% blastocyst rates. The differences found among heifers and cows are probably related to their metabolism under heat stress, compromising oocyte number and quality. Also, although RB had similar number of viable oocytes than heifers, these oocytes are probably compromised, leading to poorer results at IVP, as observed. Fazendas Santa Rita e Campestre, Vida Reprodutiva, LMMD, SAMVET, VITROGEN, OuroFino Saúde Animal, FAPESP (proc09/00938-3).

179 COMPARISON OF OOCYTE AND EMBRYO PRODUCTION AMONG BOS TAURUS, BOS INDICUS, AND INDICUS-TAURUS DONOR COWS

In recent years, Brazil has become the leading country in the world for the number of embryos produced in vitro (Thibier M 2009 IETS Embryo Transfer Newsletter 22, 12-19). This is partly due to the large numbers of Bos indicus animals in Brazil, making up about 80% of the total cattle. The mean oocyte production per ultrasound-guided follicular aspiration from Bos indicus is higher than those for European breeds (Pontes JHF et al. 2009 Theriogenology 71, 690-697). In the present study, we analyzed 5407 ovum pick ups (OPU) and compared the average production of total (n = 90,086) and viable (n = 64,826) oocytes and the number of embryos produced in vitro from Gir (Bos taurus indicus), Holstein (Bos taurus taurus), 1/4 Holstein × 3/4 Gir, and 1/2 Holstein-Gir crossbreed cows. To obtain oocytes, OPU was repeated from 4 to 7 times (mean = 5.7 ± 2.4) in each donor cow aged from 3 to 7 years (mean = 5.0 ± 2.3) during a 12-mo period. COCs (n = 90,086) obtained were classified according to the presence of cumulus cells and the oocyte cytoplasm aspect (homogeneous or heterogeneous/fragmented). The viable oocytes (n = 64,826) were in vitro matured for 24 h at 38.8°C in an atmosphere of 5% CO2 in air. Since this was a commercial programm, frozen sexed semen (2 × 106 mL-1) from Gir (n = 8) or Holstein (n = 7) sires previously tested for high efficiency was used for IVF. Fertilization was carried out (18-20 h) and the presumed embryos were cultured for 7 days in the same conditions as were used for IVM. Data were analyzed by ANOVA. On average, 16.7 ± 6.2 oocytes were obtained per OPU/IVF procedure and 71.96% were considered viable. The mean numbers of total oocytes per OPU/IVF procedure were 17.1 ± 4.4 for Gir cows (n = 617), 11.4 ± 3.9 for Holstein cows (n = 180), 20.4 ± 5.8 for 1/4 Holstein × 3/4 Gir (n = 44), and 31.4 ± 5.6 for 1/2 Holstein-Gir crossbreed females (n = 37, P < 0.01). The mean numbers of viable oocytes per OPU/IVF procedure were 12.1 ± 3.8 for Gir cows, 8.0 ± 2.6 for Holstein cows, 16.8, ± 5.0 for 1/4 Holstein × 3/4 Gir, and 24.3 ± 4.7 for 1/2 Holstein-Gir crossbreed females (P < 0.01). The average number of embryos produced by OPU/IVF were 3.2 (n = 12,243/3378) for Gir cows, 2.2 (n = 2426/1138) for Holstein cows, 3.9 (n = 1033/267) for 1/4 Holstein × 3/4 Gir, and 5.5 (n = 1222/224) for 1/2 Holstein-Gir. The average number of embryos produced per IVF session from 1/2 taurus × indicus donor cows was greater (P < 0.01) than from Bos indicus cows. The number of recoverable and viable oocytes and the number of embryos produced in vitro from Bos indicus donors were higher than from Bos taurus females. Therefore, the highest oocyte yield and the greatest embryo production were obtained from 1/2 taurus × indicus females. This work was supported by In Vitro Brasil.

293 EFFECT OF FOLLICULAR WAVE SYNCHRONIZATION ON IN VITRO EMBRYO PRODUCTION IN COWS

In order to achieve the ideal time of ovum pickup (OPU) for in vitro embryo production (IVP) in cows regarding number and quality of oocytes recovered, this study investigated the effect of synchronization of wave emergence with estradiol benzoate (EB) injected 7 days prior to follicular aspiration. In a Latin square design, 12 crossbred beef cows were randomly divided into three groups, with three replicates each. Cows were synchronized with a norgestomet ear implant for 7 days followed by an i.m. prostuglandin F2� (PGF2�) injection and aspiration of all ovarian follicles larger than 3 mm in diameter. After that, follicles from cows in group 2X were aspirated twice a week with 4- and 3-day intervals, and follicles from groups 1X and 1X-EB were aspirated once a week. Cows from group 1X-EB also received an im injection of 2 mg of EB immediately after OPU. Throughout the study cows were kept with an ear norgestomet implant that was replaced every 2 weeks. Ultrasound evaluations of numbers of follicles greater than 3 mm in diameter and size of the largest follicle at the time of OPU were performed. Recovered oocytes were evaluated for quality, and the viable ones (Grades I, II, and III) were in vitro-fertilized on Day 0. Cleavage rate was evaluated on Day 2 and blastocyst production on Day 7. Continuous variables were compared by ANOVA and binomial data were compared by chi-square. For the 2X group, only data from the OPU performed 3 days after the last OPU were used for analysis. Results are presented as percentages or mean � SEM. Size of the largest follicle was greater (P < 0.05) in 1X coes (12.9 � 0.2 mm) than in 1X-EB cows (11.1 � 0.3 mm), which was greater than in 2X (9.6 � 0.4 mm) cows. The 1X cows had more follicles at OPU than 2X cows (17.5 � 0.7 vs. 14.1 � 0.9), whereas the 1X-EB group (15.9 � 0.7) was intermediate and not different from the others. There was no difference in the mean number of recovered oocytes among 2X (9.6 � 0.6), 1X (12.7 � 0.8) and 1X-EB (12.3 � 1.0) cows, and the mean number of viable oocytes among groups (5.8 � 0.5, 7.3 � 0.5, and 7.0 � 0.6) for 2X, 1X, and 1X-EB cows, respectively). The rate of viable oocytes was also similar among groups [58.8% (191/325) for 2X, 58.4% (267/457) for 1X, and 57.2% (231/404) for 1X-EB cows]. Cleavage [68.6% (131/191), 65.2% (174/267), and 68.4% (158/231)] and blastocyst [38.7% (74/191), 43.8% (117/267), and 44.2% (102/231)] rates were also not different among 2X, 1X, and 1X-EB groups, respectively. Although the use of 2 mg of EB in association with a norgestomet implant 7 days prior to OPU altered the follicular wave profile, it was not enough to improve number and quality of the oocytes recovered. Moreover, this study failed to demonstrate a positive effect of OPU earlier after wave emergence, when the effect of dominance should be less pronounced, on IVP in cows. The first author was supported by the fellowship 141077/2004-2 of CNPq, Brazil.

Effect of methoxychlor on various life stages of Xenopus laevis.

The toxicological effects of the organochlorine pesticide methoxychlor were evaluated at various life stages of the South African clawed frog, Xenopus laevis, in an effort to determine stage-specific sensitivity. A battery of four separate assays, including a short-term (4-day) early embryo-larval assay (FETAX) (NF stages 8-46 [Nieuwkoop and Faber, 1994]), 30-day hind limb development assay (NF stages 8-54), 18-day metamorphic climax assay (NF stages 58-66), and 30-day adult reproduction assay were performed. Test concentrations for the FETAX, hind limb development, metamorphic climax, and reproductive assays ranged from 0.0001-1.0 mg/l, 0.0001-0.1 mg/l, 0.0001-0.1 mg/l, and 0.001-0.1 mg/l, respectively. Results from the short-term embryo-larval assay indicated that increased embryo-lethality, malformation, and growth inhibition were not induced at </=1.0 mg/l methoxychlor (maximum soluble concentration). The 30-day hind limb development studies indicated methoxychlor exposure >/=0.01 mg/l delayed hind limb digit differentiation. Follicular hyperplasia of the thyroid glands was noted in specimens exposed to 0.1 mg/l methoxychlor. Results from the 18-day metamorphic climax assay indicated that methoxychlor inhibited the rate of tail resorption in a concentration-dependent manner. Whole body tissue triiodothyronine (T(3)) profiles showed a reduced and delayed surge during climax compared to controls. For the reproductive assessment, adult female X. laevis were super-ovulated and both female and male were then exposed to varying concentrations of methoxychlor. A concentration-dependent reduction in ovary weight and the number of viable oocytes was observed. In exposed male specimens, a concentration-dependent reduction in testis weight and sperm count was found. Methoxychlor was found to accumulate in the ovary, and to a lesser extent in the testis. Based on breeding studies in which exposed females were bred with control males and exposed males bred with control females, the frequency of amplexus, fertilization, and embryo viability was also affected by adult female methoxychlor exposure, and to a lesser extent by male exposure. Overall, these results suggested that sensitivity to methoxychlor is most dramatic during the reproductive and metamorphic phases of the life cycle and least sensitive during early embryo-larval development.

Thermolability of mouse oocytes is due to the lack of expression and/or inducibility of Hsp70

Eukaryotic and prokaryotic cells have been shown to respond to physical and chemical stress by the induction of proteins called heat shock proteins. Heat shock protein 70 (Hsp70), is the most ubiquitous of these proteins. Although heat shock proteins are generally thought to protect cells from physiologically stressful stimuli, it cannot be assumed that this is so, because several cases exist in which thermotolerance is acquired without the production of heat shock proteins, and in several other cases the hyperproduction of these heat shock proteins does not produce thermotolerance. In this study we show that unfertilized mouse oocytes are sensitive to elevated temperatures, and that the synthesis of Hsp70 cannot be induced in these oocytes. Furthermore, our data demonstrate that the expression of Hsp70 in mouse oocytes is sufficient for the acquisition of thermotolerance. Mouse oocytes were injected with mRNA for Hsp70, and the viability of these oocytes was determined after heating. The number of viable oocytes was significantly higher in the group injected with Hsp70 mRNA and then heated compared with oocytes injected with Hsp70 antisense mRNA and sham-injected controls treated in an identical manner. No significant differences in the number of viable oocytes were found between the group that had been injected with Hsp70 mRNA, heated, and then allowed to recover for 3 hr and the group maintained at 37 degrees C throughout.(ABSTRACT TRUNCATED AT 250 WORDS)