Semen deposition by cervical, transcervical and intrauterine route for fixed-time artificial insemination (FTAI) in the ewe.

Semen deposition through the cervix into the uterus is a difficult technique in ewes and represents the main limiting factor for insemination in this species. The objective of this study was to evaluate the pregnancy rate achieved with a new transcervical insemination method in comparison with conventional cervical and laparoscopic intrauterine techniques. A total of 586 multiparous Corriedale ewes were synchronized for fixed time artificial insemination (FTAI) performed by cervical, transcervical, or intrauterine route at 46-50 h or 52-56 h after progesterone device removal in a 3 × 2 factorial design. Pregnancy rate was affected by the insemination technique and by the moment of FTAI (P < 0.05), without interaction (P= NS). Overall, the fertility was improved as semen deposition was deeper and insemination was delayed. For transcervical insemination, pregnancy rate was intermediate (42.3%; P= NS) between cervical and intrauterine route (36.0% and 50.2%; P < 0.05), and was greater for those ewes inseminated beyond 4 cm into the cervix (60.0% versus 35.1% for insemination beyond or within 4 cm into the cervix, respectively; P < 0.05). Semen deposition beyond 4 cm into the cervix was achieved only in 28.8% of the females receiving transcervical insemination. This method was more time-consuming than cervical or laparoscopic insemination (11.4 ± 1.6 versus 85.5 ± 7.5 and 56.8 ± 5.6 ewes inseminated per hour, respectively; P < 0.05). In summary, greater pregnancy rate using FTAI is obtained when semen is placed into the uterus, which was achieved in all females only through laparoscopy. Further improvements are required for transcervical insemination to be applied in large-scale FTAI programs in Corriedale ewes.

Intracytoplasmic sperm injection improves in vitro embryo production from poor quality bovine oocytes.

The objective was to use subzonal sperm injection (SUZI) to understand sperm penetration patterns and to use intracytoplasmic sperm injection (ICSI) to improve production of bovine embryos using poor quality gametes. In experiment 1, poor versus good quality oocytes were fertilized with sperm from two bulls, A and B, with poor and good sperm vigor, respectively. The blastocyst rate was higher for good versus poor quality oocytes (23.3% vs. 11.1%, P < 0.05), regardless of the bull used. There was no significant difference in blastocyst rate for bull A (low vigor) regardless of oocyte quality, and for bull B (high vigor), blastocyst rate was better for good versus poor quality oocytes (25.7% vs. 9.2%, P < 0.05). In experiment 2, poor quality oocytes were subjected to SUZI. The oocyte penetration rate was lower for bull A than for bull B (29.6% vs. 53.8%, P < 0.05), when SUZI was performed within 1 hour after sperm processing. However, when SUZI was performed 2 to 3 hours after sperm processing, penetrating capacity was similar between bulls, but for bull B, penetrating capacity significantly decreased after 3 hours of sperm processing. In an attempt to overcome sperm penetrating disorders, poor and good quality oocytes were subjected to ICSI (experiment 3). Irrespective of the bull or of the oocyte quality grade, there were no differences in cleavage or blastocyst rates. Both bulls had distinct IVF embryo production rates, which we inferred were because of particular individual sperm characteristics. In conclusion, ICSI was an effective means to achieve in vitro production of bovine embryos with gametes of variable quality.

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.

In vitro development of cloned bovine embryos produced by handmade cloning using somatic cells from distinct levels of cell culture confluence.

The relationship between the level of cell confluence near the plateau phase of growth and blastocyst yield following somatic cell cloning is not well understood. We examined the effect of distinct cell culture confluence levels on in vitro development of cloned bovine embryos. In vitro-matured bovine oocytes were manually bisected and selected by DNA staining. One or two enucleated hemi-cytoplasts were paired and fused with an adult skin somatic cell. Cultured skin cells from an adult Nellore cow harvested at three distinct culture confluence levels (70-80, 80-90, and >95%) were used for construction of embryos and hemi-embryos. After activation, structures were cultured in vitro as one embryo (1 x 100%) or as aggregates of two hemi-embryos (2 x 50%) per microwell. Fusion, cleavage and blastocyst rates were compared using the chi(2) test. The fusion rate for hemi-embryos (51.4%) was lower than for embryos (67.6%), with no influence of degree of cell confluence. However, blastocyst rates improved linearly (7.0, 17.5, and 29.4%) with increases in cell confluence. We conclude that degree of cell culture confluence significantly influences subsequent embryo development; use of a cell population in high confluence (>90%) for nuclear transfer significantly improved blastocyst yield after cloning.

Improved superovulatory response in beef cattle following ovarian follicular ablation using a simplified transvaginal device.

The influence of the timing for the ablation of dominant follicle(s) prior to superovulatory treatment, and its effect on ovarian follicular growth and embryo yield, still remain elusive in cattle. The present study was designed to evaluate the effects of: (1) the day of the estrous cycle, at mid-diestrus, for the onset of superstimulation of follicular development, (2) the presence or absence of large ovarian follicles (ovary status) and (3) the time of follicular ablation, in hours, prior to the superovulatory treatment, on the superovulatory response in cattle. From a total of 244 superovulatory treatments and embryo collections in nulliparous and multiparous females, 76 were conducted after follicular ablation using a simplified transvaginal puncture cannula. Results from the present study indicated that the presence of large palpable follicle(s) at the onset of superstimulation of follicular development markedly reduced the superovulatory response. In addition, follicular ablation at 0 h or at 24 h prior to the onset of the superstimulation treatment significantly increased the number of total viable embryos. However, superovulatory responses were not affected by the day of the estrous cycle for the onset of follicular superstimulation and by the animal category (heifers or cows). In conclusion, the ablation of palpable follicle(s) 24 h or immediately prior to the onset of gonadotropin treatment, from days 8 to 12 of the estrous cycle (day 0, behavioral estrus), increased the total number of transferable embryos per flushing in cattle.

Calves born after open pulled straw vitrification of immature bovine oocytes.

The aim of this study was to evaluate the developmental capacity of immature bovine oocytes after vitrification with 20% ethylene glycol (EG)+20% dimethyl sulfoxide (Me(2)SO) and 0.5M sucrose (SUC), by open pulled straw (OPS) technology. The effect of treatment with cytochalasin D before vitrification was also examined. No differences were observed in cleavage and blastocyst rates among the group vitrified without cytochalasin D treatment (Vitri) (49.0% and 6.1%) and that with cytochalasin D treatment before vitrification (CDVitri) (46.4% and 3.6%), but both were lower (P<0.05) than the unvitrified control group (85.1 and 45.9%). Calves were obtained after transfer of fresh and vitrified blastocysts from the Vitri group and after transfer of vitrified blastocysts from the CDVitri group. Cytochalasin D treatment does not improve the development of immature bovine vitrified oocytes. The results show that a small proportion of immature oocytes vitrified with this technology are fully competent to produce blastocysts, which may be transferred immediately or vitrified before transfer, and go on to develop healthy offspring.

Avaliaçäo de diferentes concentraçöes de glicerol e sacarose no congelamento ultra-rápido de mórulas Mus musculus / Ultrarapid freezing of Mus musculus morulae with different concentrations of glycerol and sucrose

Mórulas Mus musculus da cepa nCF1 Suiço Albina, colhidas 76 a 78h após a administraçäo de hCG, foram congeladas em PBS modificado, contendo glicerol 3,0 ou 4,0 M, associado a sacarose 0,25 ou 0,5 M. Cento e sessenta e quatro mórulas excelentes (Grau I) ou boas (Grau II) foram colocadas em 0,05 ml da soluçäo de congelamento, em grupos de 5 a 11, para um período de desidrataçäo de 5 minutos, à temperatura ambiente (20 - 23§C), durante o qual foi processado o envase em palhetas de 0,25 ml. As palhetas contendo os embriöes foram mantidas em vapor de nitrogênio (N2) durante 2 minutos, antes da imersäo em N2 líquido. Após o descongelamento em banho-maria, a 37§C, por 20 segundos, a diluiçäo do crioprotetor foi efetuada em 0,5 ml de uma soluçäo de sacarose 0,5 M, durante 5 minutos, à temperatura ambiente. Os embriöes viáveis foram cultivados a 37§C, em PBS modificado + 20 SFB e avaliados 24 e 48 h após o início do cultivo. Os índices de sobrevivência foram semelhantes (P>0,05) com a utilizaçäo de glicerol 3,0 a 4,0 M, nos três momentos de avaliaçäo. Na avaliaçäo efetuada logo após a diluiçäo dos crioprotetor, näo foram observadas diferenças entre as concentraçöes 0,25 e 0,5 M (P>0,05), embora os índices de sobrevivência o//btidos com sacarose 0,25 M tenham sido superiores aos verificados com sacarose 0,5 M, após (P<0,05) e 48 h de cultivo (P<0,01). Uma diminuiçäo da viabilidade foi verificada a partir do tempo zero de avaliaçäo, até 24 h de cultivo, embora os índices de sobrevivência tenham se mantido constantes, entre 24 e 48h de cultivo

Avaliaçäo de diferentes concentraçöes de sacarose e lactose associadas a glicerol 2.0 M no congelamento ultra-rápido de mórulas Mus musculus / Ultra-rapid freezing of Mus musculus morulae with different concentrations of sucrose and lactose associated to 2.0 M glycerol

Seiscentos e vinte e oito (628) mórulas Mus musculus da cepa CF1 Suiço Albina, colhidas 76 a 78 h após a administraçäo de HCG, foram congeladas em soluçäo de PBS modificado contendo glicerol 2,0 M associado a três níveis crescentes de sacarose e lactose (0,125, 0,25 e 0,5 M). Mórulas excelentes (Grau I) e boas (Grau II) foram colocadas em 0,05 ml da soluçäo de congelamento, em grupos de 4 a 11, para um período de desidrataçäo de 5 minutos, a temperatura ambiente (20-23§C), durante o qual foi processado o envase em palhetas de 0,25 ml. Depois de seladas, as palhetas foram mantidas em vapor de nitrogênio (N2) durante 2 minutos, e logo após imersas em N2 líquido. O descongelamento foi efetuado em banho-maria a 37§C, por 20 segundos, sendo a remoçäo do crioprotetor efetuada em 0,5 ml de uma soluçäo contendo o mesmo açucar utilizado no congelamento, nas concentraçöes 0,25 e 0,5 M, durante 5 minutos, a temperatura ambiente. Os embriöes viáveis foram cultivados em PBS modificado + 20//SFB, a 37§C, sendo avaliados 24 e 48 h após o início do cultivo. Foi observada uma reduçäo dos índices de sobrevivência com a utilizaçäo de sacarose e lactose 0,5 M, sendo mais acentuada entre 24 e 48 h de cultivo. No entanto, os índices de sobrevivência se mantiveram praticamente constantes, neste mesmo intervalo, com as concentraçöes 0,125 e 0,25 M de sacarose e lactose. O índice médio de sobrevivência obtido com lactose foi superior ao obtido com sacarose, após 48 h de cultivo (P<0,05). As concentraçöes 0,25 e 0,5 M de sacarose e de lactose foram igualmente efetivas (P>0,05) no processo de remoçäo do crioprotetor. Após a transferência in vivo de mórulas congeladas em glicerol 2,0 M + sacarose 0,25 M, foi obtido um percentual de 50//de implantaçöes e 10//de fetos. Devido ao baixo índice de desenvolvimento fetal observado, a mistura de glicerol 2,0 M + sacarose 0,25 M foi considerada inadequada para o congelamento ultra-rápido de mórulas de camundongos, nas condiçöes deste experimento