A. D. Vieira

Microbiological and functional evaluation of an alternative device (OB®) for estrous synchronization in ewes

The use of synthetic progestagens released by vaginal devices is an important tool to overcome the reproductive seasonality in sheep, but cost and/or subsequent vaginitis are limiting factors for their use. To identify economic, simple and innocuous alternative vaginal devices for estrous synchronization/induction protocols in sheep, this study aimed to evaluate the microbiological and functional viability of the human vaginal tampons (OB®) impregnated with medroxyprogesterone acetate (MAP) on reproductive performance of ewes. The study compared them with commercial vaginal inserts (CIDR®) and polyurethane sponges impregnated with MAP. In Experiment 1, the device loss rate, the degree of vaginitis during the device removal, the count and identification of bacterial colonies at the device insertion and removal, and efficiency in estrous synchronization and estrus temporal distribution were evaluated. Pubertal ewes at the beginning of the breeding season were randomly allocated to three experimental groups: CIDR®, PSP (polyurethane sponge) and OB®. No device losses occurred in any group, but the use of OB® caused milder signs of vaginitis than polyurethane sponges, with a similar vaginal bacterial growth and microbiota than the CIDR group. The estrus distribution was more disperse in the CIDR than PSP or OB groups. In Experiment 2, pregnancy rates using CIDR® or OB® devices were compared, with estrus manifestation (85.4% and 89.8%) and pregnancy rates (58.3% and 49.0%) being similar between groups (P>0.05), respectively. In conclusion, the use of human intra-vaginal tampons (OB®) impregnated with MAP was proven highly hygienic, practical and effective as a low-cost alternative for estrous synchronization and AI in sheep.

Internal artificial vagina (IAV) to assess breeding behavior of young Bos taurus and Bos indicus bulls

Bull breeding soundness evaluation (BBSE) usually neglects the libido and mating ability evaluation. The internal artificial vagina (IAV) permits semen sampling, as well as mating ability evaluation. Few studies have been performed using IAV with young bulls and there are none with Bos indicus bulls. The present study evaluated sexual behavior, mating ability and semen quality in young Bos taurus (Devon) and B. indicus (Nellore) bulls using the IAV device. In the first experiment, 52 Devon bulls, 18-25 months old were observed, and the behavior and mating ability recorded over a 10-min period within a restrained mount-cow with an IAV inserted. In the second experiment, 20 Nellore bulls, 20-30 months old were evaluated over a 20 min period. Of the 52 Devon bulls, 45 (86.5%) had semen recovered with the IAV, 31 (69.0%) were considered satisfactory. Nellore bulls exhibited a different sexual behavior, with 10 bulls not showing any interest in the females. Four bulls demonstrated sexual interest only once, e.g., sniffing, two showed interest on more than one occasion, and four had more than two mounts or mounting attempts. None out of the Nellore bulls was collected with IAV. The IAV was an effective and welfare-promoting animal technology for the evaluation of semen quality and mating ability of B. taurus bulls. However, the IAV was not adequate for young Nellore bulls, probably due to their quiescent sexual behavior and delayed sexual maturity. Further studies are needed to evaluate the performance of the IAV for older Nellore bulls.

Selection of porcine oocytes in vitro through brilliant cresyl blue staining in distinct incubation media.

Staining with brilliant cresyl blue (BCB) may be used for oocyte selection, but BCB staining itself and the most commonly used selection medium (DMPBS) may compromise the development of porcine oocytes in vitro. This study evaluated DNA fragmentation, nuclear maturation, the area of migration of cortical granules (CG) and embryo development for stained (BCB+) and unstained (BCB-) oocytes incubated in DMPBS and in a modified medium (ReproPel) tested for the first time. Unexposed (UN), BCB+ and BCB- oocytes were incubated composing six groups: DMPBS/UN; DMPBS/BCB+; DMPBS/BCB-; ReproPel/UN; ReproPel/BCB+; and ReproPel/BCB-. There were more BCB+ oocytes in ReproPel than in DMPBS (P < 0.05). The DNA fragmentation was evaluated for oocytes in DMPBS/BCB+, DMPBS/BCB-, ReproPel/BCB+, ReproPel/BCB- and in porcine follicular fluid (control). The frequency of oocytes with no DNA fragmentation was greatest (64.6%) in DMPBS/BCB+ and lowest in ReproPel/BCB+ and ReproPel/BCB- (26.8 and 34.1%, respectively) (P < 0.05). Nuclear maturation rates were greater (P < 0.05) for DMPBS/BCB+ (63.1%), ReproPel/UN (55.1%) and ReproPel/BCB+ (50.2%) than for DMPBS/UN (40.8%) and ReproPel/BCB- (35.5%). The area of CG was greater (P < 0.05) for ReproPel/BCB- (80.7%) and DMPBS/UN (77.6%) than for ReproPel/UN (34.7%). Cleavage rates for DMPBS/BCB+ and ReproPel/BCB+ were greater than for DMPBS/UN (P < 0.05). Blastocyst development rates were greatest (P < 0.05) for ReproPel/UN and ReproPel/BCB+. In both media, BCB staining was apparently unable to select competent oocytes, which likely occurred due to toxicity. Despite the similar nuclear maturation and area of CG compared with DMPBS, oocytes selected in ReproPel presented impaired DNA integrity.

Expression of growth and differentiation Factor 9 and cognate receptors during final follicular growth in cattle

Mutations in growth and differentiation factor 9 (GDF9) gene are associated to sterility or, paradoxically, increased ovulation rate in ewes. Despite its importance, the exact function of GDF9 in ovarian physiology is still poorly understood. This study aimed to investigate GDF9 function during dominant follicle growth and its regulation in follicular fluid. The regulation of GDF9 receptors in GnRH/LH-stimulated granulosa cells was also investigated. In a first experiment, a new follicular wave was induced and the intrafollicular GDF9 treatment into the largest growing follicle (8.5-9.5 mm) at both 100 (n = 3) and 1000ng/ml (n = 4) had no effect on follicular growth, estrus manifestation and ovulation compared to control (PBSinjected) follicles (n = 3). In a second experiment, follicles were obtained just after follicular deviation (day 4 after follicular emergence) and the abundance of GDF9 in follicular fluid did not differ between healthy dominant (n = 4) and atretic subordinate follicles (n = 4), as assessed by western blot analysis. Finally, mRNA expression of BMPR2 and TGFBR1 receptors was evaluated in granulosa cells obtained from preovulatory follicles (>12 mm diameter) obtained 0, 3, 6, 12 or 24 h after i.m. GnRH administration (n = 4-5 follicles/moment). Both receptors were significantly up regulated 12 h after GnRH treatment. Present results do not confirm the hypothesis that GDF9 inhibits dominant follicle growth and suggests a minor role in determining follicle fate. In the other hand, GDF9 receptors regulation in GnRH/LH-stimulated granulosa cells provides the first in vivo evidence of its involvement in the complex cascade of events that culminates in ovulation and luteinization in cattle.

The Use of Antibiotics in Cryopreservation of Ram Sperm

Antibiotics are used in extenders for sperm cryopreservation, however no specific regulation has been established for ram sperm. This study evaluates the effects of antibiotics in ram sperm extenders for bacterial control and its effect on sperm viability. Sperm from five rams was collected, cooled and frozen using extenders with the following antibiotics: 100,000 IU/mL penicillin and 100 µg/mL streptomycin (PES); 500 µg/mL gentamycin, 100 µg/mL tylosin, 300 µg/mL lincomycin and 600 µg/mL spectinomycin (GTLS); 50 µg/mL ceftiofur sodium (CEF); and 1,000 µg/mL enrofloxacin (ENR). Bacillus sp., Corynebacterium sp., Klebsiella sp. and Staphylococcus sp. were isolated from fresh sperm and perpetual area. For cooled sperm, the antibiotic PES, GTLS and ENR were able to reduce the number of colony forming units per mL (CFU/mL), however, CEF was inefficient in all tested concentrations. Motility of cooled sperm was reduced when GTLS and ENR were used (P 0.05), however, motility was reduced with ENR treatment at doses greater than 50% (P < 0.05). The antibiogram revealed resistance of Staphylococcus sp . and Klebsiella sp. to the penicillin/streptomycin association and to tylosin. Both PES and GTLS were able to control bacterial growth on cooled sperm. However, attention should be given to the antibiotics added to the extenders, which may impair sperm motility.

69 A HOMEMADE N2 SUPERCOOLING DEVICE (NITROCOOLER) ENHANCES VIABILITY AFTER VITRIFICATION OF BOVINE OOCYTES BUT NOT OF IN VITRO-PRODUCED EMBRYOS

Increasing the cooling rate is a common strategy to enhance vitrification efficiency of bovine oocytes and in vitro-produced (IVP) embryos. Under vacuum conditions, liquid N2 (LN2) temperature decreases, increasing the cooling rate during the vitrification procedure. However, commercially available brands of equipment to supercool nitrogen are expensive. The aim of this study was to verify the effectiveness of a low-cost homemade nitrogen supercooling apparatus (Nitrocooler) for vitrification of bovine oocytes and IVP embryos. The device consists of a vacuum pump coupled to a close-tight-lidded flask with a styrofoam cup filled with 300 mL of LN2. After 5 to 7 min of vacuum pumping, LN2 goes through the slushing phenomenon, turning solid. After the lid is opened, the N2 turns liquid again, but in a stable, supercooled physical state lasting for approximately 10 min, boiling off when objects are plunged into it. Nitrocooler was tested for vitrified oocytes (Experiment I), vitrified oocytes in different containers (Experiment II), and embryos in 2 different vitrification solutions (Experiment III). In Experiment I (Ciencia Rural, 2006 36, 1501–1506), immature (IM, n = 172) or in vitro-matured (IVM, n = 174) oocytes were exposed to 10% ethylene glycol (EG) + 10% DMSO for 30 s, followed by 20% EG + 20% DMSO + 0.5 m sucrose for 20 s, loaded in open-pulled straws (OPS), and plunged into LN2 or Nitrocooler-pumped LN2. For both IM (8.8%) and IVM (10.6%) oocytes, Nitrocooler-pumped LN2 increased blastocyst rates (Bonferroni, P 0.05) to normal atmosphere rates using EG + DMSO (50.1%) or EG + PROP (56.0%). Under these experimental conditions, our results suggest that the greater cooling rates obtained favored an increase in oocyte viability after vitrification, whereas no such beneficial effects were detected for vitrified IVP embryos. In conclusion, Nitrocooler was proven effective as a low-cost device to supercool LN2, increasing viability after vitrification of bovine oocytes, but not of IVP embryos. Table 1.Effect of Nitrocooler apparatus on cryopreservation of bovine oocytes and embryos