Use of logistic models to evaluate the response of superovulation treatment and embryo production in Santa Inês ewes.

The study aimed to verify the influence of the FecGE mutation in superovulated ewes and to evaluate the probability of logistic models to determine the response capacity of these ewes to superovulatory treatment. Santa Inês ewes (n = 29) were genotyped for the FecGE mutation and separated for their genotype group in carriers of the mutant E allele (FecGE/E, FecG+/E) and non-carrier (FecG+/+) alleles. The ewes underwent hormonal treatment for superovulation. Aside from the genotypes, variables included in the statistical model were reproductive status (empty, early lactation, or late lactation), age (> or < 6 years), and number of births (nulliparous, primiparous, multiparous). The carriers of the mutation could be discriminated from the non-carriers based on the number of corpora lutea, rate of frozen embryos, and fecundity. Recovery rate was significantly higher (P < 0.05) in FecGE/E (94.31%) compared to FecG+/E (63.15%) and FecG+/+ (61.90%) (P < 0.05), whereas fecundity rate of FecG+/+ ewes (50.76%) was significantly higher than FecG+/E (18.96%) and FecGE/E (32.53%) (P < 0.05). We determined in this study that the response to superovulation and embryo production can be discriminated between FecGE/E and FecG+/E ewes in relation to the FecG+/+ genotype. Logistic models that included reproductive status and mutation, or reproductive status and age, or reproductive status and number of births were effective in predicting the response to superovulatory treatment.

Correlations of corpus luteum blood flow with fertility and progesterone in embryo recipient mares.

The aim of this study was to evaluate the correlation between the corpus luteum vascularization with the concentration of progesterone and the fertility of embryo recipient mares. Mangalarga Marchador mares (n = 33) were distributed into groups according to the days (D) after ovulation, as follows: D3 (n = 8), D4 (n = 8), D5 (n = 9), and D6 (n = 8). The evaluations of the corpus luteum, endometrium, and blood collection to quantify the progesterone concentration were carried out on D3, D4, D5, and D6. Among the parameters evaluated, only progesterone concentration on D6 differed from the other groups (P <0.05). A positive correlation (P <0.05) between the diameter and the area of the corpus luteum, and the objective and subjective methods of the corpus luteum vascular perfusion, was identified. Likewise, a positive correlation (P <0.05) was observed between the objective and subjective methods of the vascular perfusion in the corpus luteum and the progesterone concentration. The pregnancy rate obtained in this study (54.54%) was not affected (P> 0.05) by the day of embryo transfer, whose percentages were 37.50% (3/8) on D3, 50% (4/8) on D4, 66.70% (6/9) on D5, and 62.50% (5/8) on D6. It was estimated that with each increase on the day of embryo transfer, the pregnancy rate increases. The results allow to conclude that the corpus luteum vascularization in mares, evaluated by Doppler ultrasound, correlates with progesterone concentration and the embryo transfer day.

Housekeeping genes for RT-qPCR in ovine preimplantation embryos.

Housekeeping genes (HKG) are paramount for accurate gene expression analysis during preimplantation development. Markedly, quantitative reverse transcription polymerase chain reaction (RT-qPCR) in ovine embryos currently lacks HKGs. Therefore, we tested 11 HKGs for RT-qPCR normalization during ovine parthenogenetic preimplantation development. Seven HKGs reached the qPCR efficiency threshold (97.20-105.96%), with correlation coefficients ranging from -0.922 to -0.998 and slopes from -3.22 to -3.59. GeNorm ranked glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and TATA-binding protein (TBP) as the best HKG pair, while H3 histone, family 3A (H3F3A) was the third HKG. Relative gene expression was measured for zinc finger protein X-linked (ZFX) and developmental pluripotency-associated 3 (DPPA3) transcripts during ovine parthenogenetic preimplantation development. ZFX did not show any transcript abundance fluctuation among oocytes, cleavage-stage embryos, and morulae. DPPA3 transcript abundance was also similar among all developmental stages, therefore suggesting that it may not display a maternal gene expression profile. In silico analysis of ovine DPPA3 mRNA and protein showed high conservation to bovine orthologues. However, DPPA3 orthologues differed in regulatory motifs. In conclusion, GAPDH, TBP and H3F3A are stable HKGs in ovine parthenogenetic embryos and allow accurate RT-qPCR-based gene expression analysis.

Application of platelet-rich plasma in the in vitro production of bovine embryos.

The aim of this study was to replace fetal bovine serum (FBS) with platelet-rich plasma (PRP) for in vitro production of bovine embryos. The maturation media (TCM-199 medium) for the cumulus-oocyte complexes (COCs) was supplemented with 5% (G5) and 10% (G10) PRP or 10% FBS (GC). After fertilization, the presumed zygotes were randomly distributed in culture medium supplemented with 5% (G5) and 10% (G10) PRP or 10% FBS (GC) for 7 days. Cumulus cell (CC) expansion was greater (P < 0.05) in the GC (88.9%) group than in G5 (34.1%) or G10 (50.0%). Nevertheless, the expansion of CCs in group G10 was greater than in G5 (P < 0.05). Cleavage was higher in group G5 (86.0%) than in G10 (79.0%) (P < 0.05) and did not differ from group GC (82.0%). The percentage of blastocysts in group G5 (50.0%) was higher than in CG (40.2%) and G10 (34.2%) (P < 0.05). In addition, the number of blastomeres was higher in G5 (159.0 ± 4.18) than in GC (132.4 ± 4.11) and in G10 (127.1 ± 5.88) (P < 0.05). The addition of PRP into the oocytes maturation medium is not beneficial. On the other hand, the PRP addition into the embryo culture medium at 5% concentration is recommended where it increased the quantity and quality of in vitro-produced bovine embryos.

Reproductive efficiency of nellore (Bos indicus) cows subject to both FTAI and homeopathic supplementation / Eficiência reprodutiva em vacas nelore (bos indicus) submetidas à IATF e a suplementação homeopática

The aim of this work was to determine the effect of homeopathic supplementation on both ovarian dynamics and conception rate in Nellore cows subjected to fixed-time artificial insemination (FTAI). Cows (n = 150) were randomly distributed to the control (CG) and the homeopathy group (HG). The HG cows were supplemented with Pró-cio in the mineral salt for 60 days and both experimental groups were further subjected to FTAI. Cows were evaluated for ovarian dynamics (n = 16), progesterone (P4) concentration (n = 16), and conception rates (n = 150). Ovarian dynamics determined by ultrasonography and showed similar findings for CG and HG, respectively. Thus follicular diameter (8.7 ± 1.0 mm vs. 10.0 ± 0.8 mm), mean pre-ovulatory follicle volume (0.46 ± 0.15 mL vs. 0.61 ± 0.12 mL), and mean follicular growth (3.65 ± 1.41 mm vs. 4.60 ± 1.21 mm) did not differ between groups. Moreover, corpus luteum diameter was similar between groups (CG: 16.28 ± 0.7 mm vs. HG: 15.6 ± 0.8 mm; P > 0.05), although P4 levels did differ (CG: 2.55 ± 0.85 ng mL-1 vs. HG: 6.52 ± 1.19 ng mL-1; P < 0.05). The conception rate after FTAI was not affected by homeopathic supplementation (CG: 74.67 %, and did HG: 77.33 %; P > 0.05). In conclusion, the homeopathic supplementation Pró-cio increases P4 concentrations but does improve the reproductive efficiency of Nellore cows subject to FTAI.

O objetivo foi determinar o efeito da suplementação homeopática na dinâmica ovariana e taxa de concepção em vacas Nelore cows submetidas à inseminação artificial em tempo fixo (IATF). As vacas (n = 150) foram distribuídas aleatoriamente nos grupos controle (GC) e grupo homeopático (GH). As vacas do GH foram suplementadas com Pró-cio® no sal mineral mineral por 60 dias. Ambos os grupos foram submetidos à IATF. As vacas foram avaliadas quanto à dinâmica ovariana (n = 16), concentração de progesterona (P4; n = 16) e taxa de concepção (n = 150). A dinâmica ovarina foi determinada por ultra-sonografia e mostrou resultados semelhantes para o GC e o GH, respectivamente. Portanto, para diâmetro folicular (8,7 ± 1,0 mm vs. 10,0 ± 0,8 mm), volume médio do folículo pré-ovulatório (0,46 ± 0,15 mL vs. 0,61 ± 0,12 mL) e crescimento folicular médio (3,65 ± 1,41 mm vs. 4,60 ± 1,21 mm) não diferiram entre os grupos. Além disso, o diâmetro do corpo lúteo foi semelhante entre os grupos (CG: 16,28 ± 0,7 mm vs. HG: 15,6 ± 0,8 mm; P > 0.05), apesar dos níveis de P4 diferirem (CG: 2,55 ± 0,85 ng mL-1 vs. GH: 6,52 ± 1,19 ng mL-1; P < 0.05). A taxa de concepção após a IATF não foi afetada pela suplementação homeopática (GC:74.67 % vs. GH: 77.33 %; P > 0.05). Em conclusão, a suplementação homeopática com Pró-cio aumenta a concentração de P4 mas não melhora a eficiência reprodutiva de vacas Nelore cows submetidas à IATF.

Freezing of Stallion Semen: In Vitro Evaluation of Motility and Acrosin Activity in Sperm Cells Cryopreserved Using Different Semen Extenders.

The work described here aimed to verify the efficiency of different extenders for cryopreservation of equine semen using sperm motility and acrosin activity as spermatic parameters. The semen was fractioned into two equal parts and resuspended in an 11% lactose solution in a 1:1 proportion, where it remained for 20 minutes at room temperature. The semen was centrifuged at 600 g for 10 minutes, and after the second centrifugation, each pellet received the freezing extender (Merck or Zorlesco) and was loaded into 4 mL straws. Each straw was placed in liquid nitrogen vapor steam for 15 minutes and further immersion in liquid nitrogen at -196°C for long-term storage. After thawing, semen samples were initially evaluated for sperm motility, both total and progressive, and acrosin activity. Moreover, semen was incubated at 37°C and further assessed at 60 and 120 minutes in a thermoresistance test (TRT) for sperm motility and acrosin activity. Immediately after thawing, both progressive and total motility, and acrosin activity were lower (p < 0.05) in thawed semen than in fresh semen. During the TRT, total sperm motility and acrosin activity after 60 minutes were lower (p < 0.05) than those obtained after thawing. Similarly, total sperm motility and acrosin activity were lower (p < 0.05) after 120 minutes than at 60 minutes of the TRT. The analysis of motility and acrosin activity allowed the conclusion that both extenders have a similar capacity to preserve the integrity of sperm cells subject to freezing and thawing.

Conceptus loss in Santa Inês ewes carrying twin pregnancies by natural mating or embryo transfer.

Commercial application of reproductive biotechnologies such as multiple ovulation and embryo transfer depends on its overall efficiency. Sheep embryo transfer is gradually gaining wider adoption, but pregnancy rates after embryo transfer remain lower than those derived from natural mating for most breeds. The work was aimed to evaluate embryonic and fetal losses in Santa Inês ewes carrying twin pregnancies by natural mating or embryo transfer. Ewes were subjected to synchronized natural mating by ram effect or used as recipients for embryo transfer. Ewes diagnosed as carrying twin pregnancies at day 25 were used in the experiment (n = 42). Conceptus viability was monitored by ultrasonography on days 30, 35, 40, 45, 50, and 55 after conception. Conceptus loss was similar (P > 0.05) within natural mating 11/42 (26.19%) and embryo transfer 14/42 (33.34%). However, overall embryonic loss (80.0%) was greater (P < 0.05) than fetal loss (20.0%), with no difference within groups The results allow the conclusion that conceptus loss after embryo transfer is similar to natural mating and occurs predominantly during the embryonic stages.

Full-term potential of goat in vitro produced embryos after different cryopreservation methods.

Cryopreservation of preimplantation embryos represents a major challenge due to their shape and relatively large cells. Embryo source and cryopreservation method are key factors to cryotolerance efficiency and few reports have investigated more promising protocols for goat embryos. The study was aimed to compare different cryopreservation methods for goat in vitro produced (IVP) embryos. Goat blastocysts were subjected to conventional freezing (CF), Dimethyl sulfoxide vitrification (DMSO-V) and Dimethylformamide vitrification (DMF-V). Cryopreserved blastocysts were assessed for re-expansion, cell viability and in vivo development rates. Blastocyst re-expansion after cryopreservation was similar between groups, but cell viability was lower for DMF-V (32%) than CF (68%) and DMSO-V (60%). Pregnancy and delivery rates were similar for CF (60% and 50%) and DMSO-V (50% and 45%) and higher then DMF-V (20% and 15%), respectively. Finally, kidding rates were also indistinguishable for CF (40%) and DMSO-V (35%), but higher then DMF-V (12.5%). In conclusion, conventional freezing and vitrification using DMSO have similar efficiencies for cryopreservation of goat IVP embryos and cryoprotectant for vitrification affects its outcome.

Influence of biostimulation and temporary weaning on follicular dynamics and pregnancy rates in Nelore cows (Bos taurus indicus).

The objective of this study was to evaluate the effect of biostimulation and temporary weaning on the follicular dynamics and pregnancy rates in Nelore cows. We used three groups of 75 cows: a control group without biostimulation and suckling calves (WB), a group that was biostimulated and had suckling calves (BE) and a group that was biostimulated and subjected to temporary weaning for 56 h (BETW). Ovarian dynamics were examined using ultrasonography. All groups showed follicular atresia. The interval between beginning of the treatment and wave emergence was 3.25 ± 0.30 days for BE, 3.40 ± 0.27 days for BETW and 3.37 ± 0.50 days for WB. The time between implant removal and ovulation was 64.50 ± 1.88 h for BE, 66.75 ± 1.35 h for BETW and 60.85 ± 3.10 h for WB. Eight cows were submitted to ultrasound analysis, and the percentages of cows that had ovulatory follicles of the new follicular wave with maximum diameters greater than 0.80 cm were 100 % (8/8) in BE (1.28 ± 0.12 cm), 100 % (8/8) in BETW (1.52 ± 0.07 cm) and 87.5 % (7/8) in WB (1.21 ± 0.10 cm). The pregnancy rate was 52 % (39/75) in BE, 69.3 % (52/75) in BETW and 37.3 % (28/75) in WB. The association of biostimulation and temporary weaning increased follicular development, ovulation synchronisation and, consequently, the pregnancy rate in beef cows.

Use of retinyl acetate, retinoic acid and insulin-like growth factor-I (IGF-I) to enhance goat embryo production.

Experiments were carried out to investigate the beneficial effects of retinyl acetate (RAc) and retinoic acid (RA) on goat oocyte maturation as well as the effects of insulin-like growth factor-I (IGF-I), RAc and RA during embryo culture under chemically defined conditions. In Experiment 1, in vitro maturation (IVM) was performed in a chemically defined basic maturation medium (bMM) supplemented with 0.3 µM RAc or 0.5 µM RA. Presumptive zygotes and embryos (2-4 cells) were cultured in droplets of potassium simplex optimised medium (KSOM); however, none of the embryos reached the blastocyst stage. In Experiment 2, oocytes were matured in bMM + RAc or bMM + RA. Presumptive zygotes and 2- to 4-cell embryos were placed in fresh KSOM droplets supplemented with RAc, RA, IGF-I, RAc+IGF-I or RA+IGF-I. In Experiment 1, addition of RAc and RA to bMM increased (P < 0.05) the proportion of 2- to 4-cell embryos reaching the morula stage as compared to the control. In Experiment 2, supplementation of embryo culture media with retinoids and IGF-I increased (P < 0.05) the proportion of 2- to 4-cell stage embryos developing to the morula and blastocyst stage. Our data demonstrate that goat embryo production in chemically defined media could be improved by exogenous RAc or RA and by the interaction between retinoids and IGF-I, and that goat embryos can be produced in vitro from oocytes following protocols similar to those currently used for cattle.

Ultrasonographic fetal sex identification in pregnant sheep derived from natural mating and embryo transfer.

The objective of this study was to identify the migration period of the genital tubercle and the period of visualization of external genital structures in fetuses of the Dorper breed of sheep derived from natural mating and from fresh, frozen and vitrified embryo transfer. Transrectal ultrasound was performed using a double-frequency linear transducer (6.0 and 8.0 MHz) to monitor 130 ewe fetuses distributed in the four treatments regarding embryo origin. The accuracy of the ultrasound was 100% in this experiment. The fetuses originated from controlled natural mating (NM) and from fresh (FrE), frozen (FE) and vitrified (VE) embryo transfer, with embryos collected 7 days after breeding. Migration of the genital tubercle occurred earlier (P<0.05) in NM (42.21+/-2.86 days) than in FrE (43.98+/-3.00 days), FE (44.97+/-1.83 days) and VE (44.58+/-1.97 days). Visualization of the scrotal bag, prepuce and vulva occurred, respectively, earlier (P<0.05) in NM (45.22+/-1.25, 45.95+/-1.53 and 45.01+/-1.03 days) than in FrE (48.91+/-1.92, 48.52+/-1.41 and 47.41+/-1.41 days), FE (49.97+/-1.08, 49.18+/-2.00 and 47.64+/-1.82 days) and VE (50.12+/-1.66, 49.27+/-1.61 and 47.93+/-1.92 days). The results show that fetal sexing can be accomplished from the 50th day onward in fetuses produced by natural mating and from the 55th day onward in fetuses derived from fresh, frozen and vitrified embryos. It can also be concluded that real-time ultrasonography is a reliable tool for fetal sex determination in sheep taking into account both the location of the genital tubercle and the identification of external genital structures.

Migration time of the genital tubercle in caprine and ovine fetuses: comparison between breeds, sexes and species.

The aim of this work was to determine the ideal moment to sex goat and sheep fetuses, to compare the average time of genital tubercle (GT) migration between sexes, breeds and species, and to evaluate the accuracy of fetal sexing between sexes. A total of 317 fetuses of 219 pregnant females were monitored at 24-hour interval, from days 30 to 60 of pregnancy in ewes, and from days 40 to 60 in goats. Examinations were performed using transrectal ultrasound equipped with a linear transducer of double frequency. Fetuses were identified as male when the GT was next to the umbilical cord and female when the GT was next to the tail. The average time of GT migration in ewes (41.3 +/- 3.1 days) was shorter (P < 0.05) than in goats (47.2 +/- 2.3 days)? In goats, the average time of GT migration of Saanen fetuses was later (P < 0.05) than in fetuses of other breeds, with no difference in the average time of GT migration between male (46.9 +/- 2.2) and female fetuses (47.4 +/- 2.4). In ewes, the average time of GT migration did not differ (P > 0.05) among breeds and sexes. In goat and sheep, no difference was noticed in the accuracy of fetal sexing between males and females (P > 0.05). The results show that fetal sexing in ewes must be done earlier than in goats, fetal sexing in Saanen goats must be performed later, and fetal sex does not influence the time of GT migration in either of the two species.

Early fetal sexing of Saanen goats by use of transrectal ultrasonography to identify the genital tubercle and external genitalia.

OBJECTIVE: To define the optimum period for sexing of Saanen goat fetuses by use of transrectal ultrasonography. ANIMALS: 82 Saanen goats pregnant with 124 fetuses. PROCEDURES: Fetal sexing was performed on the basis of the final location of the genital tubercle or identification of external genitalia. In experiment 1, fetuses (n = 78) were monitored every 48 hours from days 40 to 60 of gestation, whereas for experiment 2, 46 fetuses were examined only once between days 47 and 77 of gestation. RESULTS: For experiment 1, accuracy of fetal sexing was 20 of 20 (100%) for a single fetus, 39 of 42 (92.8%) for twin fetuses, and 10 of 16 (62.5%) for triplet fetuses. Diagnostic accuracy was significantly lower for triplet fetuses than that for single or twin fetuses. Final location of the genital tubercle was detected between 45 and 55 days of gestation (mean +/- SEM, 48.9 +/- 1.8 days). For experiment 2, accuracy of fetal sexing for a single fetus (24/24 [100%]) was significantly higher than the accuracy for twin fetuses (16/22 [72.7%]). Considering all fetuses that were born, accuracy of diagnosis was 69 of 78 (88.4%) for experiment 1 and 40 of 46 (86.9%) for experiment 2. Accuracy did not differ significantly between experiments. CONCLUSIONS AND CLINICAL RELEVANCE: Real-time ultrasonography after day 55 of gestation is a suitable method for determination of sex of Saanen goat fetuses by observation of the genital tubercle or identification of external genitalia.

Sexagem fetal em ovelhas Santa Inês por ultra-sonografia / Fetal sexing in Santa Inês ewes by ultrasonography

O presente estudo teve a finalidade de identificar o sexo e de determinar o dia da migração do tubérculo genital (TG) de fetos ovinos através da ultra-sonografia em tempo real. O sexo foi identificado no Experimento I (EI) levando-se em consideração a localização do TG e no Experimento II (EII), a presença do pênis, prepúcio e bolsa escrotal no feto macho e das tetas, vulva e clitóris no feto fêmea. No EI, as fêmeas (n=17) foram monitoradas em intervalos de 12 horas, do 35o ao 46o dia de gestação, por via transretal com transdutor linear (6,0 e 8,0 MHz). No EII, as fêmeas (n=30) com gestação de 55 a 75 dias foram examinadas apenas uma vez, utilizando-se o mesmo transdutor e via de exame do EI. Das 17 fêmeas do EI, 11 (64,6 por cento) tiveram seus fetos corretamente sexados, independente da gestação ter sido simples (7/11), dupla (3/11) ou tríplice (1/11). Nas 6 (35,4 por cento) gestações restantes, 3 (17,7 por cento) foram duplas, sendo impossível sexar um feto de cada gestação. Nas outras 3 (17,7 por cento) gestações, os fetos foram corretamente sexados, apesar dos nascimentos não coincidirem com a quantificação. Num feto macho de uma gestação simples, a migração ocorreu no 37º dia e até o 46º, todos os fetos das outras gestações estavam corretamente sexados. Das 30 fêmeas do EII, 16 (53,4 por cento) apresentaram gestações simples e a acurácia da sexagem foi de 100 por cento. Nas 14 (46,6 por cento) restantes, as gestações foram duplas, sendo impossível, em quatro casos, determinar o sexo de, pelo menos, um dos gêmeos. De todos os fetos nascidos, a acurácia geral da sexagem foi de 88,0 por cento (EI) e 90,9 por cento (EII), não sendo observada diferença (P>0,05) entre ambos os experimentos. Os resultados permitem concluir que a ultra-sonografia em tempo real é um método eficiente para diagnosticar o sexo fetal pela visualização do TG, assim como pela identificação do pênis, prepúcio e bolsa escrotal no feto macho e das tetas, vulva e clitóris no feto fêmea, desde que os exames sejam realizados a partir do 50o dia de gestação.

Cocultura de embriões Mus musculus com monocamadas de células de linhagens primária e contínua em um sistema sem fluxo externo de CO2 / Coculture of Mus musculus embryos with monolayers of primary and continuous lineages in a system without external CO2 flow

A eficiência de três monocamadas celulares (linhagem contínua - células Madin and Darby Bovine Kidney/ MDBK;linhagem primária - células de útero e de oviduto bovino) foi testada para verificar a existência de especificidade celular através do desenvolvimento de embriões, desde o estádio de duas células até o de mórula compacta, em um sistema de cocultura sem fluxo externo de CO2. Depois da seleçâo, os embriões (n = 343) foram aleatoriamente distribuídos em diferentes tubos de ensaio, os quais foram colocados a 37°C durante 72 horas. Após o período de cocultura, as porcentagens de mórulas compactas obtidas foram de 87,7 por cento em células de oviduto, 86,2 por cento na monocamada de células uterinas e 88,3 por cento na de células MDBK. Não foi observada diferen- ça significativa entre esses valores e, por isso mesmo, conclui-se que a especificidade celular não é importante para o desenvolvimento in vitro de embriões Mus musculus.

The efficiency of three monolayers (continuous lineage - Madin and Darby Bovine Kidney/ MDBK; primary lineage - bovine celisfrom uterus and oviduct) hás been tested to verify the celular specificity through development of two cells embryos until compact morulaes stage in a coculture system without continuous CO2 flow. The selected mouse embryos (n=343) were randomiy divided into three experimental groups andplaced in closed culture tubes mantained aí 37°C during 72 hours. After the co-culture period, the porcentages of compact morulaes were 87.7 percent in oviduct cells, 86.2 percent in uterus monolayer and 88.3 percent in MDBK cells. It was not observed significative difference between these results and it is possible to condude that celular specificity is not importam to enhance the In vitro development of Mus musculus embryos.

Renovação parcial do meio de cultura como perspectiva de aumentar a eficiência de um sistema de cocultoura de embriões sem fluxo contínuo de CO2 / Partial replacement of the culture medium in order to enhance a embryos coculture system without continuous CO2 flow

Substituindo-se parcialmente (30%) os meios de cultura TC M 199/NaHCO3 (199/NaHCO3) e TC M 199/25mM HEPES (199/HEPES) a intervalos de 24, 48 ou 72 horas, testou-se a possibilidade de incrementar a obtenção de blastocistos expandidos (BLE) a partir de embriões Mus musculus no estádio de duas células incubados sem fluxo contínuo de CO2. Após distribuição aleatória dos embriões em tubos de ensaio contendo 2ml de meio de cultura e monocamada celular de oviduto bovino, os referidos tubos foram hermeticamente fechados e colocados em estufa bacteriológica a 37°C durante 96 horas. A renovação do meio de cultura em qualquer período não incrementou a porcentagem de embriões que alcançou o estádio de BLE, todavia, o número total de BLE obtido com o 199/HEPES (79,0%) foi superior (P £ 0,01) ao verificado com o 199/NaHCO3 (65,0%).

It has been evaluated, in a coculture system without continuous CO2 flow, the possibility to enhance the percentage of blastocists obtained from two cells mouse embryos by partial (30%) replacement (each 24, 48 and 72 hours of intervals) of two culture media (TCM 199/NaHCO3 and TCM 199/25 mM HEPES). The embryos, randomiy allocated in closed cultures tubes with 2 ml of medium and bovine oviduct monolayer, were maintained at 37°C during 96 hours. No diferences between spreaded blastocists were verifica when the media were replaced at ali intervals, however, the medium TCM 199/25mM HEPES was significantiy more effective (P £ 0.01) then TCM 199/NaHCO3.

Preparação de rufiões caprinos pela fixação da curvatura caudal da flexura sigmóide do pênis / Preparation of teaser goats by fixation of the caudal curvature of penis sigmoid flexure

Foram utilizados 15 caprinos sem raça definida com idade variando entre 06 e 36 meses objetivando testar uma técnica cirúrgica para preparo de rufiões por fixação da curvatura caudal da flexwa sigmóide do pênis. Para avaliação dos resultados, os animais eram colocados na presença de fêmeas em estro, a partir do décimo dia da intervenção cirúrgica, durante um período mínimo de seis meses. Os rufiões, quando testados, não apresentaram alteração da libido e mostraram-se incapazes de exteriorizar o pênis, permitindo concluir que a técnica descrita, além de ser rápida e de fácil execução, pode ser utilizada com eficiência na prática de preparo de rufiões caprinos.

In the present study fifteen. undefined breed type bucks, with age varing between six and thirty-six months, were used to test a surgery technique by fixation of the caudal curvature of penis sigmoid flexure for preparation of teaser. To evaluate the results, the teasers buck were placed with goats in oestrus for a minimum period of six months after 10 (ten) days of surgery. During evaluations, the teasers did not present any alteration of the sexual behavior and they were unable to project the penis for outside of the prepuce. There fore, it was concluded that the surgery technique used in these investigation can be easily applied and utilized with effïciency in the preparation of teasers buck.