Effects of antifreeze protein from Lolium perenne L. (LpAFP) in the vitrification of in vitro-produced bovine embryos.

In the present study, the cryoprotective effects of Lolium perenne antifreeze protein (LpAFP) on the vitrification of bovine embryos were evaluated. In vitro-produced blastocysts were divided into two groups: the control group (CG) without the addition of LpAFP and the treatment group (TG) with the addition of 500 ng/ml of LpAFP in the equilibrium and vitrification solution. Vitrification was carried out by transferring the blastocysts to the equilibrium solution [7.5% ethylene glycol (EG) and 7.5% dimethyl sulfoxide (DMSO)] for 2 min and then to the vitrification solution (15% EG, 15% DMSO and 0.5M sucrose). The blastocysts were deposited on a cryotop device and submerged in liquid nitrogen. Warming was carried out in three steps in solutions with different sucrose concentrations (1.0, 0.5, and 0.0 M, respectively). Embryos were evaluated for re-expansion/hatching, the total cell count, and ultrastructural analysis. There was no significant difference in the re-expansion rate 24 h after warming; however, there was variation (P < 0.05) in the hatching rate in the TG and the total number of cells 24 h after warming was higher in the TG (114.87 ± 7.24) when compared with the CG (91.81 ± 4.94). The ultrastructural analysis showed changes in organelles related to the vitrification process but, in the TG, there was less damage to mitochondria and rough endoplasmic reticulum compared with the CG. In conclusion, the addition of 500 ng/ml of LpAFP during the vitrification of in vitro-produced bovine embryos improved the hatching rate and total cell number of blastocysts after warming and mitigated intracellular damage.

Influência da somatotropina recombinante bovina no desenvolvimento folicular e na coleta de embriões em éguas / Influence of bovine recombinant somatotropin in the follicular development and embryo colletion in mares

Dez éguas, sem raça definida, foram submetidas a avaliações ultrassonográficas durante o intervalo interovulatório, avaliando-se folículos ≥ 5mm. Cinco éguas foram tratadas com 500mg de r-bST no primeiro e no 14º dia pós-ovulação (grupo GT), e as demais com soro fisiológico (grupo GC). Quando o folículo dominante atingiu diâmetro ≥ 40mm, foram induzidas com hCG e inseminadas 24 horas após, sendo submetidas à coleta de embrião seis dias após a ovulação. Os dados foram agrupados de acordo com o diâmetro do folículo dominante nas fases de emergência, divergência, dominância, pré-ovulatória, indução, inseminação e ovulação. Todas as éguas foram usadas duas vezes, no mesmo grupo. O GT apresentou crescimento folicular precoce para as fases de emergência, divergência, dominância e pré-ovulatória, assim como para o seu maior folículo subordinado, que cresceu mais cedo. As taxas de recuperação foram de 90% (GC) e 70% (GT), em 16 estruturas coletadas, obtendo-se uma não fecundada e um blastocisto inicial para o grupo GC; os demais, no estágio de mórula, apresentaram comportamento semelhante entre os grupos. Conclui-se que a r-bST influencia a dinâmica folicular de éguas, levando a uma antecipação do desenvolvimento folicular, que pode ser utilizada para encurtar o ciclo estral.(AU)

Ten undefined mare breeds were submitted to ultrasonographic evaluations during the interovulatory interval, evaluating follicles measuring ≥ 5mm. Five mares were treated with 500mg r-bST on the first and the 14th day after ovulation (TG group), and the others with saline (CG group). When the dominant follicle reached a diameter ≥ 40mm the ovulation was induced with hCG, and the mares were inseminated 24 hours later and submitted to embryo collection six days after ovulation. The data were grouped according to the diameter of the dominant follicle in the emergence, divergence, dominance, preovulatory, induction, insemination and ovulation phases. All mares were used twice, in the same group. The GT showed early follicular growth for the emergence, divergence, dominance and pre-ovulatory phases, as well as for its greater subordinate follicle, growing earlier. The recovery rates were 90% (CG) and 70% (TG), and 16 structures were collected, obtaining an unfertilized embryo and an initial blastocyst for the CG group, the others in the morula stage behaved similarly between the groups. It can be concluded that r-bST influences the follicular dynamics of the mares, leading to an anticipation of the follicular development that can be used to shorten the estrous cycle.(AU)

Evaluation of morphology, morphometry and follicular dynamics in FecGE genotyped ewes.

This study aimed to evaluate the effect of FecGE mutation on the development of ovarian follicles. To this end, 42 Santa Inês ewes were genotyped for FecGE mutation and classified as wild-type (FecG+/+), heterozygous (FecG+/E) or mutant homozygous (FecGE/E). Ovarian fragments were processed, and the follicles were analyzed with regard to the morphology and morphometry using classical histology. For the evaluation of follicular dynamics, ewes underwent oestrous synchronization and were monitored throughout an interovulatory period. A higher (P < 0.05) percentage of morphologically normal follicles in the primordial stage was identified in FecGE/E (90.0%) and FecG+/E (88.1%) ewes than in the FecG+/+ (73.0%) ewes. There was also a significantly greater (P < 0.05) number of morphologically normal follicles in the FecGE/E (87.3%) and FecG+/E (83.3%) ewes than in FecG+/+ (76.8%) ewes in the transitional stage. A smaller (P < 0.05) diameter was observed in the secondary follicles in FecGE/E (93.8 µm) ewes than in FecG+/E (171.8 µm) ewes. Regarding follicular dynamics, FecGE/E ewes showed a greater (P < 0.05) number of ovulations (2.5 ±â€¯0.2) than FecG+/+ ewes (1.5 ±â€¯0.3) ewes. Ovulatory follicles were smaller (P < 0.05) in the FecGE/E (5.1 mm) and FecG+/E (5.2 mm) ewes than in FecG+/+ (5.8 mm) ewes. Santa Inês nulliparous ewes carrying the FecGE mutation showed a greater proportion of morphologically normal follicles in the primordial and transitional stages than those not carrying the mutation. FecGE/E ewes demonstrated a higher number of ovulated follicles and that FecGE/E and FecG+/E ewes presented ovulatory follicles with a smaller diameter.

Ultrasonographic monitoring of 103 recipient mares of different reproductive status during the first 30 days after embryo transfers.

Ten pluriparous mares were used as donors to supply embryos which were transferred into 103 recipients, 31 of which were nulliparous, 34 were pluriparous and lactating, and 38 were pluriparous and non-lactating. The embryos were recovered eight days after ovulation and pregnancy was confirmed by ultrasound six days after the transfer; the length of the embryos was measured ultrasonographically on days 12, 14, 16, 18, 20, 25 and 30 after the embryo transfer. One hundred and fifteen of 200 flushes provided embryos, 12 being degenerate and 103 being viable embryos. From the 103 embryo transfers carried out, 51 pregnancies were confirmed by ultrasound within 30 days; 16 of the 31 nulliparous recipients became pregnant, 16 of the 34 pluriparous lactating recipients and 19 of the 38 pluriparous non-lactating recipients. There were no significant differences between the groups of mares in the mean (sd) rate of growth of their embryos between 12 and 30 days of gestation.

The use of steroid hormones in superovulation of Nelore donors at different stages of estrous cycle.

The objective of the present study was to evaluate the superovulatory response and ova/embryo recovery from Nelore donors following treatment with a controlled internal drug releasing device and estradiol benzoate (CIDR-B program) at different stages of the estrous cycle. The control group (TI; n=40) received a standard superovulation protocol with females of this group being between days 9 and 12 of the estrous cycle (estrus = day 0). The donors that received a CIDR-B program containing 1.9 g progesterone and an intramuscular injection of estradiol benzoate (2 mg) were at day 0 (TII; n=30), between days 2 and 6 (TIII; n=30), days 7 and 12 (TIV; n=30), days 13 and 16 (TV; n=30) and days 17 and 20 (TVI; n=30) of the estrous cycle. Superovulation was induced with 400 IU of p-FSH, divided into eight decreasing doses (80/80; 60/60; 40/40; 20/20) at intervals of 12h. The donors received PGF2alpha (Cloprostenol) 48 h after beginning the treatment and CIDRs were removed 12h later. Artificial inseminations (AI) were performed 12 and 22 h after the initiation of estrus and embryos were collected 7 days after AI. The mean numbers (+/-S.E.M.) of total ova and embryos, viable (transferable) and degenerated embryos were 14.2+/-11.3, 7.4+/-6.9 and 3.2+/-3.5 (TI), 13.3+/-10.4, 7.1+/-6.2 and 3.3+/-4.3 (TII), 13.5+/-7.0, 8.1+/-6.7 and 2.3+/-3.0 (TIII), 17.4+/-9.9, 9.4+/-6.9 and 4.0+/-4.4 (TIV), 16.9+/-8.8, 9.8+/-8.1 and 2.7+/-2.5 (TV) and 13.0+/-7.8, 7.2+/-6.9 and 2.3+/-2.5 (TVI), with no significant differences (P>/=0.05) among groups. Pregnancy rates of 67.1% (TI; n=86/128), 60.8% (TII; n=59/97), 62.5% (TIII; n=73/115), 64.1% (TIV; n=84/131), 72.3% (TV; n=81/112) and 60.6% (TVI; n=63/104) were obtained with embryos transferred from these collections and did not differ significantly (P>/=0.05) among groups. The results of the present study allow us to conclude that a combination of steroid hormones may be used prior to superovulation in Nelore donors, at any stage of the estrous cycle without affecting the efficiency of embryo transfer programs.