Phospholipid composition and resistance to vitrification of in vivo blastocyst of a Brazilian naturalized porcine breed / Composição de fosfolipídios e resistência à vitrificação de blastocistos produzidos in vivo de uma raça de suíno naturalizada brasileira

Piau porcine blastocysts were submitted to MALDI-TOF to identify the main phospholipids (PL). After that, in vivo blastocysts (D6) were vitrified (n=52), non-vitrified were used as control (n=42). After warming, blastocysts were in vitro cultured to assess re-expansion and hatching at 24 and 48 hours. Finally, at 48 hours, hatched blastocysts were submitted to RT-qPCR searching for BCL2A1, BAK, BAX and CASP3 genes. For MALDI-TOF, the ion intensity was expressed in arbitrary units. Blastocyst development was compared by Qui-square (P< 0.05). Among the most representative PL was the phosphatidylcholine [PC (32:0) + H]+; [PC (34:1) + H]+ and [PC (36:4) + H]+. Beyond the PL, MALDI revealed some triglycerides (TG), including PPL (50:2) + Na+, PPO (50:1) + Na+, PLO (52:3) + Na+ and POO (52:2) + Na. Re-expansion did not differ (P> 0.05) between fresh or vitrified blastocysts at 24 (33.3%; 32.7%) or 48 hours (2.4%; 13.5%). Hatching rates were higher (P< 0.05) for fresh compared to vitrified at 24 (66.7%; 15.4%) and 48 hours (97.6%; 36.0%). BAX was overexpressed (P< 0.05) after vitrification. In conclusion, Piau blastocysts can be cryopreserved by Cryotop. This study also demonstrated that the apoptotic pathway may be responsible for the low efficiency of porcine embryo cryopreservation.(AU)

Blastocistos de suínos foram submetidos ao MALDI-TOF para se identificarem os principais fosfolipídios (PL). Depois, parte destes embriões (D6) foram vitrificados (n=52), ou permaneceram frescos (grupo controle, n=42). Após o aquecimento, os blastocistos foram cultivados in vitro para se avaliar a reexpansão e a eclosão (BE) às 24 e 48 horas. Finalmente, às 48 horas, os BE foram submetidos ao RT-qPCR em busca dos genes BCL2A1, BAK, BAX e CASP3. No MALDI-TOF, a intensidade do íon foi expressa em unidades arbitrárias. O desenvolvimento embrionário foi comparado por qui-quadrado (P<0,05). Entre os PL mais representativos estavam as fosfatidilcolinas [PC (32: 0) + H] +; [PC (34: 1) + H] + e [PC (36: 4) + H] +. Além do PL, o MALDI revelou alguns triglicerídeos (TG), incluindo PPL (50: 2) + Na +, PPO (50: 1) + Na +, PLO (52: 3) + Na + e POO (52: 2) + Na. A reexpansão não diferiu (P>0,05) entre blastocistos frescos ou vitrificados às 24 (33,3%, 32,7%) e 48 horas (2,4%, 13,5%). As taxas de eclosão foram maiores (P<0,05) para o grupo fresco comparado ao vitrificado às 24 (66,7% x 15,4%) e 48 horas (97,6% x 36,0%). O BAX estava mais expresso (P<0,05) após a vitrificação. Concluindo, os blastocistos Piau podem ser criopreservados por Cryotop. Este estudo também demonstrou que a via apoptótica pode ser responsável pela baixa eficiência da criopreservação de embriões suínos.(AU)

Phospholipid composition and resistance to vitrification of in vivo blastocyst of a Brazilian naturalized porcine breed / Composição de fosfolipídios e resistência à vitrificação de blastocistos produzidos in vivo de uma raça de suíno naturalizada brasileira

Piau porcine blastocysts were submitted to MALDI-TOF to identify the main phospholipids (PL). After that, in vivo blastocysts (D6) were vitrified (n=52), non-vitrified were used as control (n=42). After warming, blastocysts were in vitro cultured to assess re-expansion and hatching at 24 and 48 hours. Finally, at 48 hours, hatched blastocysts were submitted to RT-qPCR searching for BCL2A1, BAK, BAX and CASP3 genes. For MALDI-TOF, the ion intensity was expressed in arbitrary units. Blastocyst development was compared by Qui-square (P< 0.05). Among the most representative PL was the phosphatidylcholine [PC (32:0) + H]+; [PC (34:1) + H]+ and [PC (36:4) + H]+. Beyond the PL, MALDI revealed some triglycerides (TG), including PPL (50:2) + Na+, PPO (50:1) + Na+, PLO (52:3) + Na+ and POO (52:2) + Na. Re-expansion did not differ (P> 0.05) between fresh or vitrified blastocysts at 24 (33.3%; 32.7%) or 48 hours (2.4%; 13.5%). Hatching rates were higher (P< 0.05) for fresh compared to vitrified at 24 (66.7%; 15.4%) and 48 hours (97.6%; 36.0%). BAX was overexpressed (P< 0.05) after vitrification. In conclusion, Piau blastocysts can be cryopreserved by Cryotop. This study also demonstrated that the apoptotic pathway may be responsible for the low efficiency of porcine embryo cryopreservation.(AU)

Blastocistos de suínos foram submetidos ao MALDI-TOF para se identificarem os principais fosfolipídios (PL). Depois, parte destes embriões (D6) foram vitrificados (n=52), ou permaneceram frescos (grupo controle, n=42). Após o aquecimento, os blastocistos foram cultivados in vitro para se avaliar a reexpansão e a eclosão (BE) às 24 e 48 horas. Finalmente, às 48 horas, os BE foram submetidos ao RT-qPCR em busca dos genes BCL2A1, BAK, BAX e CASP3. No MALDI-TOF, a intensidade do íon foi expressa em unidades arbitrárias. O desenvolvimento embrionário foi comparado por qui-quadrado (P<0,05). Entre os PL mais representativos estavam as fosfatidilcolinas [PC (32: 0) + H] +; [PC (34: 1) + H] + e [PC (36: 4) + H] +. Além do PL, o MALDI revelou alguns triglicerídeos (TG), incluindo PPL (50: 2) + Na +, PPO (50: 1) + Na +, PLO (52: 3) + Na + e POO (52: 2) + Na. A reexpansão não diferiu (P>0,05) entre blastocistos frescos ou vitrificados às 24 (33,3%, 32,7%) e 48 horas (2,4%, 13,5%). As taxas de eclosão foram maiores (P<0,05) para o grupo fresco comparado ao vitrificado às 24 (66,7% x 15,4%) e 48 horas (97,6% x 36,0%). O BAX estava mais expresso (P<0,05) após a vitrificação. Concluindo, os blastocistos Piau podem ser criopreservados por Cryotop. Este estudo também demonstrou que a via apoptótica pode ser responsável pela baixa eficiência da criopreservação de embriões suínos.(AU)

Produção de embriões em ovinos Morada Nova e Somalis Brasileira / Embryo production in Morada Nova and Somalis Brasileira ewes

Superovulatory response and embryo yield in 19 Morada Nova and 20 Somalis Brasileira ewes was analyzed. All animals were synchronized with the insertion of an intravaginal device (CIDR®) on Day 0, replaced by a new device on Day 7, which remained in place until Day 14 and superovulated with 133mg of porcine FSH (pFSH) in decreasing doses at 12h intervals from Day 12 until Day 15 of the treatment, and a single dose of equine chorionic gonadotropin (eCG, 200UI) on Day 14 (i.e., administered in CIDR removal). Fifty hours after CIDR® removal, females were inseminated by laparoscopy. All embryos were recovered by laparotomy 5 days after insemination. Sheep which responded to the superovulation protocol (P>0.05) included 74% of the Morada Nova ewes and 50% of the Somalis Brasileira ewes. Morada Nova showed better results (P<0.05) than Somalis Brasileira in number of ovulations (15.38 ± 5.24 vs. 10.56 ± 2.83), total structures (11.00 ± 7.55 vs. 3.33 ± 1.94) and embryo yields (6.79 ± 5.35 vs. 2.90 ± 2.18). Despite the high fertilization rate, degenerate embryo rate was high for both breeds, with an overall rate of 39% (57/145). In conclusion, superovulatory response and embryo yields in Morada Nova ewes were considered sufficient to justify the use of this procedure in genetic resources conservation programs. However, improvements to embryo quality and control of precocious regression of corpus luteum are necessary to produce better results in the MOET program, with minimal variations and maximum embryo yield in Morada Nova and Somalis Brasileira ewes.(AU)

Produção de embriões em ovinos Morada Nova e Somalis Brasileira / Embryo production in Morada Nova and Somalis Brasileira ewes

Superovulatory response and embryo yield in 19 Morada Nova and 20 Somalis Brasileira ewes was analyzed. All animals were synchronized with the insertion of an intravaginal device (CIDR®) on Day 0, replaced by a new device on Day 7, which remained in place until Day 14 and superovulated with 133mg of porcine FSH (pFSH) in decreasing doses at 12h intervals from Day 12 until Day 15 of the treatment, and a single dose of equine chorionic gonadotropin (eCG, 200UI) on Day 14 (i.e., administered in CIDR removal). Fifty hours after CIDR® removal, females were inseminated by laparoscopy. All embryos were recovered by laparotomy 5 days after insemination. Sheep which responded to the superovulation protocol (P>0.05) included 74% of the Morada Nova ewes and 50% of the Somalis Brasileira ewes. Morada Nova showed better results (P<0.05) than Somalis Brasileira in number of ovulations (15.38 ± 5.24 vs. 10.56 ± 2.83), total structures (11.00 ± 7.55 vs. 3.33 ± 1.94) and embryo yields (6.79 ± 5.35 vs. 2.90 ± 2.18). Despite the high fertilization rate, degenerate embryo rate was high for both breeds, with an overall rate of 39% (57/145). In conclusion, superovulatory response and embryo yields in Morada Nova ewes were considered sufficient to justify the use of this procedure in genetic resources conservation programs. However, improvements to embryo quality and control of precocious regression of corpus luteum are necessary to produce better results in the MOET program, with minimal variations and maximum embryo yield in Morada Nova and Somalis Brasileira ewes.(AU)

Behavioral characteristics of calving in Curraleiro Pé-duro cows

Studies were conducted to characterize the labor behavior in Curraleiro Pé-duro cows, a Locally adapted breed found in the semiarid region of Brazil, which is considered rustic and fertile. Eleven Curraleiro Pé-duro cows were kept in pasture and were Observed every 4 h to assess the beginning of birth, and subsequently were constantly monitored until the complete expulsion of the placenta, accordingly to the three different stages of labor. The cows were evaluated according to the following events: staring into the flank (SIF); licking vulva and lifting the tail (LVUT); head-Butts to the flank (HF); dripping colostrum (DC); lying down and standing (restlessness; LDS); duration of the first phase (Dur1P); amount of initial contractions (IC); time to onset of appearing of feet (TOAF); duration of calving (DC) and duration time from end of calving til l the expulsion of the placenta (DurExpP). The results are presented as mean ± standard deviation: SIF (4.00 ± 2.37); LVUT (2.38 ± 1.06); HF (1.80 ± 0.45); DC (1.20 ± 0.45); LDS (2.56 ± 1.33);IC (6.00 ± 2.9); Dur1F (71 ± 40 min); TOAF (7± 8 min); DC (25 ± 24 min); and DurExpP (228 ± 76 min). These results allow us to characterize the events that precede and accompany the moment of labor in Curraleiro Pé-duro cows.(AU)

Ovulation induction in ewes using GnRH in long and short-term synchronization protocols

This study was done to evaluate the efficiency of GnRH along with long-term and short-term synchronization protocols on ovulation induction and corpus luteum development. Ewes underwent four protocols: Long+GnRH (n = 11) with vaginal sponge (60 mg MAP) for 12 days along with 300 IU of eCG on day 12 and 0.025 mg of GnRH 27 h after sponge removal; Long (n = 10) with vaginal sponge for 12 days along with 300 IU of eCG on day 12; Short GnRH (n = 10) with vaginal sponge for 7 days along with 37.5 μg of D-cloprostenol on day 5 and 300 IU of eCG on day 7, plus 0.025 mg of GnRH used 27 h after sponge removal; and Short (n = 10) with vaginal sponge for 7 days. D-cloprostenol (37.5 μg) was administered on day 5 and eCG (300 IU) was administered on day 7.Ovulation was evaluated 52, 56, 60, 66, 72, 76 h after sponge removal. Blood was collected twelve days after sponge removal to measure progesterone concentration. On this same day, the corpus luteum was measure and counted. When GnRH was used, all ewes ovulated, while 70 and 80% of ewes ovulated in protocols that had not received GnRH (Long and Short, respectively). The GnRH accelerated ovulation (P< 0.05) in relation to sponge removal in both protocols and induced ovulation in approximately 28 h. The GnRH Was effective in inducing ovulation without decreasing the corpus luteum volume and progesterone concentration.

Behavioral characteristics of calving in Curraleiro Pé-duro cows

Studies were conducted to characterize the labor behavior in Curraleiro Pé-duro cows, a Locally adapted breed found in the semiarid region of Brazil, which is considered rustic and fertile. Eleven Curraleiro Pé-duro cows were kept in pasture and were Observed every 4 h to assess the beginning of birth, and subsequently were constantly monitored until the complete expulsion of the placenta, accordingly to the three different stages of labor. The cows were evaluated according to the following events: staring into the flank (SIF); licking vulva and lifting the tail (LVUT); head-Butts to the flank (HF); dripping colostrum (DC); lying down and standing (restlessness; LDS); duration of the first phase (Dur1P); amount of initial contractions (IC); time to onset of appearing of feet (TOAF); duration of calving (DC) and duration time from end of calving til l the expulsion of the placenta (DurExpP). The results are presented as mean ± standard deviation: SIF (4.00 ± 2.37); LVUT (2.38 ± 1.06); HF (1.80 ± 0.45); DC (1.20 ± 0.45); LDS (2.56 ± 1.33);IC (6.00 ± 2.9); Dur1F (71 ± 40 min); TOAF (7± 8 min); DC (25 ± 24 min); and DurExpP (228 ± 76 min). These results allow us to characterize the events that precede and accompany the moment of labor in Curraleiro Pé-duro cows.

Ovulation induction in ewes using GnRH in long and short-term synchronization protocols

This study was done to evaluate the efficiency of GnRH along with long-term and short-term synchronization protocols on ovulation induction and corpus luteum development. Ewes underwent four protocols: Long+GnRH (n = 11) with vaginal sponge (60 mg MAP) for 12 days along with 300 IU of eCG on day 12 and 0.025 mg of GnRH 27 h after sponge removal; Long (n = 10) with vaginal sponge for 12 days along with 300 IU of eCG on day 12; Short GnRH (n = 10) with vaginal sponge for 7 days along with 37.5 μg of D-cloprostenol on day 5 and 300 IU of eCG on day 7, plus 0.025 mg of GnRH used 27 h after sponge removal; and Short (n = 10) with vaginal sponge for 7 days. D-cloprostenol (37.5 μg) was administered on day 5 and eCG (300 IU) was administered on day 7.Ovulation was evaluated 52, 56, 60, 66, 72, 76 h after sponge removal. Blood was collected twelve days after sponge removal to measure progesterone concentration. On this same day, the corpus luteum was measure and counted. When GnRH was used, all ewes ovulated, while 70 and 80% of ewes ovulated in protocols that had not received GnRH (Long and Short, respectively). The GnRH accelerated ovulation (P< 0.05) in relation to sponge removal in both protocols and induced ovulation in approximately 28 h. The GnRH Was effective in inducing ovulation without decreasing the corpus luteum volume and progesterone concentration.(AU)

Situação atual da superovulação em ovinos / Current status of superovulation in ewes

Esta revisão apresenta uma visão geral e crítica da base técnica dos procedimentos de superovulação,expondo as limitações e os últimos avanços dessa tecnologia na produção de embriões ovinos. Entre osresponsáveis pelas limitações dos protocolos comumente empregados, destacam-se a variabilidade no grau depurificação das gonadotrofinas derivadas da hipófise, a falta de controle da dinâmica folicular, as falhas noprocesso de fertilização e o elevado índice de regressão prematura do corpo lúteo. Algumas abordagens foramempregadas visando ao controle desses limitantes e obteve-se aceitável resposta superovulatória, como: autilização de gonadotrofinas recombinantes; a superestimulação no dia, ou em até 24 h, da emergência de umaonda folicular; a administração de GnRH e seus agonistas, como indutores de ovulação, principalmente quandofor realizada inseminação artificial com sêmen congelado; e a aplicação de hCG, GnRH ou inibidores daprostaglandina F2α após a ocorrência das ovulações múltiplas. Essas abordagens resultaram em melhoriasrelevantes nos programas de MOTE em ovinos.(AU)

The present paper reviews an overview and critique of the technical basis of the procedures forsuperovulation, exposing the limitations and recent advances in this technology in production of sheep embryos.Among those responsible for the limitations of commonly used protocols, those that stand out are the variability inthe degree of purification of pituitary gonadotropins, the lack of control of follicular dynamics, failures in thefertilization process and the high rate of premature regression of the corpus luteum. Some approaches have beenemployed for the control of these impediments, enhancing superovulatory response, such as the use of recombinantgonadotropins; superstimulation on the day or within 24 h of emergence of a follicular wave; and the administrationof GnRH agonists to induce ovulation, especially when artificial insemination is carried out with frozen semen; andthe administration of hCG, GnRH or prostaglandin F2α inhibitors after the occurrence of multiple ovulations.These approaches have resulted in significant improvements to MOET programs in sheep.(AU)

Situação atual da superovulação em ovinos / Current status of superovulation in ewes

Esta revisão apresenta uma visão geral e crítica da base técnica dos procedimentos de superovulação,expondo as limitações e os últimos avanços dessa tecnologia na produção de embriões ovinos. Entre osresponsáveis pelas limitações dos protocolos comumente empregados, destacam-se a variabilidade no grau depurificação das gonadotrofinas derivadas da hipófise, a falta de controle da dinâmica folicular, as falhas noprocesso de fertilização e o elevado índice de regressão prematura do corpo lúteo. Algumas abordagens foramempregadas visando ao controle desses limitantes e obteve-se aceitável resposta superovulatória, como: autilização de gonadotrofinas recombinantes; a superestimulação no dia, ou em até 24 h, da emergência de umaonda folicular; a administração de GnRH e seus agonistas, como indutores de ovulação, principalmente quandofor realizada inseminação artificial com sêmen congelado; e a aplicação de hCG, GnRH ou inibidores daprostaglandina F2α após a ocorrência das ovulações múltiplas. Essas abordagens resultaram em melhoriasrelevantes nos programas de MOTE em ovinos.

The present paper reviews an overview and critique of the technical basis of the procedures forsuperovulation, exposing the limitations and recent advances in this technology in production of sheep embryos.Among those responsible for the limitations of commonly used protocols, those that stand out are the variability inthe degree of purification of pituitary gonadotropins, the lack of control of follicular dynamics, failures in thefertilization process and the high rate of premature regression of the corpus luteum. Some approaches have beenemployed for the control of these impediments, enhancing superovulatory response, such as the use of recombinantgonadotropins; superstimulation on the day or within 24 h of emergence of a follicular wave; and the administrationof GnRH agonists to induce ovulation, especially when artificial insemination is carried out with frozen semen; andthe administration of hCG, GnRH or prostaglandin F2α inhibitors after the occurrence of multiple ovulations.These approaches have resulted in significant improvements to MOET programs in sheep.