Blastocyst morphology, actin cytoskeleton quality and chromosome content are correlated with embryo quality in the pig.

Embryo survival rates obtained after transfer of in vitro produced porcine blastocysts are very poor. This is probably related to poor quality of the embryos. The aim of the present study was to determine markers for good quality blastocysts. Therefore, we tried to link blastocyst morphology to several morphological and cell biological properties, and evaluated the survival of in vitro produced, morphologically classified, blastocysts following non-surgical transfer. In vitro and in vivo produced blastocysts were allocated to two groups (classes A and B) on the basis of morphological characteristics. The quality of their actin cytoskeleton, their total cell number, their ability to re-expand after cytochalasin-B treatment and the occurrence of numerical chromosome aberrations were studied and compared. In vivo produced blastocysts were used as a control. Our results indicate that the ability of blastocysts to re-expand after cytochalasin-B-induced actin depolymerization was positively correlated with the morphology of the blastocyst, and associated with the quality of the actin cytoskeleton. Chromosome analysis revealed that mosaicism is inherent to the in vitro production of porcine embryos, but also that in vivo produced blastocysts contained some non-diploid cells. In non-surgical embryo transfer experiments more recipients receiving class A blastocysts were pregnant on Day 20 than those receiving class B blastocysts. One recipient gave birth to six piglets from class A in vitro produced blastocysts, providing a verification of the enhanced viability of blastocysts that were scored as 'good' on the basis of their morphology.

Reproduction results and offspring performance after non-surgical embryo transfer in pigs.

Increased interest in transfer of valuable genetic material around the world with minimal health risks has stimulated the development of non-surgical embryo transfer (nsET) technologies in pigs. Experimental evidence shows that nsET without sedation of the recipients is now feasible. The goal of this study, therefore, was to evaluate a method of nsET under commercial conditions. The experiment included 135 donor gilts and 45 multiparous recipient sows. Ovulation was induced in both donors and recipients, and nsET was performed using the Swinlet catheter. Donor gilts averaged 16.5 (7-45) corpora lutea, but this depended on age of the donor (P < 0.05). An average of 10.1 transferable blastocysts was recovered per donor, and the recovery rate was 84%. For 44 nsET, 14 recipients (31%) came into estrous before Day 23 after ovulation, 7 recipients (16%) came into estrous between Days 23 and 30, 3 recipients (6.8%) came into estrous between Days 39 and 48, 2 recipients (4.5%) had a late abortion. Finally, 18 of 44 recipients (41%) resulted in successful births, with an average liter size of 7.2 +/- 2.8. Birth weight of nsET piglets were 0.2 kg more than control piglets, but depended on litter size ((P < 0.05). The sex-ratio was not different from 50%. No anatomical abnormalities were observed in the offspring of nsET. Of the recipients that did not become pregnant from nsET, 91% became pregnant after insemination in the next estrous. Gilts born from nsET gave on average 12.4 +/- 3.0 total born piglets in their first pregnancy. In conclusion, the nsET procedure used in this study can be applied in practice without the need for special facilities, such as surgical and anesthesia equipment.

Quality of porcine blastocysts produced in vitro in the presence or absence of GH.

GH receptor (GHR) mRNA is expressed in bovine in vitro produced embryos up to the blastocyst stage and GH improves the quality of bovine embryos by increasing blastocyst cell numbers and reducing the incidence of apoptosis as evaluated by DNA strand-break labelling. Porcine in vitro produced blastocysts have lower cell numbers than in vivo blastocysts and exhibit higher incidences of apoptosis. Therefore we investigated the effects of 100 ng GH/ml NCSU23 medium during in vitro culture of presumptive in vitro fertilized sow zygotes on embryo development and blastocyst quality (defined by diameter, cell number, apoptosis and survival after non-surgical transfer). In vivo produced blastocysts were analysed concurrently as a reference value. GHR was expressed in embryos from the 2-cell to blastocyst stages. GH had no effect on blastocyst development or cell numbers, but increased the mean blastocyst diameter. The incidence of apoptosis, detected by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL), was decreased by GH, but when non-TUNEL-labelled apoptotic fragmented nuclei were included, no difference was seen. GH appeared to slow down the progression of apoptosis though. In vivo produced blastocysts presented no apoptotic nuclei, and contained higher cell numbers and larger diameters. Pregnancy rates on day 11 were similar for all groups, but survival was poorer for in vitro than in vivo produced blastocysts. In this study GH appeared to be beneficial only from the blastocyst stage, but the presence of GHR from early cleavage stages nevertheless indicates a role for GH throughout porcine embryo development and deserves further investigation.

Development, DNA fragmentation and cell death in porcine embryos after 24 h storage under different conditions.

For practical applications of porcine embryo transfer (ET) it is important to develop feasible embryo storage conditions. The aim of the present study was to evaluate the effect of short-term storage (24 h) on the quality of in vivo produced porcine embryos. Three temperatures 18, 25 and 38 degrees C and three different media: Dulbecco's phosphate buffered saline (DPBS), TCM199 and Emcare, were tested for two different embryo ages: D4 embryos (collected 144 h after hCG treatment) and D5 embryos (collected 168 h after hCG). After slaughter of the donor gilts, embryos were collected and transported at 25 degrees C to the lab where morulas and blastocyst were selected (D4 n = 222; D5 n = 167) and randomly used as controls or distributed over the treatment groups. Developmental stage and embryo diameter were assessed by normal light microscopy, while total number of cells and incidence of apoptosis were assessed using a fluorescent embryo quality staining technique that combines three different dyes: Ethidium Homodimer (EthD-1), TUNEL and Hoechst 33342. Following 24 h storage, D5 embryos had higher rates of hatching (24%) and degeneration (9%) compared to D4 embryos (10 and 4%, respectively; P < 0.05). Embryos stored at 38 degrees C had higher rates of hatching (37%) compared to those ones stored at 25 degrees C (13%) or 18 degrees C (0%; P < 0.01). More embryos hatched when stored in medium Dulbecco's phosphate buffered saline (DPBS) or in TCM199 compared to those stored in Emcare (P < 0.05). A higher percentage of embryos stored at 18 degrees C degenerated compared to those stored at 25 or 38 degrees C (P < 0.01). No significant increase in apoptosis was observed after storage compared to the rates of apoptosis at 0 h (controls) or between the different storage groups. Based on the results we conclude that D4 porcine embryos produced in vivo, selected under normal light microscopy and stored at 25 degrees C in a serum free medium for 24 h will have a suitable developmental stage for ET and a high embryo quality.

Duration of estrus in relation to reproduction results in pigs on commercial farms.

This research was conducted to determine factors that influence duration of estrus, AI strategy, and reproduction results between and within commercial swine farms that use AI. Data from 15,186 sows and gilts on 55 farms for a period of 6.1+/-4.2 mo per farm were used in this study. The average duration of estrus was 48.4+/-1.0 h, ranging from 31 to 64 h, and was consistent from month to month within a farm (repeatability of 86%). Differences in duration of estrus between farms accounted for 23% of the total variation in duration of estrus. On most farms (n = 45), gilts showed a shorter (P < .05) duration of estrus than sows (40.8+/-1.1 h vs 48.5+/-1.0 h). The duration of first estrus after weaning was longer (P < .0001) compared with that of repeat-breeder sows (50.2+/-1.0 h vs 46.8+/-1.0 h). Duration of estrus decreased (P < .05) when interval from weaning to estrus increased from 4 to 6 d (56.0 +/- 1.2 h vs 45.8 +/-1.2 h). The regression of interval from onset to estrus to first AI and interval from weaning to estrus varied between farms and ranged from -7.4 to +1.3 h/d; four farms had a positive relationship. Farrowing rate decreased (P < .05) from 89.7+/-2.7% to 78.2+/-5.74 when the interval from weaning to estrus increased from 4 to 10 d. The litter size decreased (P < .05) from 11.7 to 10.6 pigs when the interval from weaning to estrus increased from 4 to 7 d. Compared with a single AI, double AI in sows and gilts resulted in a 4.3 and 7.0% higher (P < .05) farrowing rate, respectively. When the first AI was performed after expected ovulation, reproduction results were lower than when AI was performed before or at expected ovulation in sows. Duration of estrus was not related to farrowing rate or litter size in individual pigs. Number of inseminations per estrus, time of AI, and duration of estrus were correlated, which made it difficult to assess which of these factors was primarily related to the farrowing rate or litter size. Knowledge of average duration of estrus on farms and of factors that influence the duration of estrus on commercial farms can help to improve the efficiency of the AI strategy specific for each farm.

Semen backflow after insemination and its effect on fertilisation results in sows.

The aim of the present study was to investigate the volume of and number of spermatozoa in semen backflow during and after insemination, and the effect of backflow on fertilisation results assessed at day 5 of pregnancy. Multiparous sows (n = 140) were artificially inseminated with either (1, 3 or 6) x 10(9) mixed spermatozoa from three boars in a constant volume of 80 ml. Backflow of semen was measured three times: during insemination (M1); during the first half hour after insemination (M2); and from 0.5 h until about 2.5 h after insemination (M3). Transrectal ultrasonography was performed at intervals of 4 h to determine the time of ovulation. Sows were sacrificed at 120 +/- 0.4 h after ovulation to assess the results of fertilisation. Every sow had some backflow and the variation in volume, and number of spermatozoa within the backflow was high. The average semen backflow within 2.5 h after insemination was 70 +/- 3.4% of the volume and 25 +/- 1.4% of the spermatozoa of the inseminated dosage. The concentration of the backflow (% of the inseminated dosage) decreased with time after insemination from 65% at M1 to 40% and 26% at M2 and M3, respectively. The correlations between volume and number of spermatozoa were high: r = 0.97, r = 0.73 and r = 0.81 in M1, M2 and M3, respectively. More than 5% of the inseminated spermatozoa in backflow during insemination affected fertilisation negatively in those sows inseminated with 1 x 10(9) spermatozoa (P < 0.05). Backflow after insemination had no effect on fertilisation results (P > 0.05). Timing of insemination relative to ovulation and oestrus were not related to backflow during or after insemination (P > 0.05). Of the sows which had backflow, those of parity 1 tended to have the highest proportion of sows with more than 5 ml backflow (47%; n = 8 of 17) compared with sows from parity 2 and higher (24%; n = 14 of 59) (P = 0.075). It was concluded that excessive backflow of semen during insemination had a negative effect on fertilisation results when sows where inseminated with only 1 x 10(9) spermatozoa. Causes of variation in backflow between sows were not clearly identifiable.

Influence of insemination-ovulation interval and sperm cell dose on fertilization in sows.

This experiment was conducted to determine the effects of sperm dose at insemination on fertilization rates and accessory sperm cells attached to day 5 embryos. Multiparous sows (n = 115) were artificially inseminated once with 1 x 10(9), 3 x 10(9) or 6 x 10(9) sperm cells between 3 h and 48 h before ovulation. Transrectal ultrasonography was performed at intervals of 4 h to determine the time of ovulation and sows were killed at 120 +/- 5 h after ovulation to assess the results of fertilization. The insemination-ovulation interval had a major influence on the fertilization rate and accessory sperm count. A nonsignificant but consistent increase in fertilization rate and in number of accessory sperm cells due to the sperm dose was observed. During the insemination-ovulation interval of 12-24 h, the median fertilization rates were 95%, 100% and 100%, and the median accessory sperm counts were 11, 17 and 31 for the 1 x 10(9), 3 x 10(9) and 6 x 10(9) doses, respectively. During the insemination-ovulation interval of 24-36 h, the median fertilization rates were 88%, 95% and 97%, and the median accessory sperm counts were 6, 8 and 11 for the 1 x 10(9), 3 x 10(9) and 6 x 10(9) doses, respectively. No direct relationship was detected between embryo quality and the accessory sperm count but there was a relationship between insemination-ovulation interval and accessory sperm count. The fertilization rate was positively correlated with the breeding value for litter size of the sows. In conclusion, the effects of sperm dose on fertilization rate and on accessory sperm count in sows were small and nonsignificant, indicating only small effects of sperm dose on the functioning of the sperm reservoir in the sow.

Multiple herd evaluation of the effects of maternal lineage on yield traits of Holstein cattle.

Effects of maternal lineage on yield traits were examined by using animal models. Data were 6054 multiparous records of 2264 cows from six herds in North Carolina and the breeding herd of Iowa State University. Separate analyses were performed by using first lactation records from North Carolina, all records from North Carolina, and pooled records from North Carolina and Iowa. Traits were mature equivalent yields of milk, fat, and protein; percentages of fat and protein; and milk energy concentration and yield. Cattle were assigned to maternal lineages on the basis of the earliest female ancestor recorded. Fixed effects in the models were herd-year-season, parity, and maternal lineage; random effects were animal, permanent environment, and residual. All additive genetic relationships were considered. For all analyses, maternal lineage was associated with significant differences in fat percentage and milk energy concentration. Differences between maternal lineages for yield traits were not significant. Variance components were also obtained with REML using the same data and models, but with lineage as a random effect. Based on records pooled from Iowa and North Carolina, maternal lineage accounted for 2.7% of the variance in fat percentage. Otherwise, < 1.2% of the variance of any trait was associated with maternal lineage.