Repeatability and heritability of response to superovulation in Holstein cows

The objective of this study was to estimate the relative effects of genetic and phenotypic factors on the efficacy and efficiency of superovulation for Holstein-Friesian cows reared in Brazil. A database, established by the Associacao Brasileira de Criadores de Bovinos da Raca Holandesa, consisting of a total of 5387 superovulations of 2941 cows distributed over 473 herds and sired by 690 bulls was used for the analysis. The records were analyzed by MTDFREML (Multiple Trait Derivative-Free Restricted Maximum Likelihood), using a repeatability animal model. The fixed effects included in the model were contemporaneous group (veterinarian, herd, year and season of the superovulation); number of semen doses; cow age; and superovulation order. The estimated repeatability of the number of the transferable embryos was low (0.13), and the estimated heritability was 0.03. These results indicate that environmental factors play a critical role in the response of a cow to a superovulation treatment. There is little evidence that future responses to superovulation by individual females can be predicted by previous treatment(s) or that superovulation response is an heritable trait.

Detecção de Leptospira pomona em sêmen bovino por eletroforese capilar fluorescente

This study was performed in order to evaluate the detection limit of PCR with fluorescent capillary electrophoresis for Leptospira pomona diagnosis in bovine semen. Negative bovine semen samples were artificially contaminated with Leptospira pomona (10 0 to 10 7 bacteria/ ml) and DNA was extracted by phenol/chloroform protocol. DNA fragments visualization was done by three electrophoresis methods: under UV light in 2 % agarose gel, silver staining 8% polyacrylamide gel and fluorescent capillary electrophoresis. The detection limit of capillary electrophoresis for Leptospira pomona was 10 2bacteria/ml. Under UV light, in 2 % agarose gel, the detection limit was of 10 4 bacteria/ ml while for silver stained 8 % polyacrylamide gel it was 10 2 bacteria/ ml. PCR with fluorescent capillary electrophoresis is an efficient and rapid diagnostic test for DNA detection of Leptospira in bovine semen and this can be an important tool for herd and semen sanitary control in artificial insemination centers.

The effect of type of vaginal insert and dose of pLH on embryo production, following fixed-time AI in a progestin-based superstimulatory protocol in Nelore cattle

The objective was to analyze and report field data focusing on the effect of type of progesterone-releasing vaginal insert and dose of pLH on embryo production, following a superstimulatory protocol involving fixed-time artificial insemination (FTAI) in Nelore cattle (Bos taurus indicus). Donor heifers and cows (n = 68; 136 superstimulations over 2 years) received an intravaginal, progesterone-releasing insert (CIDR® or DIB®, with 1.9 or 1.0 g progesterone, respectively) and 3-4 mg of estradiol benzoate (EB) i.m. at random stages of the estrous cycle. Five days later (designated Day 0), cattle were superstimulated with a total of 120-200 mg of pFSH (Folltropin-V®), given twice daily in decreasing doses from Days 0 to 3. All cattle received two luteolytic doses of PGF2α at 08:00 and 20:00 h on Day 2 and progesterone inserts were removed at 20:00 h on Day 3 (36 h after the first PGF2α injection). Ovulation was induced with pLH (Lutropin-V®, 12.5 or 25 mg, i.m.) at 08:00 h on Day 4 with FTAI 12, 24 and in several cases, 36 h later. Embryos were recovered on Days 11 or 12, graded and transferred to synchronous recipients. Overall, the mean (±S.E.M.) number of total ova/embryos (13.3 ± 0.8) and viable embryos (9.4 ± 0.6) and pregnancy rate (43.5%; 528/1213) did not differ among groups, but embryo viability rate (overall, 70.8%) was higher in donors with a DIB (72.3%) than a CIDR (68.3%, P = 0.007). In conclusion, the administration of pLH 12 h after progesterone removal in a progestin-based superstimulatory protocol facilitated fixed-time AI in Nelore donors, with embryo production, embryo viability and pregnancy rates after embryo transfer, comparable to published results where estrus detection and AI was done. Results suggested a possible alternative, which would eliminate the need for estrus detection in donors. © 2006 Elsevier Inc. All rights reserved.

LH surge in Nelore cows (Bos indicus), after induced estrus or after ovarian superestimulation

Considering that there is limited information about the preovulatory LH surge in Zebu cattle (Bos indicus), the purpose of the present work was to assess the LH surge in Nelore cows during the estrous cycle and after ovarian superestimulation of ovarian follicular development with FSH. This information is particularly important to improve superovulatory protocols associated with fixed-time artificial insemination. Nelore cows (n = 12) had their estrus synchronized with an intravaginal device containing progesterone (CIDR-B ®) associated with estradiol benzoate administration (EB, 2.5 mg, i.m., Day 0). Eight days later all animals were treated with PGF2α (Day 8) in the morning (8:00 h) and at night, when CIDR devices were removed (20:00 h). Starting 38 h after the first PGF2α injection, blood sampling and ovarian ultrasonography took place every 4 h, during 37 consecutive hours. Frequent handling may have resulted in a stress-induced suppression of LH secretion resulting in only 3 of 12 cows having ovulations at 46.7 ± 4.9 and 72.3 ± 3.8 h, respectively, after removal of CIDR-B. Thirty days later, the same animals received the described hormonal treatment associated with FSH (Folltropin ®, total dose = 200 mg) administered twice a day, during 4 consecutive days, starting on Day 5. Thirty-six hours after the first injection of PGF2α, to minimize stress, only seven blood samples were collected at 4 h interval each, and ultrasonography was performed every 12 h until ovulation. In 11 of 12 cows (92%) the LH surge and ovulation were observed 34.6 ± 1.6 and 59.5 ± 1.9 h, respectively, after removal of progesterone source. The maximum values for LH in those animals were 19.0 ± 2.6 ng/ml (mean ± S.E.M.). It is concluded that, in Nelore cows submitted to a ovarian superstimulation protocol, the LH surge occurs approximately 35 h after removal of intravaginal device containing progesterone, and approximately 12 h before the LH surge observed after an induced estrus without ovarian superstimulation. © 2008 Elsevier B.V. All rights reserved.

Efeito do eCG ou benzoato de estradiol associado ao norgestomet na taxa de concepção de vacas de corte submetidas à IATF no pós-parto

This study aimed to compare conception rates at the postpartum period (PPP) in beef cows which were administered either Equine Chorionic Gonadotrophin (eCG) or Estradiol Benzoate (EB) after Norgestomet and submitted to fixed-time artificial insemination (FTAI). The hypothesis was that the administration of eCG or EB enhances the conception rate. Lactating Nelore cows (n=138) and Brangus (n = 63), between 26 and 118 days of post partum period (PPP) were divided into three homogeneous groups, and subdivided in two blocks, inside each group one of them with a PPP d'' 45 days (PPP1; n=107) and another with a PPP > 45 days (PPP2; n=94). All cows received an auricular implant containing 3mg Norgestomet (Crestar®), followed by the administration of 5mg Estradiol Valerate. The auricular implants were kept during 10 days. Cows received 1mL saline solution (n=68, Control Group) or 500IU eCG (Folligon®; n=67; eCG Group) or 1mg Estradiol Benzoate (Index, n=66; EB Group) 24 hours after the removal of the implant. FTAI was made 54 hours after the implant removal. The pregnancy diagnosis was carried through ultrasonography 30 days after FTAI. There was an interaction between treatments and PPP. In the PPP1, the conception rate was higher in the eCG Group than in the EB Group (47.22% vs. 15.38%; Pd''0.01). In the PPP2, the conception rates of the eCG and EB Groups were higher than in the Control Group (41.93%, 44.44% vs. 22.22%: Pd''0.01). It was concluded that in cows up to 45 days of PPP, the eCG associated with Norgestomet enhances the conception rates.

Use of LH or eCG in the last day of superestimulatory treatment

In the last decades several hormonal treatments to induce multiple ovulation and embryo transfer (MOET) have been developed. Tight control of the time of ovulation allowed the use of fixed-time artificial insemination (FTAI) in embryos donors, facilitating animal management. Although, protocols that allow FTAI have evolved and yield as much embryo as conventional protocols that requires estrus detection, substantial increase in viable embryo production has not been observed in superestimulated bovine cattle. The present mini-review put emphasis on superstimulatory protocols in which the last two doses of pFSH are replaced by eCG or LH. Recent results indicate that an extra LH stimulus (using eCG or LH), on the last day of P-36 superestimulatory treatment, seems to improve transferable embryo yield in both Bos taurus and Bos indicus cattle.

Reproductive technology in domestic carnivorous

Background: The delay in development of artificial reproduction techniques on carnivorous could be due to countless reasons, but the lack of commercial interest is probably the most important one. The majority of canines are small structures, canidae are extremely fertile and a great number of species are adapted to domestication or captivity. Finally, the canine gamete physiology presents a difficult adaptation of technology knowledge obtained from other species. Furthermore, domestic felines are animals of company and there is no interest in reproducing them in a large scale, as it has been observed in other domestic animals, however, besides of being a valuable model for the development of in vitro techniques, the domestic cat is also used as an embryo receptor for different species of small wild felines due to physiological similarities among them, in vitro embrionary development, Review: It was reviewed the main insights about the reproductive physiology in female dogs, in vitro oocytary maturation (IVM), pregnancy and conception rate with dogs' frozen/unfrozen semen and PIV in domestic cats. The majority of mammal oocytes restart meiosis spontaneously after ovulation and reaches MII in artificial environment; in an in vitro maturation system in bovines, around 90% of oocytes complete their maturation, although its development capacity can be reduced subsequently. The success of IVM in canidae have been limited, with maturation rate varying from 0 to 58%, usually around 20%. The greatest difficulties include oocyte quality, hormonal environment, protein supplementation, cumulus / oocyte cell interaction, donor breed and age, culture systems, oxygen tension, amino acids, growth factor and sequential means. The freezing process reduces the quality of the semen, firstly because it reduces the number of living sperms and secondly because freezing produces cell modifications that could alter the sperm motility, longevity, integrity of membranes and its fertilizing capacity. Conclusion: Nowadays, several researches are being performed with the aim of increasing viability after dogs' and cats' semen is unfrozen, using extenders, cryoprotectors, freezing and unfreezing curves, addition of antioxidant substances. The aim of this text is to inform about the improvements obtained on the artificial reproduction techniques, emphasizing the oocytary maturation in female dogs, semen cryopreservation and artificial insemination in domestic dogs and cats.

Use of bayesian inference to correlate in vitro embryo production and in vivo fertility in Zebu Bulls

The objective of this experiment was to test in vitro embryo production (IVP) as a tool to estimate fertility performance in zebu bulls using Bayesian inference statistics. Oocytes were matured and fertilized in vitro using sperm cells from three different Zebu bulls (V, T, and G). The three bulls presented similar results with regard to pronuclear formation and blastocyst formation rates. However, the cleavage rates were different between bulls. The estimated conception rates based on combined data of cleavage and blastocyst formation were very similar to the true conception rates observed for the same bulls after a fixed-time artificial insemination program. Moreover, even when we used cleavage rate data only or blastocyst formation data only, the estimated conception rates were still close to the true conception rates. We conclude that Bayesian inference is an effective statistical procedure to estimate in vivo bull fertility using data from IVP. © 2011 Mateus José Sudano et al.

How FTAI and FTET impact reproductive efficiency of Brazilian dairy herds

Background: Throughout dairy cows evolution, milk production was always the key point to select the superior animal. Currently, several evidences has shown that high milk production have intensively contributed to the decline of dairy cattle fertility. Beyond milk production, dairy cows have their reproductive performance impaired by another factors, heat stress and repeat-breeding. Methods like fixed time artificial insemination and embryo transfer were developed to minimize the effects of these factors, and improve dairy herds profitability. This review aims to show some key-point experiments conducted to improve the efficiency of the self-appointed protocols for artificial insemination and embryo transfer in Brazil, overcoming several reproductive problems. Our goal is to develop cheap and easy self-appointed programs that facilitate animal handling and maximize their reproductive outcomes all over the year. Review: Failure in estrus detection is the mainly limiting factor for the use of artificial insemination in high-production dairy herd. An excellent alternative to overcome the need of estrus detection is fixed time artificial insemination. Many protocols with and without the use of estradiol have been developed to that end. Among the protocols for fixed time artificial insemination without estradiol, DoubleOvsynch has been extensively used recently in American dairy herds. In Brazil, similar pregnancy rate was obtained compared to progesterone-estradiol based protocols for fixed time artificial insemination. Particularities of progesterone-estradiol based protocols as (1) new progesterone device or devices previously used for eight days; (2) different doses of eCG; and (3) the use of estradiol cypionate for fixed time artificial insemination have been studied in Brazil. The use of self-appointed artificial insemination also enabled the reduction of the interval calving-conception compared to cows inseminated following the standing estrus. Regarding the low fertility of repeat breeders and the effect of heat stress at early pregnancy, other methods like embryo transfer became important tools to enhance reproductive efficiency of Brazilian dairy herds. Protocols were also developed to allow fixed time embryo transfer, eliminating the need of estrus detection and improving the reproductive efficiency of lactating recipients. As well as described for fixed time artificial insemination treatments, there is a large variety of hormone combination for fixed time embryo transfer (with and without estradiol). An experiment conducted in Brazil demonstrated that protocols for fixed time embryo transfer without estradiol can be as good as with estradiol to synchronize high-producing Holstein recipients, essentially during summer. Particularities related to embryos cryopreservation, synchronization of the estrus cycle of donors and recipients and the site of embryo release into the uterine horn were also investigated. Greater conception rates were achieved when fresh embryos were transferred compared to frozen-thawed ones. Also, the tight synchronization between donor and recipient (same day of estrus) resulted more pregnancies than when recipients were one day later or in advantage in relation to donors. Moreover, the site of embryo release into the uterine horn (ipsilateral to the corpus luteum) had no effect on pregnancy rates after in vivo produced embryo transfer. Conclusion: Both fixed time artificial insemination and fixed time embryo transfer are important tools to improve reproductive efficiency of high-producing dairy cows. These biotechnologies help bypassing some of the greatest challenges of dairy cattle reproduction: the difficulties of estrus detection, and the low fertility associated to heat stress and repeat breeding.

Effect of superstimulatory treatments on the expression of genes related to ovulatory capacity, oocyte competence and embryo development in cattle.

Multiple ovulation (superovulation) and embryo transfer has been used extensively in cattle. In the past decade, superstimulatory treatment protocols that synchronise follicle growth and ovulation, allowing for improved donor management and fixed-time AI (FTAI), have been developed for zebu (Bos indicus) and European (Bos taurus) breeds of cattle. There is evidence that additional stimulus with LH (through the administration of exogenous LH or equine chorionic gonadotrophin (eCG)) on the last day of the superstimulatory treatment protocol, called the 'P-36 protocol' for FTAI, can increase embryo yield compared with conventional protocols that are based on the detection of oestrus. However, inconsistent results with the use of hormones that stimulate LH receptors (LHR) have prompted further studies on the roles of LH and its receptors in ovulatory capacity (acquisition of LHR in granulosa cells), oocyte competence and embryo quality in superstimulated cattle. Recent experiments have shown that superstimulation with FSH increases mRNA expression of LHR and angiotensin AT(2) receptors in granulosa cells of follicles >8 mm in diameter. In addition, FSH decreases mRNA expression of growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15) in oocytes, but increases the expression of both in cumulus cells, without diminishing the capacity of cumulus-oocyte complexes to generate blastocysts. Although these results indicate that superstimulation with FSH is not detrimental to oocyte competence, supplementary studies are warranted to investigate the effects of superstimulation on embryo quality and viability. In addition, experiments comparing the cellular and/or molecular effects of adding eCG to the P-36 treatment protocol are being conducted to elucidate the effects of superstimulatory protocols on the yield of viable embryos.

Progesterone-based strategies to induce ovulation in prepubertal Nellore heifers

Four experiments were conducted to evaluate hormonal strategies to induce ovulation in Nellore heifers. In experiment 1, heifers (N = 1039) received a controlled internal drug release (CIDR) of fourth use (CIDR-4) on Day -12 or no CIDR (CIDR-0). The CIDR was removed on Day 0 in the CIDR-4 treatment, and estrus detection and AI were performed from Days 1 to 7. On Day 8, heifers not detected in estrus were evaluated for CL presence and received the same treatment again, followed by estrus detection and AI from Days 21 to 27. All heifers in experiments 2 (N = 896), 3 (N = 839), and 4 (N = 948) received the CIDR-4 treatment on Day -12. In experiment 2, heifers were randomly assigned to a control group (no additional treatment) or to receive equine chorionic gonadotropin (eCG; 200 IU eCG im) on Day 0. In experiment 3, heifers received the same treatments as in experiment 2, or a treatment that included eCG and estradiol cypionate (ECP) (eCG+ECP; 200 IU im eCG plus 0.5 mg ECP im) on Day 0. In experiment 4, heifers received the treatments described in experiment 3 or only ECP (0.5 mg) on Day 0. In experiments 2 and 3, estrus detection and AI was performed from Days 1 to 7 and on Day 8, heifers not detected in estrus were evaluated for CL presence. In experiment 4, heifers were evaluated for presence of a CL between Days 10 and 14. In experiment 1 heifers treated with CIDR-4 had greater estrus detection, ovulation induction, and pregnancy rates than in the CIDR-0 group. In experiment 2, heifers treated with eCG had greater estrus detection, ovulation induction, and pregnancy rates in 7 days than heifers in the control group. In experiment 3, heifers treated with eCG+ECP had greater estrus detection, ovulation induction, and pregnancy rates than the control and eCG treatments. In experiment 4, ovulation induction was greater for heifers treated with eCG and eCG+ECP relative to control, but did not differ from the ECP treatment. In conclusion, the use of a CIDR of fourth use for 12 days and the addition of eCG and/or ECP at CIDR removal efficiently induced ovulation and increased pregnancy rates in prepubertal Nellore heifers. © 2013 Elsevier Inc.

Effects of vaccination against reproductive diseases on reproductive performance of beef cows submitted to fixed-timed AI in Brazilian cow-calf operations

The objectives were to assess incidence of pregnancy losses, associate this outcome with immunization programs against reproductive diseases, and evaluate the effects of vaccination against bovine herpesvirus-1 (BoHV-1), bovine viral diarrhea virus (BVDV), and Leptospira spp., on reproductive efficiency of Brazilian cow-calf operations. In experiment 1, 7614 lactating Nelore cows from 18 ranches were assigned to the same estrus synchronization and fixed-time AI protocol (ESFTAI; Days -11 to 0). Pregnancy status was determined with transrectal ultrasonography on Days 30 and 120 after AI. Pregnancy loss was deemed to have occurred when cows were pregnant on Day 30 but nonpregnant on Day 120. Incidence of pregnancy loss across all ranches was 4.1%; pregnancy losses were detected (P < 0.10) in 14 ranches but not detected (P > 0.11) in four ranches. Pregnancy loss was lower (P ≤ 0.02) in ranches that vaccinated against BoHV-1, BVDV, and Leptospira spp. compared with ranches that did not vaccinate, or only vaccinated against Leptospira spp. In experiments 2 and 3, lactating Nelore cows (N = 1950 and 2793, respectively) from ranches that did not have a history of vaccinating against reproductive diseases (experiment 2), or only vaccinated against Leptospira spp. (experiment 3), were assigned to the same ESFTAI used in experiment 1. Within each ranch, cows received (VAC) or not (CON) vaccination against BoHV-1, BVDV, and Leptospira spp. at the beginning of the ESFTAI (Day -11) and 30 days after (Day 41) AI. In experiment 2, VAC cows had greater (P ≤ 0.05) pregnancy rates compared with CON on Days 30 and 120. In experiments 2 and 3, pregnancy loss was reduced (P ≤ 0.03) in primiparous VAC cows compared with CON cohorts. In experiment 4, 367 primiparous, lactating Nelore cows previously vaccinated against Leptospira spp. were assigned to the same ESFTAI used in experiment 1. Cows received VAC, or the same vaccine 30 days before (Day -41) and at the beginning (Day -11) of the ESFTAI (PREVAC). Pregnancy rates on Days 30 and 120 were greater (P ≤ 0.05) in PREVAC cows compared with VAC cows. In conclusion, pregnancy losses affected reproductive and overall efficiency of Brazilian cow-calf operations, and might be directly associated with BoHV-1, BVDV, and Leptospira spp. infections. Hence, vaccinating cows against these pathogens, particularly when both doses are administered before fixed-time AI, improved reproductive performance in Brazilian cow-calf systems. © 2013 Elsevier Inc.

Ovarian response to porcine FSH in association with ablation-induced or spontaneous follicular wave development during the estrous cycle in crossbred and Brazilian Warmblood mares

The primary objective of this study was to examine the follicular and ovulatory responses following treatment with pFSH in association with ablation-induced or spontaneous follicular wave emergence or follicle deviation during diestrus in crossbred (Mangalarga × Arabian) and Brazilian Warmblood mares with a propensity for spontaneous multiple ovulations; secondary considerations were given to the collection of embryos In Experiment 1, crossbred mares were administered (im) saline (control, n= 7) or pFSH (25 mg) when the largest follicle of the ablation-induced follicular wave reached ≥13 mm (n= 7) or ≥20 mm (n= 7) or, after pre-treatment ovulation (Day 0) on Day 6 (n= 7) In Experiment 2, crossbred mares were administered (im) saline (control, n= 10) or a larger dose of pFSH (50 mg, n= 7) when the largest follicle of the ablation-induced follicular wave reached ≥13 mm In Experiment 3, Brazilian Warmblood mares were administered (im) saline (control, n= 7), pFSH (25 mg, n= 7 or 50 mg, n= 5) or EPE (12.5 mg, n= 7) as a positive control on Day 6 Ultrasonic technology was used to ablate all follicles ≥8 mm and to monitor follicular development and detect ovulation Treatment with pFSH or EPE was done twice daily until the largest follicle reached ≥32 mm; thereafter, hCG (2500 IU) was administered (iv) when the largest follicle reached ≥35 mm Artificial insemination was done 12 h after hCG and embryo collections were done 8 d after post-treatment ovulations In Experiments 1 and 2, treatment of crossbred mares with pFSH post-ablation in association with the expected time of wave emergence or follicle deviation did not (P> 0.05) enhance the follicular or ovulatory responses or collection of embryos compared to controls In Experiment 3, although the enhanced ovulatory response of mares to EPE at the expected time of spontaneous wave emergence was not different (P> 0.05) from controls, it was greater (P< 0.05) than the response to pFSH In conclusion, the novelty of using follicle ablation prior to pFSH treatment at the time of wave emergence or follicle deviation did not enhance the follicular or ovulatory responses or collection of embryos to treatment in crossbred mares In addition, the hypothesis that Brazilian Warmblood mares with a greater propensity for spontaneous multiple ovulations are as responsive to pFSH compared to EPE was not supported Thus, the combined experimental results of the present study continue to support the general consensus that pFSH is relatively ineffective for follicular superstimulation/superovulation in mares © 2012 Elsevier B.V.

Effects of vaccination against reproductive diseases on reproductive performance of lactating dairy cows submitted to AI

Four experiments evaluated the effects of vaccination against bovine herpesvirus-1 (BoHV-1), bovine viral diarrhea virus (BVDV), and Leptospira spp. on reproductive performance of lactating dairy cows without (experiments 1, 2, and 3) or with previous vaccination against these diseases (experiment 4). Cows were assigned to a fixed-time AI protocol (FTAI; d -11 to 0) in all experiments, as well as AI 12. h upon estrus detection in experiment 3. Pregnancy status was determined with transrectal ultrasonography on d 30 and 71 (d 60 for experiment 3) after AI. Pregnancy loss was considered in cows pregnant on d 30 but non-pregnant on the subsequent evaluation. In experiment 1, 853 cows received (VAC) or not (CON) vaccination against BoHV-1, BVDV, and Leptospira spp. at the beginning of the FTAI (d -11) and 30. d after AI. Pregnancy loss was reduced (P=0.03) in VAC cows compared with CON. In experiment 2, 287 cows received VAC or CON 30. d prior to (d -41) and at the beginning (d -11) of the FTAI. Pregnancy rates on d 30 and 71 were greater (P≤0.03) in VAC cows compared with CON. In experiment 3, 1680 cows with more than 28. d in milk were randomly assigned to receive VAC or CON with doses administered 14. d apart, and inseminated within 15-135. d after the second dose. Pregnancy rates on d 30 and 60 were greater (P≤0.02) in VAC cows compared with CON. In experiment 4, 820 cows received (REVAC) or not (CON) revaccination against BoHV-1, BVDV, and Leptospira spp. at the beginning of the FTAI protocol (d -11). Pregnancy rates and loss were similar (P≥0.54) between treatments. Hence, vaccinating naïve cows against BoHV-1, BVDV, and Leptospira spp. improved reproductive efficiency in dairy production systems, particularly when both doses were administered prior to AI. © 2013 Elsevier B.V.