Differential hepatic oxidative status in steers with divergent residual feed intake phenotype

Oxidative stress occurs when oxidant production exceeds the antioxidant capacity to detoxify the reactive intermediates or to repair the resulting damage. Feed efficiency has been associated with mitochondrial function due to its impact on cell energy metabolism. However, mitochondria are also recognized as a major source of oxidants. The aim of this study was to determine lipid and protein oxidative stress markers, and gene and protein expression as well as activity of antioxidant enzymes in the liver of steers of divergent residual feed intake (RFI) phenotypes. Hereford steers (n = 111) were evaluated in post-weaning 70 days standard test for RFI. Eighteen steers exhibiting the greatest (n = 9; high-RFI) and the lowest (n = 9; low-RFI) RFI values were selected for this study. After the test, steers were managed together under grazing conditions until slaughter when they reached the slaughter body weight. At slaughter, hepatic samples were obtained, were snap-frozen in liquid nitrogen and stored at −80°C until analyses. Hepatic thiobarbituric acid reactive species and protein carbonyls were greater (P = 0.05) and hepatic 4-hydroxynonenal protein adducts tended (P = 0.10) to be greater for high- than low-RFI steers. Hepatic gene expression glutathione peroxidase 4, glutamate–cysteine ligase catalytic subunit and peroxiredoxin 5 mRNA was greater (P ≤ 0.05) and glutathione peroxidase 3 mRNA tended (P = 0.10) to be greater in low- than high-RFI steers. Hepatic protein expression and enzyme activity of manganese superoxide dismutase and glutathione peroxidase enzyme activity tended (P ≤ 0.10) to be greater for low- than high-RFI steers. High-efficiency steers (low-RFI) probably had better hepatic oxidative status which was strongly associated with greater antioxidant ability near to the oxidant production site and, therefore, reduced oxidative stress of the liver. Decreased hepatic oxidative stress would reduce maintenance requirements due to a lower protein and lipid turnover and better efficiency in the use of energy.     

Geographic distribution of haplotype diversity at the bovine casein locus

The genetic diversity of the casein locus in cattle was studied on the basis of haplotype analysis. Consideration of recently described genetic variants of the casein genes which to date have not been the subject of diversity studies, allowed the identification of new haplotypes. Genotyping of 30 cattle breeds from four continents revealed a geographically associated distribution of haplotypes, mainly defined by frequencies of alleles at CSN1S1 and CSN3. The genetic diversity within taurine breeds in Europe was found to decrease significantly from the south to the north and from the east to the west. Such geographic patterns of cattle genetic variation at the casein locus may be a result of the domestication process of modern cattle as well as geographically differentiated natural or artificial selection. The comparison of African Bos taurus and Bos indicus breeds allowed the identification of several Bos indicus specific haplotypes (CSN1S1*C-CSN2*A²-CSN3*A_I/CSN3*H) that are not found in pure taurine breeds. The occurrence of such haplotypes in southern European breeds also suggests that an introgression of indicine genes into taurine breeds could have contributed to the distribution of the genetic variation observed.     

Dynamics of follicular growth and progesterone concentrations in cyclic and anestrous suckling Nelore cows (Bos indicus) treated with progesterone,equine chorionic gonadotropin,or temporary calf removal

The objective of this study was to investigate the effects of eCG and temporary calf removal (TCR) associated with progesterone (P4) treatment on the dynamics of follicular growth, CL size, and P4 concentrations in cyclic (n = 36) and anestrous (n = 30) Nelore cows. Cyclic (C) and anestrous (A) cows were divided into three groups. The control group received 2 mg of estradiol benzoate via intramuscular (IM) injection and an intravaginal device containing 1.9 g of P4 on Day 0. On Day 8, the device was removed, and the animals received 12.5 mg of dinoprost tromethamine IM. After 24 hours, the animals received 1 mg of estradiol benzoate IM. In the eCG group, cows received the same treatment described for the control group but also received 400 UI of eCG at the time of device removal. In the TCR group, calves were separated from the cows for 56 hours after device removal. Ultrasound exams were performed every 24 hours after device removal until the time of ovulation and 12 days after ovulation to measure the size of the CL. On the same day as the CL measurement, blood was collected to determine the plasma P4 level. Statistical analyses were performed with a significance level of P ≤ 0.05. In cyclic cows, the presence of the CL at the beginning of protocol resulted in a smaller follicle diameter at the time of device removal (7.4 ± 0.3 mm in cows with CL vs. 8.9 ± 0.4 mm in cows without CL; P = 0.03). All cows ovulated within 72 hours after device removal. Anestrous cows treated with eCG or TCR showed follicle diameter at fixed-timed artificial insemination (A-eCG 10.2 ± 0.3 and A-TCR 10.3 ± 0.5 mm) and follicular growth rate (A-eCG 1.5 ± 0.2 and A-TCR 1.3 ± 0.1 mm/day) similar to cyclic cows (C-eCG 11.0 ± 0.6 and C-TCR 12.0 ± 0.5 mm) and (C-eCG 1.4 ± 0.2 and C-TCR 1.6 ± 0.2 mm/day, respectively; P ≤ 0.05). Despite the similarities in CL size, the average P4 concentration was higher in the A-TCR (9.6 ± 1.4 ng/mL) than in the A-control (4.0 ± 1.0 ng/mL) and C-TCR (4.4 ± 1.0 ng/mL) groups (P < 0.05). From these results, we conclude that eCG treatment and TCR improved the fertility of anestrous cows by providing follicular growth rates and size of dominant follicles similar to cyclic cows. Additionally, TCR increases the plasma concentrations of P4 in anestrous cows.     

Cattle demographic history modelled from autosomal sequence variation

The phylogeography of cattle genetic variants has been extensively described and has informed the history of domestication. However, there remains a dearth of demographic models inferred from such data. Here, we describe sequence diversity at 37 000 bp sampled from 17 genes in cattle from Africa, Europe and India. Clearly distinct population histories are suggested between Bos indicus and Bos taurus, with the former displaying higher diversity statistics. We compare the unfolded site frequency spectra in each to those simulated using a diffusion approximation method and build a best-fitting model of past demography. This implies an earlier, possibly glaciation-induced population bottleneck in B. taurus ancestry with a later, possibly domestication-associated demographic constriction in B. indicus. Strikingly, the modelled indicine history also requires a majority secondary admixture from the South Asian aurochs, indicating a complex, more diffuse domestication process. This perhaps involved multiple domestications and/or introgression from wild oxen to domestic herds; the latter is plausible from archaeological evidence of contemporaneous wild and domestic remains across different regions of South Asia.     

Mitochondrial abundance and function in muscle from beef steers with divergent residual feed intakes

The objective of this study was to evaluate the relationship between muscle mitochondrial function and residual feed intake (RFI) in growing beef cattle. A 56-day feeding trial was conducted with 81 Angus × Hereford steers (initial BW = 378 ± 43 kg) from the University of California Sierra Foothills Research Station (Browns Valley, CA, USA). All steers were individually fed the same finishing ration (metabolizable energy = 3.28 Mcal/kg DM). Average daily gain (ADG), DM intake (DMI) and RFI were 1.82 ± 0.27, 8.89 ± 1.06 and 0.00 ± 0.55 kg/day, respectively. After the feeding trial, the steers were categorized into high, medium and low RFI groups. Low RFI steers consumed 13.6% less DM (P < 0.05) and had a 14.1% higher G : F ratio (P < 0.05) than the high RFI group. No differences between RFI groups were found in age, ADG or BW (P > 0.10). The most extreme individuals from the low and high RFI groups were selected to assess mitochondrial function (n = 5 low RFI and n = 6 high RFI). Mitochondrial respiration was measured using an oxygraph (Hansatech Instruments Ltd., Norfolk, UK). State 3 and State 4 respiration rates were similar between both groups (P > 0.10). Respiratory control ratios (RCRs, i.e., State 3 : State 4 oxygen uptakes) declined with animal age and were greater in low RFI steers (4.90) as compared to high RFI steers (4.26) when adjusted for age by analysis of covariance (P = 0.003). Mitochondrial complex II activity levels per gram of muscle were 42% greater in low RFI steers than in high RFI steers (P = 0.004). These data suggest that skeletal muscle mitochondria have greater reserve respiratory capacity and show greater coupling between respiration and phosphorylation in low RFI than in high RFI steers.     

Protein metabolism,body composition and oxygen consumption in young bulls divergent in residual feed intake offered two contrasting forage-based diets

Protein metabolism and body composition have been identified as major determinants of residual feed intake (RFI) in beef cattle fed high-starch fattening diets. This study aimed to evaluate if these two identified RFI determinants in beef cattle are the same across two contrasting silage-based diets. During two consecutive years, an 84-day feed efficiency test (Test A) immediately followed by a second 112-day feed efficiency test (Test B) was carried out using a total of 100 animals offered either one of two diets (either corn silage- or grass silage-based) over 196 days. At the end of Test A, the 32 animals most divergent for RFI (16 extreme RFI animals per diet, eight low RFI and eight high RFI) were identified and evaluated during Test B for their i) N use efficiency (NUE; N retention/N intake) calculated either from a 10-d nitrogen balance trial or from estimations based on body composition changes occurring during the whole experiment (Test A and Test B; 196 days), ii) carcass and whole-body protein turnover rates analysed through the 3-methyl-histidine urinary excretion and the N isotopic turnover rates of urine, respectively, and iii) body composition measured at the slaughterhouse at the end of Test B. Oxygen consumption was measured during Test B for the 100 animals by two GreenFeed systems. Irrespective of the diet, efficient RFI animals tended (P = 0.08) to improve their NUE when N retention was estimated for 196 days or when considering their lower urinary urea-N to total N ratio (P = 0.03). In contrast, NUE calculated during the 10-d N balance showed no differences (P = 0.65) across RFI groups suggesting that this method may not be suitable to capture small NUE differences. Efficient RFI individuals presented higher dressing percentage and muscle deposition in the carcass (P = 0.003) but lighter rumen (P = 0.001), and a trend for lower oxygen consumption (P = 0.08) than inefficient RFI animals irrespective of the diet. Lower protein degradation rates of skeletal muscle and lower protein synthesis rates of plasma proteins were found in efficient RFI cattle but only with the corn silage-based diet (RFI × Diet; P = 0.02). The higher insulinaemia associated with the corn silage-based diet (P = 0.001) seemed to be a key metabolic feature explaining the positive association between protein turnover and RFI only in this diet. Feed N was more efficiently used for growth by efficient RFI animals regardless of the diet but lower protein turnover rates in efficient RFI animals were only observed with corn silage-based diets.     

How do SNP ascertainment schemes and population demographics affect inferences about population history?

Background

The selection of variable sites for inclusion in genomic analyses can influence results, especially when exemplar populations are used to determine polymorphic sites. We tested the impact of ascertainment bias on the inference of population genetic parameters using empirical and simulated data representing the three major continental groups of cattle: European, African, and Indian. We simulated data under three demographic models. Each simulated data set was subjected to three ascertainment schemes: (I) random selection; (II) geographically biased selection; and (III) selection biased toward loci polymorphic in multiple groups. Empirical data comprised samples of 25 individuals representing each continental group. These cattle were genotyped for 47,506 loci from the bovine 50 K SNP panel. We compared the inference of population histories for the empirical and simulated data sets across different ascertainment conditions using F_ST and principal components analysis (PCA).

Results

Bias toward shared polymorphism across continental groups is apparent in the empirical SNP data. Bias toward uneven levels of within-group polymorphism decreases estimates of F_ST between groups. Subpopulation-biased selection of SNPs changes the weighting of principal component axes and can affect inferences about proportions of admixture and population histories using PCA. PCA-based inferences of population relationships are largely congruent across types of ascertainment bias, even when ascertainment bias is strong.

Conclusions

Analyses of ascertainment bias in genomic data have largely been conducted on human data. As genomic analyses are being applied to non-model organisms, and across taxa with deeper divergences, care must be taken to consider the potential for bias in ascertainment of variation to affect inferences. Estimates of F_ST, time of separation, and population divergence as estimated by principal components analysis can be misleading if this bias is not taken into account.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1469-5) contains supplementary material, which is available to authorized users.     

When and how did Bos indicus introgress into Mongolian cattle?

The Mongolian cattle are one of the most widespread breeds with strictly Bos taurus morphological features in northern China. In our current study, we presented a diversity of mitochondrial DNA (mtDNA) D-loop region and Y chromosome SNP markers in 25 male and 8 female samples of Mongolian cattle from the Xinjiang Uygur autonomous region in Western China, and detected 21 B. taurus and four Bos indicus (zebu) mtDNA haplotypes. Among four B. indicus mtDNA haplotypes, two haplotypes belonged to I1 haplogroup and the remaining two haplotypes belonged to I2 haplogroup. In contrast, all 25 male Mongolian cattle samples revealed B. taurus Y chromosome haplotype and no B. indicus haplotypes were found. Historical and archeological records indicate that B. taurus was introduced to Xinjiang during the second millennium BC and B. indicus appeared in this region by the second century AD. The two types of cattle coexisted for many centuries in Xinjiang, as depicted in clay and wooden figurines unearthed in the Astana cemetery in Turfan (3rd–8th century AD). Multiple lines of evidence suggest that the earliest B. indicus introgression in the Mongolian cattle may have occurred during the 2nd–7th centuries AD through the Silk Road around the Xinjiang region. This conclusion differs from the previous hypothesis that zebu introgression to Mongolian cattle happened during the Mongol Empire era in the 13th century.     

Ovum pick up, in vitro embryo production, and pregnancy rates from a large-scale commercial program using Nelore cattle (Bos indicus) donors

The objective was to clarify in vitro production of bovine embryos in Brazil. Data from 656 ovum pick-up/in vitro production (OPU/IVP) procedures, performed on 317 Nelore (Bos indicus) donors, without hormone stimulation or control of ovarian follicular waves, were analysed. Donors were subjected to OPU from one to nine times (no specific schedule), with < 15 d between consecutive procedures. There were 20,848 oocytes, of which 15,747 (75.53%) were considered viable, 5,446 embryos were obtained, 5,398 embryos were immediately transferred, resulting in 1,974 pregnancies (36.57%) at Day 30 and 1,788 (33.12%) pregnancies at Day 60. The average number of total and viable oocytes produced per OPU session was (mean ± SEM) 30.84 ± 0.88 and 23.35 ± 0.7 (average of 8.1 ± 0.3 embryos and 3.0 ± 0.1 pregnancies per OPU-IVP procedure). Since oocyte production varied widely among donor, they were designated as very high, high, intermediate, and low, with 58.94 ± 2.04, 32.61 ± 0.50, 22.13 ± 0.50, and 10.26 ± 0.57 oocytes, respectively, produced by 78, 80, 79, and 80 donors. The number of viable oocytes recovered ranged from 0 to 128; since donors with numerous viable oocytes produced many viable embryos and pregnancies, oocyte production was useful for donor selection. However, there was no significant effect of the number of OPU sessions per donor on mean numbers of oocytes produced. In conclusion, we confirmed field reports of high oocyte production by some Nelore donors and demonstrated individual variation in oocyte yield, which was associated with embryo production and pregnancy rates.     

Predicting feed intake using modelling based on feeding behaviour in finishing beef steers

Current techniques for measuring feed intake in housed cattle are both expensive and time-consuming making them unsuitable for use on commercial farms. Estimates of individual animal intake are required for assessing production efficiency. The aim of this study was to predict individual animal intake using parameters that can be easily obtained on commercial farms including feeding behaviour, liveweight and age. In total, 80 steers were used, and each steer was allocated to one of two diets (40 per diet) which consisted of (g/kg; DM) forage to concentrate ratios of either 494:506 (MIXED) or 80:920 (CONC). Individual daily fresh weight intakes (FWI; kg/day) were recorded for each animal using 32 electronic feeders over a 56-day period, and individual DM intakes (DMI; kg/day) subsequently calculated. Individual feeding behaviour variables were calculated for each day of the measurement period from the electronic feeders and included: total number of visits to the feeder, total time spent at the feeder (TOTFEEDTIME), total time where feed was consumed (TIMEWITHFEED) and average length of time during each visit to the feeder. These feeding behaviour variables were chosen due to ease of obtaining from accelerometers. Four modelling techniques to predict individual animal intake were examined, based on (i) individual animal TOTFEEDTIME relative expressed as a proportion of the dietary group (GRP) and total GRP intake, (ii) multiple linear regression (REG) (iii) random forests (RF) and (iv) support vector regressor (SVR). Each model was used to predict CONC and MIXED diets separately, giving eight prediction models, (i) GRP_CONC, (ii) GRP_MIXED, (iii) REG_CONC, (iv) REG_MIXED, (v) RF_CONC, (vi) RF_MIXED, (vii) SVR_CONC and (viii) SVR_MIXED. Each model was tested on FWI and DMI. Model performance was assessed using repeated measures correlations (R²_RM) to capture the repeated nature of daily intakes compared with standard R², RMSE and mean absolute error (MAE). REG, RF and SVR models predicted FWI with R²_RM = 0.1–0.36, RMSE = 1.51–2.96 kg and MAE = 1.19–2.49 kg, and DMI with R²_RM = 0.13–0.19, RMSE = 1.15–1.61 kg and MAE = 0.9–1.28 kg. The GRP models predicted FWI with R²_RM = 0.42–0.49, RMSE = 2.76–3.88 kg and MAE = 2.46–3.47 kg, and DMI with R²_RM = 0.32–0.44, RMSE = 0.32–0.44 kg, MAE = 1.55–2.22 kg. Whilst more simplistic GRP models showed higher R²_RM than regression and machine learning techniques, these models had larger errors, likely due to individual feeding patterns not being captured. Although regression and machine learning techniques produced lower errors associated with individual intakes, overall precision of prediction was too low for practical use.     

EEF1A1 transcription cofactor gene polymorphism is associated with muscle gene expression and residual feed intake in Nelore cattle

Mammalian Genome - Cis-acting effects of noncoding variants on gene expression and regulatory molecules constitute a significant factor for phenotypic variation in complex traits. To provide new...     

Non-synonymous mutations mapped to chromosome X associated with andrological and growth traits in beef cattle

Background

Previous genome-wide association analyses identified QTL regions in the X chromosome for percentage of normal sperm and scrotal circumference in Brahman and Tropical Composite cattle. These traits are important to be studied because they are indicators of male fertility and are correlated with female sexual precocity and reproductive longevity. The aim was to investigate candidate genes in these regions and to identify putative causative mutations that influence these traits. In addition, we tested the identified mutations for female fertility and growth traits.

Results

Using a combination of bioinformatics and molecular assay technology, twelve non-synonymous SNPs in eleven genes were genotyped in a cattle population. Three and nine SNPs explained more than 1% of the additive genetic variance for percentage of normal sperm and scrotal circumference, respectively. The SNPs that had a major influence in percentage of normal sperm were mapped to LOC100138021 and TAF7L genes; and in TEX11 and AR genes for scrotal circumference. One SNP in TEX11 was explained ~13% of the additive genetic variance for scrotal circumference at 12 months. The tested SNP were also associated with weight measurements, but not with female fertility traits.

Conclusions

The strong association of SNPs located in X chromosome genes with male fertility traits validates the QTL. The implicated genes became good candidates to be used for genetic evaluation, without detrimentally influencing female fertility traits.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1595-0) contains supplementary material, which is available to authorized users.     

Review: divergent selection for residual feed intake in the growing pig

This review summarizes the results from the INRA (Institut National de la Recherche Agronomique) divergent selection experiment on residual feed intake (RFI) in growing Large White pigs during nine generations of selection. It discusses the remaining challenges and perspectives for the improvement of feed efficiency in growing pigs. The impacts on growing pigs raised under standard conditions and in alternative situations such as heat stress, inflammatory challenges or lactation have been studied. After nine generations of selection, the divergent selection for RFI led to highly significant (P<0.001) line differences for RFI (−165 g/day in the low RFI (LRFI) line compared with high RFI line) and daily feed intake (−270 g/day). Low responses were observed on growth rate (−12.8 g/day, P<0.05) and body composition (+0.9 mm backfat thickness, P=0.57; −2.64% lean meat content, P<0.001) with a marked response on feed conversion ratio (−0.32 kg feed/kg gain, P<0.001). Reduced ultimate pH and increased lightness of the meat (P<0.001) were observed in LRFI pigs with minor impact on the sensory quality of the meat. These changes in meat quality were associated with changes of the muscular energy metabolism. Reduced maintenance energy requirements (−10% after five generations of selection) and activity (−21% of time standing after six generations of selection) of LRFI pigs greatly contributed to the gain in energy efficiency. However, the impact of selection for RFI on the protein metabolism of the pig remains unclear. Digestibility of energy and nutrients was not affected by selection, neither for pigs fed conventional diets nor for pigs fed high-fibre diets. A significant improvement of digestive efficiency could likely be achieved by selecting pigs on fibre diets. No convincing genetic or blood biomarker has been identified for explaining the differences in RFI, suggesting that pigs have various ways to achieve an efficient use of feed. No deleterious impact of the selection on the sow reproduction performance was observed. The resource allocation theory states that low RFI may reduce the ability to cope with stressors, via the reduction of a buffer compartment dedicated to responses to stress. None of the experiments focussed on the response of pigs to stress or challenges could confirm this theory. Understanding the relationships between RFI and responses to stress and energy demanding processes, as such immunity and lactation, remains a major challenge for a better understanding of the underlying biological mechanisms of the trait and to reconcile the experimental results with the resource allocation theory.     

Mitochondrial abundance and function in skeletal muscle and liver from Simmental beef cattle divergent for residual feed intake

Cellular mitochondrial function has been suggested to contribute to variation in feed efficiency (FE) among animals. The objective of this study was to determine mitochondrial abundance and activities of various mitochondrial respiratory chain complexes (complex I (CI) to complex IV (CIV)) in liver and muscle tissue from beef cattle phenotypically divergent for residual feed intake (RFI), a measure of FE. Individual DM intake (DMI) and growth were measured in purebred Simmental heifers (n = 24) and bulls (n = 28) with an initial mean BW (SD) of 372 kg (39.6) and 387 kg (50.6), respectively. All animals were offered concentrates ad libitum and 3 kg of grass silage daily, and feed intake was recorded for 70 days. Residuals of the regression of DMI on average daily gain (ADG), mid-test BW^0.75 and backfat (BF), using all animals, were used to compute individual RFI coefficients. Animals were ranked within sex, by RFI into high (inefficient; top third of the population), medium (middle third of population) and low (efficient; bottom third of the population) terciles. Statistical analysis was carried out using the MIXED procedure of SAS v 9.3. Overall mean ADG (SD) and daily DMI (SD) for heifers were 1.2 (0.4) and 9.1 (0.5) kg, respectively, and for bulls were 1.8 (0.3) and 9.5 (1.02) kg, respectively. Heifers and bulls ranked as high RFI consumed 10% and 15% more (P < 0.05), respectively, than their low RFI counterparts. There was no effect of RFI on mitochondrial abundance in either liver or muscle (P > 0.05). An RFI × sex interaction was apparent for CI activity in muscle. High RFI animals had an increased activity (P < 0.05) of CIV in liver tissue compared to their low RFI counterparts; however, the relevance of that observation is not clear. Our data provide no clear evidence that cellular mitochondrial function within either skeletal muscle or hepatic tissue has an appreciable contributory role to overall variation in FE among beef cattle.     

Resynchronization with unknown pregnancy status using progestin-based timed artificial insemination protocol in beef cattle

Two experiments were designed to evaluate the use of resynchronization (RESYNCH) protocols using a progestin-based timed artificial insemination (TAI) protocol in beef cattle. In experiment 1, 475 cyclic Nelore heifers were resynchronized 22 days after the first TAI using two different inducers of new follicular wave emergence (estradiol benzoate [EB; n = 241] or GnRH [n = 234]) with the insertion of a norgestomet ear implant. At ear implant removal (7 days later), a pregnancy test was performed, and nonpregnant heifers received a dose of prostaglandin plus 0.5 mg of estradiol cypionate, with a timed insemination 48 hours later. The pregnancy rate after the first TAI was similar (P = 0.97) between treatments (EB [41.9%] vs. GnRH [41.5%]). However, EB-treated heifers (49.3%) had a greater (P = 0.04) pregnancy per AI (P/AI) after the resynchronization than the GnRH-treated heifers (37.2%). In experiment 2, the pregnancy loss in 664 zebu females (344 nonlactating cows and 320 cyclic heifers) between 30 and 60 days after resynchronization was evaluated. Females were randomly assigned to one of two groups (RESYNCH 22 days after the first TAI [n = 317] or submitted only to natural mating [NM; n = 347]). Females from the NM group were maintained with bulls from 15 to 30 days after the first TAI. The RESYNC-treated females were resynchronized 22 days after the first TAI using 1 mg of EB on the first day of the resynchronization, similar to experiment 1. No difference was found in P/AI (NM [57.1%] vs. RESYNC [61.5%]; P = 0.32) or pregnancy loss (NM [2.0%] vs. RESYNC [4.1%]; P = 0.21) after the first TAI. Moreover, the overall P/AI after the RESYNCH protocol was 47.5%. Thus, the administration of 1 mg of EB on day 22 after the first TAI, when the pregnancy status was undetermined, promotes a higher P/AI in the resynchronized TAI than the use of GnRH. Also, the administration of 1 mg of EB 22 days after the TAI did not affect the preestablished pregnancy.