Papilio glaucus L. (Lepidoptera) showed no costs for melanic versus yellow in adult size, oviposition preferences, fecundity, egg viability, larval survival/growth rates, cold stress tolerance, or postdiapause emergence times. Sexual selection  had been suggested to provide a balanced polymorphism in P. glaucus, but spermatophore counts in wild females and direct field tethering studies of size‐matched pairs of virgin females (dark and yellow), show that male preferences are random or frequency‐dependent from Florida to Michigan, providing no yellow counter‐advantages. Recent frequency declines of dark (melanic/mimetic) females in P. glaucus populations are shown in several major populations from Florida (27.3°N latitude) to Ohio (38.5° N). Summer temperatures have increased significantly at all these locations during this time (1999–2018), but whether dark morphs may be more vulnerable (in any stage) to such climate warming remains to be determined. Additional potential reasons for the frequency declines in mimetic females are discussed: (i) genetic introgression of Z‐linked melanism suppressor genes from P. canadensis (R & J) and the hybrid species, P. appalachiensis (Pavulaan & Wright), (ii) differential developmental incompatibilities, or Haldane effects, known to occur in hybrids, (iii) selection against intermediately melanic (“dusty”) females  which higher temperatures can cause.Polymorphisms for melanic form of insects may provide various selective advantages. However, melanic alleles may have significant/subtle pleiotrophic “costs.” Several potential pleiotrophic effects of the W (=Y)‐linked melanism gene in  The s+ allele for suppression of b+ appears at very high frequencies in P. canadensis populations while the Z‐linked s– “enabler” allele is in very high frequencies in P. glaucus populations  at which the dark glaucus female frequency quickly goes to zero  genotype might otherwise express  does not occur in Papilio glaucus have fascinated naturalists since Linneaus described the two forms originally as different species P. turnus, and P. glaucus  morph females of P. glaucus is a putative mimic of the distasteful Aristolochia‐feeding pipevine swallowtail, Battus philenor (L.) in the eastern half of the United States  was documented by Platt and Brower (Limenitis a. astyanax (Fabr.) has also been described  of d Brower   may be vastly underestimated  and yellow (tiger‐striped) s Kunte, . Also, is Kunte, . Such stP. glaucus males typically leave a single spermatophore that persists indefinitely, the number of matings and differences between yellow and dark females can be inferred  across most of butterfly's range to the northern limits, near 40–42°N latitude within 50–150 miles from the historical hybrid zone with the univoltine P. appalachiensis types on the warm side of the hybrid zone possess the Z‐linked enabler (s–) for melanism  has introgressed into some wild males of the 1999–2012 Vermont “Late flight” recombinant hybrids ‐linked  of insect melanism, as in Drosophila  were calculated for each of more than 2000 stations in northeastern United States . The base developmental threshold was calculated as the inverse of the time required to complete development when plotted against rearing temperatures  were examined for county of capture records at many University, State, Government and Public Museums . In contrast, at the northern edge of the range where dark females have been rare or nonexistent, very intense and sustained sampling has been made by our group and others . In fact, it is almost certain that most collectors in these regions look even more intensely for “rare” specimens, such as dark females ; n = 2652 for Levy Co. FL; n = 2761 for Clarke and Oglethorpe Co. GA; n = 333 for Rabun and Habersham Cos. GA and Macon Co. NC; and n = 983 for Gallia and Lawrence Cos. OH. Specific annual sample sizes are indicated in the captions of Figs. The key populations sampled for multiple decades for change in frequencies of dark females include; Highlands Co. FL lat 27.3°N), Levy Co. FL lat. 29.1°N) Clarke and Oglethorpe Cos. in northern GA lat 34.0°N); Rabun/Habersham Cos. GA (lat. 34.0°N), and Gallia & Lawrence Cos. OH lat. 38.5°N). The total number of females sampled for these locations were: °N, Levy .0°N; Rab°N. The tP. glaucus mothers from MI, WI, OH, IN, MO, VA, GA, FL, and for primary hybrids of dark and yellow females mated to P. canadensis males (Table Field‐collected (or hand‐paired) females were individually placed in multi‐choice oviposition arenas where they could choose individual leaves as the arenas rotated in front of a bank of 60 W lights , hop tree (Ptelea trifoliate L.), white ash (Fraxinus americana L.), black cherry (Prunus serotine Ehrh.), spicebush (Lindera benzoin L. Blume), Rhamnus spp. and quaking aspen (Populus tremuloides Michx.). Data are expressed as family means for dark and yellow females from several locations and/or years  conditions were compared for families from 21 dark and 18 yellow Dane Co. Wisconsin mothers (Table Neonate larvae were randomly and gently transferred (using fine hair brushes) onto different host plant species for survival and growth bioassays. Subsequent larval rearing to pupation was in controlled environment chambers . Neonate larvae were set up in a split‐brood design on tulip tree, black cherry, and quaking aspen leaves in groups of 1–3 per dish. Data are expressed as the mean of the families of dark versus yellow from each of two locations . These conditions included 4 d in mid‐January of cold stress at –21, –18, or –13 °C. In addition a 4‐d winter warming period of 20 °C for mid‐January was included for comparison with control pupae allowed to remain at 4 °C for the entire winter Table . StressePupae of Pennsylvania reared dark and yellow morph females were put in the usual diapause storage conditions at our lab (4 °C in darkness) and weighed at mid‐winter (December). Live weights of all pupae were taken on a macroanalytical balance (and a subsample was taken to determine water content by deep freezing and subsequent drying). After 6 months the pupae were removed from diapause conditions weighed live and sacrificed for dry weight determinations Table .Severe mid‐winter conditions were simulated by exposing diapausing pupae of dark and yellow morph females to 10‐, 20‐, 35‐d durations at –18 °C  compared to the control group (undisturbed diapausers). Spring emergences were allowed after 6 months and the times for development into eclosing adults from pupae were determined Table .P. glaucus of both color morphs, it had previously been proposed that the preference of wild males for the yellow (nonmimetic) morphs might negate the higher bird predation on the yellow morph, preventing the dark morph from going to fixation where the B. philenor model was most abundant  as one factor influencing the distribution/frequency of dark morph P. glaucus in areas where dark female frequency declines. Sites examined for differential mate preferences included Isabella Co, MI, Lawrence/Gallia Cos. OH, Highlands and Levy Cos. FL where the natural frequencies of dark morphs ranging from 0% to 90 %  females with field‐captured or lab males. The offspring of dark and yellow mothers paired with males of s Tables  and 14. s Tables .A very informative laboratory backcross family (#18006) was created by pairing a dark morph female and a hybrid (g × c) father. The mother produced 388 eggs, 286 neonate larvae, and 112 pupae (many larvae died in host suitability assessments). Of these pupae 20 females (17 dark and intermediate and 3 yellow) and 39 male adults developed and emerged under long‐day (18 : 6 h) lab conditions. Four males and four females  emerged in year 2, and one male and four females emerged in year 3 . Many recombinations of diagnostic traits were observed in these offspring (below), but one in particular is that, of the 20 direct developing  females, 19 had the Ldh‐100 allele (showing close linkage between od– and Ldh‐100). Of these 20 females, 17 were dark or intermediate . Previously, only yellow morph females had been reported south of Highlands Co. Florida , and loss of mating/ reproductive potential (if too late). In thermally constrained regions , hop tree (Ptelea trifoliata of Rutaceae), white ash (Fraxinus americana of Oleaceae), black cherry (Prunus serotina of Rosaceae), spicebush (Lindera benzoin of Lauraceae), Rhamnus spp. (Rhamnaceae), and quaking aspen . The rank order of preferences was also the same across all locations  offspring of interspecific crosses using dark and yellow morph P. glaucus mothers from PA with P. canadensis fathers both exhibit excellent survival and growth, with those from dark mothers appearing to have a slightly higher survival on all three hosts, although with only four families, the differences did not reach statistical significance (Table The ability of neonate larvae to survive on tulip tree (TT), black cherry (BC) and quaking aspen (QA) do not differ between offspring of different color morphs of ce Table . The totce Table .P. glaucus and P. canadensis , where the yellow female frequency was 55%–70%; and P. glaucus. males preferred dark morphs for 76% of copulations in Ohio where population frequency was 75%–85% dark females; Table When offered field‐tethered pairs of size‐matched virgin females of both yellow and dark morphs, wild male mating preferences were basically frequency‐dependent . These hybrid zone males even preferred heterospecific yellow P. glaucus females over P. canadensis females of their own species  failed to show differences as the population ages , which was not different than wild dark females with 1.40 (n = 76).Spermatophores inside females sampled in Levy Co. Florida during these periods ranged from 1.38 to 2.60 for dark and 1.33 to 2.0 for yellow Tables  and 12, P. glaucus  size has remained constant during this recent 15‐year warming period across all latitudes for P. glaucus female had ever been reported from Mississippi or Alabama , suggest that a decline in dark frequencies (or increased yellow morph frequencies) may be geographically extensive  and the dark (mimetic) P. glaucus. A similarly very close geographic correspondence of the B. philenor model and the mimetic form of Limenitis has been recently documented  than dark P. glaucus females East of the Great Lakes region , A. tomentosa Sims, A. clematitis L., and A. serpentaria L.) suggests that the northern limits of plants clearly correlate with that of the Aristolochiaceae‐specialized butterfly, B. philenor, from Missouri to New England. Western species of Aristolochia   Figs. .Papilio, the extent of general bird predation (or lizard or invertebrate predation) has not been clearly demonstrated. Some preliminary experimental analyses of field predation were conducted using dark and yellow tethered females of P. glaucus. In an early‐afternoon assessments of Anolis carolinensis lizard predation in Highlands Co. Florida .While blue jays  were made in 2006 (and n = 57 pairs in 2008 at Levy Co.) using tethered pairs of dark and yellow females. As with the mate preference tethering (above), females were tied around the thorax between the forewings and hindwings with black thread so that they were able to fly on this tether and choose the resting site near where the thread was attached .Early morning bird predation assessments . The total numbers of tethered individuals in Florida missing (eaten) over this 3‐d period were eight yellow morphs and seven dark morphs . Again the predators did not appear to discriminate between the morph colors. Of 68 total pairs tethered out in Florida, missing or dead females were observed in 11 yellow and 10 dark females.In Florida, two types of tethering presentations were used in two years of evaluations. One was a paired tethering . The other was with equal numbers of dark and yellow tethered individually along two sides of the road (butterfly flyway). In 2006, six adjacent pairs (1.5 m apart) of yellow and dark morphs were tethered for one night and morning at the Levy Co. site, and three dark and three yellow morphs were eaten. Although preliminary, these results do show that dark morphs are not immune from predation. Using “pairs” on opposite sides of the road (approximately 10 m apart) in Levy Co. Florida in the spring of 2008 additional replications of tethered color morphs were made , and 1 was dead  on the tether. Of the 20 dark morph P. glaucus females, 15 were totally missing, 4 alive, and 1 dead on the tether.Butterflies tethered out in northern Michigan for two evenings  suffered intense predation, were almost completely eaten, damaged severely or totally removed by the end of the 48 h. Of the 20 yellow morph Papilio were not examined in these studies  in our interspecific primary (F‐1) hybrids were evident in the skewed sex‐ratios of crosses with a P. canadensis father (g × c), but not the reciprocal hybrid cross with P. glaucus males  than daughters (11 families) in families with P. canadensis fathers Table et al., P. glaucus  entered a permanent diapause and died  were detected for a variety of life history factors.The potential physiological and ecological “costs” for melanism have been shown to be significant for some Lepidoptera  and 4 d of warming stress (+20 °C) showed no differences in the pupal weight loss and survival of pupae from yellow mothers or dark mothers. This was observed both in families derived from the field in southeastern Pennsylvania (Lancaster Co.) and northern Georgia (Clarke Co.). However, the Georgia pupae from both color morphs exhibited high mortality at –21 °C (60%–80%) compared to those from Pennsylvania  cold stress . Slightly skewed flight peaks for the model and mimics nevertheless suggest rarity for the model and fail to explain changes in dark morph frequencies of tiger swallowtail females in Levy County, FL .Although a few stray dark morph Battus philenor  hybrids are hypothesized to be largely X‐linked. Sexually selected traits are often sex‐linked  males. However, such genetic factors may play key roles in hybrid introgressiuon, and hybrid speciation (see below).Mating “attractiveness” of female  flights in univoltine obligate diapausers . When such offspring are expressed as a proportion of females, relative to males, with the same two paternally inherited allozyme alleles , the four recombinant backcross X‐linked allozyme genotypes clearly show a shortage of females in the eight backcross families ‐chromosome between Ldh and Pgd (or separate regions near each locus) contains the genes that disrupts pupal development in combination with P. glaucus genes . Diapause  and mimicry  genes may serve as potential causes of hybrid inviability in Tiger Swallowtails: If W‐linked b+ dark allele in P. glaucus coevolved closely with its Z‐linked enabler (s–) genes, then it may not function properly with homologous genes from other species (including P. canadensis). The Z‐linked suppressor gene (s+) in P. canadensis may suppress dark expression, but may also cause other developmental dysfunction involving the P. glaucus Y‐chromosome .These data suggest that either a region of While the Haldane Effect on diapausing female pupae (permanent diapause = death) is not simply a result of the negative pleiotropic effects of possessing the b+ gene for melanism, different causes may exist regarding “Haldane effects” for sterility versus inviability  contains mostly P. canadensis‐derived genome , which is close to the 6PGD allozyme locus, has shown up in the hybrid species  seen in the cooler side of the hybrid zone has been suggested  with negative impact on developmental rates  were detected in many important life history traits described above, there are many other potential pleiotriophic costs (or perhaps benefits) of melanism that have not yet been studied. For example, one very intriguing concept is that the melanic forms of Lepidoptera may have higher parasitoid resistance  individuals  has been documented at several long‐term study populations in Highlands County of Florida, Levy County of Florida, Clarke & Oglethorpe Counties in Georgia, adjacent counties in northern Georgia (Rabun & Habersham) and Macon County North Carolina, and in southern Ohio . Other southern areas that had been essentially all dark morphs have recently experienced an increase in yellow morphs. For example, in 1999 several yellow females of P. glaucus were reported in both the June and the August flights in northern Mississippi  morph females of P. glaucus females of being all dark (possessing both the W‐linked melanism gene (b+) and the Z‐linked enabler gene (s–)] instead of being the tiger‐striped yellow morph were not evident in any of the traits examined here). These include: 1) adult size, 2) fecundity, 3) egg viability, 4) larval survival, 5) larval growth rates, 6) total developmental durations, 7) short‐term mid‐winter thermal stresses (warm and cold) on diapausing pupae, 8) postdiapause pupal development rates and thresholds, and 9) oviposition preferences in multichoice arrays. Other, unknown pleiotrophic effects that might reduce fitness of the dark morph may be involved and should be investigated .Several potential factors potentially contributing to these dark morph frequency declines across eastern North America were investigated here, including ecological, physiological, behavioral, and genetic selection pressures. However, the potential pleiotrophic costs to P. glaucus, it has been shown that males do not always favor yellow females in their courtship and matings. Evidence from spermatophore counts in wild females and direct mating behavior using tethered dark and yellow female pairs was reviewed and expanded. The results clearly suggest that the mating behavior by P. glaucus males with the two color morphs is random , making it highly unlikely that it would be responsible for the female frequency changes.While male mating preferences for yellow morph females was previously suggested as a possible factor resulting in a balanced polymorphism with the advantage of dark mimetic females of et al., et al., et al., et al., P. glaucus dark morph in the 1950s but not supported by the higher frequencies (30%–50%) from 1960 to 1987  in females appears to move more slowly than most traits on the Z‐chromosome which are shown to extensively recombine and introgress via interspecific hybridization  species has been postulated as a major selection pressure, and dark morph frequencies have been the highest in such areas where the model is most abundant  using tethered dark and yellow morph females in the field suggest that differential predation rates on yellow and dark females may not exist in Florida or Michigan (near the dark range limits). However, studies have not been done in the latitudes in between where the model abundance is highest. Nonavian predators such as spiders, mantids and Anole lizards also appear to be nondiscriminatory between dark and yellow morph P. glaucus, although increased replication is needed before any rigorous conclusions about potential mimic (or even model) avoidance by these predators can be evaluated properly.The ecological (mimetic) advantages of dark morph females in regions with high frequencies of the  Brower, . The geoP. glaucus in eastern North America remains an interesting ecological and evolutionary phenomenon. The recent discovery of this color dimorphism also occurring in the recombinant homoploid hybrid species  in the area where the parental species (P. canadensis and P. glaucus) overlap provides unique opportunities for study of genetic introgression, recombinant hybrid divergence, homoploid hybrid speciation, and evolutionary genomics •Swenson, N.G. and Howard, D.J. (2004)3.The following changes should be made to the text:•On page 11, column 1, in the heading ‘Updated distribution records for dark morph females ’, 1997–2014 has been amended to 1997–2018.•n” should be added in column 2.In Table •et al., 2013 has been corrected to Dubovskiy et al., 2013, and DOI has been amended to ‘280, 10.1098/rspb.2013.0584’.In the references list, Dubovshiy •et al., 2003’ has been amended to ‘Fordyce & Nice, 2003’.On page 18, column 2, line 7, the citation ‘Fordyce [Corrections added on 28 February 2019, after first online publication:


As a result of its complex histological structure, regeneration patterns of grey and white matter are quite different in the spinal cord. Therefore, tissue engineering scaffolds for repairing spinal cord injury must be able to adapt to varying neural regeneration patterns. The aim of the present study was to improve a previously reported spinal cord‐mimicking partition‐type scaffold by adding microchannels on a single tubular wall along its longitudinal axis, thus integrating the two architectures of a single H‐shaped central tube and many microchannels. Next, the integrated scaffold was loaded with bone marrow stromal cells (BMSCs) and transplanted to bridge the 5‐mm defect of a complete transverse lesion in the thoracic spinal cord of rats. Subsequently, effects on nerve regeneration, locomotion function recovery, and early neuroprotection were observed. After 1 year of repair, the integrated scaffold could guide the regeneration of axons appearing in the debris of degraded microchannels, especially serotonin receptor 1A receptor‐positive axonal tracts, which were relatively orderly arranged. Moreover, a network of nerve fibres was present, and a few BMSCs expressed neuronal markers in tubular lumens. Functionally, electrophysiological and locomotor functions of rats were partially recovered. In addition, we found that BMSCs could protect neurons and oligodendrocytes from apoptosis during the early stage of implantation. Taken together, our results demonstrate the potential of this novel integrated scaffold loaded with BMSCs to promote spinal cord regeneration through mechanical guidance and neuroprotective mechanisms. Eventually, these processes result in the loss of sensation, paralysis, loss of bladder and bowel control, and sexual dysfunction , multiple channels, numerous microchannels, and cylindrical sponges, have been reported to guide the extension of regenerative axons , scaffolds were utilized to bridge a 5‐mm‐long defect in rat thoracic spinal cords. The effects of repair were assessed over the course of 1 year using behavioural, electrophysiological, and histological methods. In addition, apoptosis of neurons and glial cells was observed to examine the neuroprotective role of BMSCs.22.14 D and deacetylation degree of 92.3% was dissolved in 2% acetic acid  at a ratio of 1 g per 20 ml, followed by addition of the same weight of chitin . The gel mixture was injected into a −20°C stainless steel casting mould for 2.5 hr. After removal of the outer part of the mould, scaffolds with mould core were immersed in 10% NaOH  for 2 hr. Following rinsing with purified water  to a neutral pH, scaffolds were frozen and dried at −40°C and 0.02 Pa for 1 hr using a freeze dryer . After removal of the mould core, scaffolds cast from different moulds were formed into ST with “O” shape lumen  used in this work are described in detail in 2.3All experimental procedures and animal care were by the guidelines of the Institutional Animal Care and Use Committee of Nantong University and approved ethically by the Administration Committee of Experimental Animals of Jiangsu Province, China.n = 15), ST/MC (n = 27), ST with GFP‐BMSCs , ST/MC with GFP‐BMSCs , SCI without graft as a negative control , and laminectomy only . In ST and ST/MC groups, 5‐mm scaffolds were used to bridge the spinal cord defect, followed by injection with 10 μl of phosphate‐buffered saline (PBS) into the lumen. In ST + BMSCs and ST/MC + BMSCs groups, scaffolds were implanted to bridge the gap as above, followed by injection with 10 μl of BMSC suspension (106 cells/ml in PBS) into the lumen. In addition, 10 μl of a pan‐caspase inhibitor, z‐VAD‐fmk , was injected into ST/MC as a control group (n = 12) for the apoptosis assay. Finally, the muscle and skin were stitched together layer by layer. Postoperatively, buprenorphine‐HCl (0.03–0.05 mg/kg) was subcutaneously administered twice a day for 2 days to relieve pain. Animals were then treated with penicillin (105 U/kg) for 7 days as needed, and the bladder was manually emptied twice a day until the reflex recovered.For surgeries, 129 adult female Sprague Dawley rats  were anaesthetized intramuscularly (2.5 ml/kg) with a mixture of ketamine (62.50 mg/kg), xylazine (3.18 mg/kg), and acepromazine (0.63 mg/kg). A T8–T10 laminectomy was performed, and the dura was carefully opened using a longitudinal incision. The spinal cord was transected at approximately 4.5 mm from the level of T8 towards the caudal end and removed completely, thus leaving a defect approximately 5 mm long following retraction of the rostral and caudal ends. Motor evoked potentials (MEPs) were recorded at zero to ensure complete transection of the spinal cord. Rats were then randomly divided into six groups: ST  locomotor score: Rats were allowed to walk freely in an open field, and the locomotor rating scale was determined after a 5‐min observation session  were sacrificed 1 year after injury or implantation. Rats were deeply anaesthetized and transcardially perfused with PBS, followed by 4% paraformaldehyde (PFA) in 0.1 M of phosphate buffer (PB). Spinal cords were dissected, postfixed in PFA for 1 hr, and then placed in 30% sucrose in 0.1‐M PB at 4°C. For quantitative analysis, the spinal cord  was divided into six equal segments. Longitudinal sections were used for observation of axonal regeneration, whereas transverse sections for quantitative analyses were serially cut at 15 μm on a cryostat . Every fifth section was mounted onto the same glass slide for immunolabelling. For immunohistochemistry, sections were washed in 0.01‐M PBS, permeabilized with 0.1% Triton X‐100, and blocked with bovine serum albumin and normal serum from the species of the appropriate secondary antibody. Next, sections were incubated for 12 hr with a primary antibody Table .For apoptosis analysis, specimens were collected from three rats per group  at 3 weeks post‐surgery. Cell types undergoing apoptosis were determined by terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) and cell‐specific markers. Briefly, following TUNEL staining (remaining information was detailed in Supporting Information), sections were processed for immunocytochemistry using an antibody against neuron‐specific neuronal nuclei (NeuN), oligodendrocyte‐specific 2′,3′‐cyclic‐nucleotide 3′‐phosphodiesterase (CNPase), or astrocyte‐specific glial fibrillary acidic protein (GFAP) Table . LikewisFor transmission electron microscopy (TEM), three rats randomly selected from each group at 12 months post‐surgery were perfused with saline and a solution of 4% PFA/2.5% glutaraldehyde in 0.1‐M PB. After dissection, spinal cord samples containing the lesion site were fixed successively in 2.5% glutaraldehyde solution at 4°C for 2 hr and 1% osmic acid for 2 hr. Samples were then dehydrated in an ascending graded series of ethanol (50–100%), embedded overnight in Epon 812, and polymerized at 60°C for 48 hr. Embedded tissue was cut on an ultramicrotome , and the resulting ultrathin slices were treated with lead citrate/uranyl acetate and examined by TEM . Five randomized images for each section were analysed with a Leica Qwin analytical system. Numbers of myelinated nerve fibres and the thickness of myelin sheaths for regenerated nerve fibres were calculated.2.61A) in each group 1 year after surgery, as well as caspase‐3 (p17 form), poly (ADP‐ribose) polymerase (PARP), and cleaved PARP in each group 3 weeks after surgery, were determined by electrophoresis and western blot analysis  randomly selected from each group. Spinal cord tissue 3‐mm rostral and caudal to the epicentre was collected and lysed in radioimmunoprecipitation assay buffer  for 30 min on ice. Tissue debris were pelleted by centrifugation at 12,000 rpm for 10 min at 4°C. Protein concentrations of tissue lysates were determined using a NanoPhotometer . Membranes  were probed with 5‐HT1A and NF‐200 antibodies, or caspase‐3 (p17), PARP, and cleaved PARP antibodies, followed by horseradish peroxidase‐conjugated secondary antibody (Table Amounts of neurofilament protein (NF‐200) and serotonin 1A receptor . BBB scores and CatWalk footprint parameters were analysed with repeated measurement analysis of variance. Immunocytochemistry data for caspase‐3 were analysed with a Kruskal–Wallis test, and pairwise comparisons were performed using a Wilcoxon test with Bonferroni correction. Other experimental data were analysed with one‐way or two‐way analysis of variance, followed by least significant difference test for pairwise comparison. Values of 33.1p < 0.05; Figure BBB locomotor score: At 1 week post‐surgery, the average BBB score was almost zero in all experimental groups, except the sham group. Notably, significant differences gradually emerged between the SCI group and ST, ST/MC, ST + BMSCs, and ST/MC + BMSCs groups. Animals in ST/MC + BMSCs and ST + BMSCs groups, having received scaffolds loaded with BMSCs, exhibited significantly improved BBB scores compared with SCI, ST, and ST/MC groups from 10 weeks to 12 months post‐surgery (p < 0.05) and the SCI group (p < 0.01) from 8 weeks post‐surgery , ST/MC (p < 0.05), and SCI (p < 0.01) groups; however, the sham group had the lowest scores . However, it did not begin to increase until Week 12 in the ST + BMSCs group and remained significantly lower than the ST/MC + BMSCs group until the end of the experiment (p < 0.05). Hind paw pressure was basically unchanged throughout the test period in ST, ST/MC, and SCI groups  nerve fibres regenerated into and extended throughout the implants of ST/MC + BMSCs group rats, yielding an almost linear arrangement of axons . In western blot analysis, significantly increased levels of 5‐HT1A were observed in the ST/MC + BMSCs group compared with other implantation groups . However, numbers of apoptotic cells in the ST/MC + PBS group were not significantly decreased within 3 weeks , but there was no significant difference between ST/MC + BMSCs and ST/MC + PBS groups or ST/MC + BMSCs and ST/MC + z‐VAD‐fmk groups  but was higher compared with ST/MC + z‐VAD‐fmk and sham groups (p < 0.05), in which PARP expression of each group was basically unchanged  provided partitioned regenerative regions for white and grey matter after SCI. Moreover, when the scaffold was loaded with BMSCs, the number of regenerating axons was further increased. Tissue engineering with ST/MC and BMSCs had the potential to promote spinal cord regeneration through mechanical guidance by scaffolds, antiapoptotic effects of BMSCs, and differentiation of a few BMSCs into neurons.The macrostructure and microstructure of biomaterial scaffolds, as well as their chemical properties, are important factors influencing the quality of tissue engineering and subsequent repair. In previous decades, different scaffold macroarchitectural designs were found to facilitate axonal growth across a lesion after SCI  Schematic diagram of ST design and size, a central empty area and surrounding solid ring represented the circular lumen and wall of the tube, respectively. (B) Schematic diagram of ST/MC design and size, an ‘H’ shape central lumen and surrounding solid tubular wall correspond to the spinal cord grey matter and white matter, respectively; moreover, many microchannels on the tubular wall. (C) ST and (D) ST/MC micrograph on SEM, Scale bar: 2 mmFigure S2. Scaffold with or without BMSCs bridged SCI defect for 12 months. Low power overview of the spinal cord of an animal grafted with ST (A), ST/MC (B), ST + BMSCs (C), or ST/MC + BMSCs (D) bridged to a 5 mm defect at complete transverse thoracic spinal cord for 1 year, regenerated tissue filled the spinal cord defect, integrating the rostral and caudal ends of the spinal cordFigure S3. TEM images of nerve fibers in all groups at 12 months SCI. (A) There were more axon fascicles for myelinated or unmyelinated nerve fibers in the mid‐portion of the regenerated tissues in the ST/MC + BMSCs group. (B) Higher magnifications of area boxed in (A), axons of myelinated nerve fibers wrapped by a thick, uniform and electron‐dense lamellar myelin sheath. (C) There were synapse‐like connections in the regenerated tissues of ST/MC + BMSCs. (D) There were some myelinated or unmyelinated nerve fibers of the regenerated tissues in the ST + BMSCs group, (E) higher magnifications of area boxed in (D). (F) The new capillaries with a well‐established ultrastructure of the regenerated tissues were in the ST + BMSCs and ST/MC + BMSCs groups. (G) and (H) Only a small number of newly formed axons with or without myelin sheath were surrounded by fibroblasts and collagen fibers in the ST and ST/MC groups. (I) There was only glial scar in the SCI group. (J) Bar chart showed significant higher in the number of the myelinated nerve fibers of the ST/MC + BMSCs group. (K) Bar chart showed no significant differences in the thickness of the myelin sheath among 4 implanted groups. . Scale bar: 5 μm ; 2 μm ; 1 μm ; 0.5 μm (C)Figure S4. BMSCs alleviating neural apoptosis during 1‐3 week after SCI. The number of TUNEL+ cells in the ST/MC + BMSCs and ST/MC + z‐VAD‐fmk groups was significantly lower than that in the ST/MC + PBS group at 2 and 3 weeks after surgery, respectively. The apoptosic cells in the ST/MC + BMSCs and ST/MC + z‐VAD‐fmk groups were significantly decreased at 3 weeks than that at 1 week after surgery, however, that of the ST/MC + PBS group were not significantly decreased within 3 weeks. Figure S5. BMSCs alleviating neural apoptosis during 1 to 3 week after SCI. TUNEL labeling (red) showed extensive apoptosis in each group at 1 week after SCI, and there was no significant difference in TUNEL+ cell number between 3 groups (A‐C). TUNEL+ cells in the ST/MC + BMSCs and ST/MC + z‐VAD‐fmk groups were relatively reduced during 2‐3 week after SCI, while a large number of apoptotic cells still existed in the ST/MC + PBS group . Scale bar: 50 μmClick here for additional data file.


Claviceps spp.  on florets, producing dark spur‐like sclerotia on spikes that are toxic to humans and animals, leading to detrimental impacts on agriculture and economy due to the downgrading of cereal grains, import–export barriers, reduced yield, and ecological concerns. At least seven phylogenetic lineages (phylogenetic species) were identified within the premolecular concept of C. purpurea s.l. (sensu lato) in agricultural areas and vicinities in Canada and the Western United States. Claviceps purpurea s.s (sensu stricto) remained as the most prevalent species with a wide host range, including cereal crops, native, invasive, and weedy grasses. The knowledge on genetic diversity and distribution of C. purpurea s.s. in North America is lacking. The objective of the present study was to shed light on genetic differentiation and evolution of the natural populations of C. purpurea s.s. Multilocus DNA sequences of samples from Canada and the Western USA were analyzed using a phylogenetic network approach, and population demographic parameters were investigated. Results showed that three distinct genetically subdivided populations exist, and the subdivision is not correlated with geographic or host differentiations. Potential intrinsic mechanisms that might play roles in leading to the cessation of gene flows among the subpopulations, that is, mating and/or vegetative incompatibility, genomic adaptation, were discussed. The neutrality of two house‐keeping genes that are widely used for DNA barcoding, that is, translation elongation factor 1‐α (TEF1‐α) and RNA polymerase II second largest subunit (RPB2), was challenged and discussed.The ergot diseases of agricultural and nonagricultural grasses are caused by the infection of  Claviceps purpurea, that infects wide range of agricultural and nonagricultural grasses, was subdivided in nature. The population structure shed light on the underline mechanisms that might have led to the cessation of gene flow among subdivided populations. House‐keeping gene markers were not neutral in certain circumstances.Regardless of the frequent sexuality and multiple modes of dispersal, the population of the toxigenic plant pathogen,  Host specificity studies under controlled conditions showed virulence variations between the C. purpurea s.l. isolates  released from ascostromata developed from overwintered sclerotia. While the infection of late flowering plants is primarily caused by secondary inocula in the form of asexual conidia, which is immersed in honeydew that oozes from florets and is transmitted by insect vectors, rain splash, or direct head‐to‐head contacts , Manitoba (MB), Ontario (ON), Quebec (QC), Saskatchewan (SK), and Colorado (CO), supplemented with occasional samples from British Columbia (BC), Montana (MT), Newfoundland (NL), Nova Scotia (NS), and Wyoming (WY). Sclerotia from the same host and location (<100 m2) were pooled as one sample, as such a total of 303 samples were obtained. Axenic fungal cultures were isolated and purified for 252 samples; other 51 samples were as sclerotia without pure culture  following the manufacturer's manual. All fungal isolates, sources (culture or sclerotium), hosts, and locations were provided in Table RPB2) using Claviceps specific forward primer TTTCGTGGTATTGTTCGCAGA  using EF1‐983F and EF1‐2218R  using easE996f and easE1895r, and ergot alkaloid chanoclavine I aldehyde oxidoreductase (easA) using easA547f and easA867r , and missing loci were treated as gaps. To confirm the identities of all samples, the concatenated alignment was appended with the 24 reference sequences of 14 related species, and subjected to a phylogenetic analysis using PAUP* 4.0b10  were available in BEASTv2.5, and therefore, we set GTR for TEF1‐α, and HKY for the other three genes . Other priors were set as default, 10,000,000 generations, sampling frequency 1,000, burn‐in 10%, and link trees. Resulting phylogenies were visualized using DensiTree v 2.0.0  via AMOVA between subpopulations (genetic clusters), PCoA, Mantel, and spacial autocorrelation analyses.Genetic differentiation between subpopulations  was further tested by the analysis of molecular variance  and the principle coordinate analysis (PCoA) in GenAlEx 6.5 ; 2. Eastern Canada ; and 3. Western US  ; (b) Noncrop Triticeae , and (c) Crop Triticeae . The two haplotypes on Avena (Aveneae) and other 22 haplotypes were not included in the analyses because their host species belonged to distant taxa and small sample size. Genetic differentiation among these three host groups was examined through AMOVA, PCoA, and pairwise genetic differentiation, ΦPT statistics, and compared with the estimates from genetic clusters.Besides geographic isolation, host specialization is considered another major force driving population subdivision in plant pathogen populations , Ne , I (Shannon's information index = −1* ∑ (Pi * Ln (Pi))), h (haploid genetic diversity = 1‐∑Pi2), where Pi is the frequency of the ith allele for the population; ∑Pi2 is the sum of the squared population allele frequencies. Nucleotide diversity (π), mutation rate (θ), recombination parameter (R), linkage disequilibrium, and neutrality were tested in DnaSP v6. The recombination parameters include R per gene , R per adjacent sites (Ra), and minimum recombination events (Rm). The parameter for linkage disequilibrium was the Kelly's ZnS statistic measuring the overall association between polymorphic sites , and ZZ (= ZnS − Za)  as a clade with a 94% bootstrapping support , haplotype, and nucleotide diversities , than three other genes. TEF1‐α was the least variable ; easE showed nonsignificant deviation based on Tajima's D, however, significant at 0.02 critical level based on Fu & Li' D and F; TEF1‐α and RPB2 showed significant departure from neutral based on all parameters at various critical levels . Overall, Fu & Li's statistics based on coalescence simulation, as expected, showed more sensitive than Tajima's D , 66% (29/44), and 62% (58/94), while relatively fewer private haplotypes 32% (9/28) were observed for easA , with very limited gene flow  was close to 0.5 and the PCoA did not ascertain geographic separation, indicating subpopulations were not strongly differentiated and likely belong to the same population , was much lower (0.036 versus 0.445), and gene flow level (Nm = 6.27) was much higher than among three genetic clusters. The differentiation among three host groups was not statistically significant and had a higher level of gene flow . Pairwise comparisons between Western U.S. and Canadian regions  were also statistically significant; however, the level of the differentiation as estimated by ΦPH values and Nei's D was an order of magnitude lower than those between genetic clusters  and was not significant between crops and each grass tribe  , however, p‐value = .010, indicating significant nonrandom distribution of haplotypes  Figure . However) Figure . In all d Figure . Mantel s Figure . Bromeae3.4Ne = 1.13 than GC2 (1.11) and GC3 (1.09), highest allelic diversity measured as Shannon's information index I = 0.144, and haploid genetic diversity h = 0.083. GC3 had highest total allele number (Na = 1.66), rare (frequency < 5%), and private allele frequencies , while GC3 had higher mutation rates per site and per sequence . Neutrality tests based on Tajima's D, Fu, and Li's D* and F* all suggested GC3 was under significant selective pressure. GC1 was under detectable pressure based on Fu and Li's D* , but not based on Tajima's D and Fu and Li's F*. GC2 was not under selection based on all parameters  were lower than the three genetic clusters independently. These parameters were the highest in GC2 , further supporting population subdivision. Combination of GC1 and GC2 resulted in lower values of Rg (109) and Ra (0.0465) compared with each individual genetic clusters . The same pattern was shown when GC1 and GC3 were combined (Table g (82) and Ra (0.035) were resulted, which is consistent with the result of PCoA analyses showing the incomplete separation between GC2 and GC3  were higher than the individual GCs (9–14). A likely explanation could be that Rm was affected by the sample sizes in that the combined population had a larger sample size (N = 85–156 samples) than three GCs . The estimates of LD parameters, Zns, Za, ZZ, Wall's B, and Q, were very low  suggesting frequent recombination in GCs and among GCs in general. In comparison with individual GCs, the lower values of combined populations, that is, GC1 with GC2, and GC1 with GC3, could suggest the lower recombination during selective sweeping between GC1 and GC2 and between GC1 and GC3  in Canada and western USA using multilocus genotyping. The results from the network analyses , higher rare, and private alleles and showed the deviation from neutrality, indicating GC3 is likely experiencing recent fast adaptation under selective pressure. The separation of three genetic clusters seems consistent with what was found in Oregon and Washington using simple sequence repeat markers  and LD  within and between the GCs , after chill treatment and incubation for 8–10 weeks, produced tiny buds on sclerotia, and then, these buds stopped growing up to normal stromata  and limited gene flow between the genetic clusters  that is likely maintained by high recombination rates and transposon‐mediated gene duplication, but the high recombination rate is also likely influencing the overall trend of purifying selection across the genome  were the results of a recombination event. It is possible that the combination of positive selection on secondary metabolite genes with purifying selection across the rest of the genome has resulted in a more specific, as yet undetected, niche adaptation followed by population stabilization that has resulted in the observed patterns in geographic and host overlap of the three GCs found in North America.The genomes of the members in three GCs may also be evolving and adapting to other environmental pressures, resulting in subpopulations remaining separated. A recent comparative genomic analysis of the genus 4.1easA was not significantly deviating from neutral, while the other three genes all showed significant deviation at varied critical levels  because it is common that genes involving secondary metabolite production are under selective pressure ; Formal analysis (lead); Funding acquisition ; Investigation ; Methodology (supporting); Project administration (lead); Resources (supporting); Supervision (lead); Validation ; Visualization ; Writing‐original draft (lead); Writing‐review & editing . Parivash Shoukouhi: Data curation (lead); Investigation ; Methodology (lead); Resources (supporting); Supervision (supporting); Writing‐review & editing (supporting). Kassandra Bisson: Data curation (lead); Investigation ; Methodology ; Resources (supporting); Writing‐review & editing (supporting). Stephen A. Wyka: Data curation (lead); Investigation ; Methodology ; Resources ; Writing‐review & editing . Kirk D. Broders: Data curation (supporting); Funding acquisition ; Project administration ; Supervision ; Validation ; Writing‐original draft (supporting); Writing‐review & editing . Jim G. Menzies: Data curation (supporting); Funding acquisition (supporting); Investigation ; Methodology ; Project administration (supporting); Resources (lead); Supervision ; Writing‐review & editing (supporting).Fig S1Click here for additional data file.Fig S2Click here for additional data file.Table S1Click here for additional data file.Table S2Click here for additional data file.


There are only a few cases of steroid cell tumours that have been described in the literature. Here, we present an exceptionally rare case of a steroid cell tumour arising from the ovary in early pregnancy. Steroid cell tumours, which used to be called lipid cell tumours, are rare sex cord tumours. They account for approximately 0.1% of all ovarian tumours. These tumours are subdivided into 3 types: stromal luteoma, Leydig cell tumours, and steroid cell tumours not otherwise specified (NOS). Steroid cell tumours NOS are the most common subtype accounting for about 60% of these tumours. First described by Scully et al., they cannot be categorized as either stromal luteoma or Leydig cell tumours and should be considered a diagnosis of exclusion as their significance is that the cell lineage is not defined [Here, we present the case of a steroid cell tumour in pregnancy. A 32-year-old, Vietnamese female, gravida 2, para 1, who had one previous uncomplicated normal vaginal delivery nine years ago, presented at nine weeks of gestation for antenatal care in a tertiary obstetric unit in Sydney, Australia. Her only clinical symptom was occasional left-sided, lower abdominal pain. She had no features of virilisation and had no prior gynaecological history. Her clinical examination was unremarkable. She had recently moved to Australia from Vietnam, six months prior to her pregnancy, where an initial ultrasound demonstrated an ovarian mass. An abdominal/pelvic CT scan ordered by her general practitioner, unbeknown to a very early pregnancy, showed a left ovarian cyst, 100  × 80  × 80 mm in size, with solid nodules along the cyst wall, no ascites, a normal right ovary, and no lymphadenopathy or peritoneal enhancement . The Ca The gestational dating ultrasound confirmed a 102  × 94  × 71 mm well-circumscribed, left ovarian mass with an irregular, solid periphery and a large central cystic component measuring 82  × 62  × 52 mm associated with only a small amount of free pelvic fluid and no vascularity. The consensus was to perform a laparoscopic cystectomy, ideally in her second trimester by the gynaecology oncology team. She, however, presented to the emergency department at 14 weeks of gestation with rapidly accumulating ascites and associated worsening abdominal pain, and her Ca 125 had increased to 1700 kU/L. An ultrasound-guided ascitic drainage identified no malignant cells and 1000 mL of fluid was drained. A laparoscopy was performed at 15 weeks, and upon inspection of the left adnexa, a grey, complex mass measuring 70 × 100 mm was noted to be consuming the left ovary. The intraoperative decision to convert to a midline laparotomy to avoid surgical spillage was made . A left Her postoperative recovery was uneventful, and she was referred to the high-risk antenatal clinic for ongoing perinatal care. At 38 weeks of gestation, she went into spontaneous labour and delivered a healthy 3 kg baby boy with a normal Apgar score. She will have gynaecology oncology follow-up post delivery.The sectioned surface of the left ovary on gross examination was smooth and grey with a solid, multinodular, and cystic appearance with focal haemorrhage and a thickened cyst wall of 10-20 mm. The three nodules described: a 25 × 20 × 5 mm, pale, yellow nodular area, underneath being a cystic cavity 50 × 30 × 25 mm filled with clear watery fluid, surrounded by yellowish nodules at the periphery 4-10 mm across. There was also a defined yellow, grey nodule 30 × 20 × 15 mm. The omentum and fallopian tube were normal.On microscopic examination, the paraffin sections confirmed the frozen section appearance of a solid and cystic expansion of the left ovary, with a thin attenuated rim of ovarian stroma wrapped around the lesion. Nodules of viable epithelioid tumour cells blended seamlessly into the surrounding oedematous stroma, including the large areas of tumour necrosis. The constituent cells were large and seemingly arranged in small nests with a delicate vascular supportive framework. The nuclei were round with vesicular chromatin, prominent nucleoli, irregular nuclear membranes, occasional apoptosis, and simple mitoses were seen. The cytoplasm was abundant, vaguely granular/bubbly, and eosinophilic . No ReinAn ovarian tumour presenting during pregnancy has an incidence ranging from 1 : 815 to 1 : 2200 . Among tAs in this case, steroid cell tumours should primarily be managed surgically to preserve fertility and the fetus in the second trimester . SteroidMost of these tumours are diagnosed early. There is limited data on the recurrence rate and little consensus on adjuvant therapy for advanced disease. Treatment should be based on the tumour histology, stage, and desire for fertility . The mosWhen pathologists encounter a rare tumour such as this, they should use all the clinical, histopathological, immunohistochemical, and microscopic evidence available to determine its origin . When th


Gastric carcinoma (GC) is uncommon in dogs, except in predisposed breeds such as Belgian Shepherd dogs (BSD) of the Tervuren and Groenendael varieties. When GC is diagnosed in dogs it is often late in the disease, resulting in a poorer prognosis. The aim of this prospective clinical study was to investigate possible associations of gastric mucosal pathologies with clinical signs, laboratory test results and GC in BSD. An online survey gathered epidemiological data to generate potential risk factors for vomiting as the predominant gastric clinical sign, and supported patient recruitment for endoscopy. Canine Chronic Enteropathy Clinical Activity Index (CCECAI) score and signs of gastroesophageal reflux (GER) were used to allocate BSD older than five years to either Group A, with signs of gastric disease, or Group B, without signs. Findings in the clinical history, laboratory tests and gastric histopathology of endoscopic biopsies were statistically analysed in search of associations.The online survey included 232 responses. Logistic regression analysis recognized an association of vomiting with gagging, poor appetite and change in attitude. Recruitment for endoscopy included 16 BSD in Group A ; and 11 in Group B . Seven (25.9%) of the 27 BSD  had leukopenia. Serum C-reactive protein tended to be increased with more advanced GC (P = 0.063). Frequency of GC, mucosal atrophy, mucous metaplasia, or glandular dysplasia did not differ between groups. GC was frequently diagnosed (6/27), even without clinical signs (2/11). The odds ratio for vomiting  was increased only when glandular dysplasia was present. GC was associated with mucous metaplasia (P = 0.024) and glandular dysplasia (P = 0.006), but not with mucosal atrophy (P = 1).GC can develop as an occult disease, associated with metaplasia and dysplasia of the gastric mucosa. Suggestive clinical signs, notably vomiting, should warrant timely endoscopy in BSD. Extensive endoscopic screening of asymptomatic dogs remains, however, unrealistic. Therefore, biomarkers of mucosal pathology preceding clinical illness are needed to support an indication for endoscopy and enable early diagnosis of GC. Gastric carcinoma (GC), the most common gastric neoplasm in dogs, is relatively rare, representing less than 1% of canine cancers. However, a few lineages within certain dog breeds are predisposed to GC. GC is frequently seen in Tervuren and Groenendael Belgian Shepherd dogs (BSD) and some other dog breeds, including Bouvier des Flandres, Collie, Standard Poodle, Norwegian Elkhound and Chow-Chow . A previIn dogs with GC, non-specific clinical signs of upper gastrointestinal (GI) illness tend to appear late in the course of disease, at a time point when surgical resection is seldom curative since the tumour has already metastasised , 7, 9. IIn humans, early diagnosis of gastric cancer is an important factor for long-term survival, and endoscopy with gastric mucosal biopsy is the most valuable diagnostic tool . HoweverStudies on clinical presentation and early diagnosis of GC in dogs remain scarce. Clinical signs consistently reported in association with GC are vomiting, anorexia and weight loss, but many other signs such as ptyalism, gagging, retching, apathy, lethargy and cachexia, and occasionally melaena and abdominal pain, can be present , 7, 10. There is conflicting information about the role of grass eating in upper GI diseases. In veterinary textbooks, this non-specific sign is sometimes considered a possible mechanism for self-induced vomiting in nauseated animals . In contBlood test abnormalities may be absent in dogs with GC or tend to be minor, and may include mild anaemia , hypoglycaemia, hyperproteinaemia, hypoalbuminaemia and increased liver values , 7, 10. This prospective study is a first attempt to improve the early diagnosis of canine GC by comparing clinical history with endoscopic and histological findings of gastric mucosal biopsies in Tervuren and Groenendael BSD with and without clinical signs of upper GI disease.The main objective of the study was to investigate possible associations of gastric mucosal pathology with signalment, clinical signs (specific and non-specific signs of upper GI disease) and selected standard laboratory tests .The secondary objective was to test for possible associations between gastric histolopathogical changes and GC, and their potential involvement as preneoplastic changes in dogs.Breed variety ;Age and sex;Feeding  and diet ;Incidence and frequency of vomiting (whether after eating or on empty stomach);Presence of gagging;Presence of ‘altered appetite’ including grass-eating, licking surfaces or objects, eating foreign material such as soil, faeces, wood, toys etc. (pica);Weight loss;Being regarded as nervous, or depressed, or with a change in behaviour or disposition;Previous clinical abnormalities ;Current medication.An electronic questionnaire was available at the University’s canine research webpage, and it was advertised along with links in the website of the Belgian Shepherd Dog Association of Finland. The data supplied an epidemiologic survey and facilitated the recruitment of dogs to the prospective study. Only complete responses concerning pedigree-registered dogs were considered. The enquiry included:Survey responses facilitated patient recruitment. The owners of BSD older than 5 years were actively contacted following the chronological order of response to the survey, to minimize potential bias, e.g. related to individual interest in the outcomes of the examinations or of the research. Volunteers were invited for a clinical visit. During anamnesis, the owners were specifically questioned about gastrointestinal signs such as vomiting (including frequency), and signs of gastroesophageal reflux. Dogs underwent physical examination and samples were taken for routine laboratory tests including complete blood count (CBC), serum biochemistry and faecal parasitology. The resulting information was used for patient inclusion and grouping.Individual dogs were included in accordance with prospectively defined criteria . Patient grouping was based on the Canine Chronic Enteropathy Clinical Activity Index (CCECAI), an indicator primarily targeted for enteropathies that also includes criteria referable to gastric illness such as patient behaviour, appetite, weight loss, and vomiting . Along wMalinois and Lakenois BSD were excluded from the clinical study due to the lack of evidence for predisposition to GC in these breeds . Dogs prBlood was collected in EDTA tubes for CBC including differential white blood cell count (WBC) and in serum tubes for biochemistry, including CRP. The resulting information was used to support patient inclusion, grouping and further statistical analysis. Remaining EDTA-blood and serum, as well as faecal samples, were frozen at − 20 °C for 6–18 months and then transferred to storage at − 80 °C. Serum CRP was measured 10 months later as a single batch, applying a particle-enhanced turbidimetric immunoassay  analyzed using Konelab 60i .Dogs of Groups A and B underwent a clinical study based on gastroscopy  with mucosal biopsy sampling . The procedures were performed under anaesthesia as described elsewhere .Biopsy site selection for focal lesions was facilitated by image enhancement using two subsequent techniques: 1) narrow band imaging (NBI)  and chroThe mucosal samples were placed onto wood chips then immersed in 10% formalin for fixation, using separate vials labelled in reference to the respective gastric region , or focal lesion . The biopsies were paraffin-embedded, cut into four-micrometre-thick sections and stained with haematoxylin–eosin. Special staining techniques (periodic acid-Schiff reaction) were applied at the pathologist’s discretion. Immuno-histochemical staining for epithelial cells was performed using an anti-cytokeratin AE1/AE antibody . The antigens were retrieved with 0.01 M citrate buffer at pH 6 and heated for 20 min at 99 °C. The signal was revealed according to instructions for the UltraVision Detection System HRP/DAB kit .Histological slides were examined by a board-certified pathologist following the template recommended by the World Small Animal Veterinary Association’s International Gastrointestinal Standardization Group . Mucous For data analysis of the epidemiological survey, signalment, feeding, and clinical and behavioural variables were treated as potential risk factors for vomiting, the most relevant clinical sign for gastric localization. For that purpose, a cut-off for vomiting was set at a frequency of ≥ 1 episode per month. The factors were first assessed separately with univariate logistic regression. Factors deemed significant underwent a multivariate model, where main effects were simultaneously included as fixed effects.t-test was applied in search of sex differences, both within groups and independently of grouping, concerning CCECAI score (Group A), WBC, CRP, and MSS, using Prism 8 for Windows . Presence of gastric mucosal atrophy, metaplasia, and dysplasia as well as GC were treated as binary variables. The effects of vomiting, GER and Group on histopathological variables were analysed by logistic regression. The same applied for the effects of atrophy, metaplasia, dysplasia and GC on the incidence of vomiting and serum CRP concentration, as well as the effect of vomiting on CRP. Odds ratios (OR) with 95% confidence intervals (CI) were calculated to quantify the results of the logistic regression analyses. The effects of clinical signs and histopathological variables on MSS were analysed by analysis of variance and tests of fixed effects; a calculated Pr > F value < 0.05 was deemed significant. The possible association between GC and gastric mucosal atrophy, mucous metaplasia and glandular dysplasia was tested with Fisher’s exact tests. P-values < 0.05 were considered statistically significant. Except otherwise noted, SAS System for Windows, version 9.4  was used for statistical analyses.Concerning data analysis in the endoscopic clinical study, the updated clinical information of patients was compared with findings from endoscopy and histopathology, including the calculated MSS of gastric mucosal inflammation. D’Agostino & Pearson’s test was applied for normality of distribution (confirmed when α < 0.05); Welch’s Out of 232 valid answers to the survey, 120 BSD were female (51.7%) and 112 were male (48.3%); 98 were Tervuren (42.2%), 64 Groenendael (27.6%), 65 Malinois (28%) and five Lakenois (2.2%). The mean age was 6.9 ± 3.4 years. Age followed a normal distribution.Eating history revealed that about 88% of the dogs were fed treats, 87.5% were eating grass and 34% were eating foreign material. Vomiting once a month or more often was reported in 43% of the dogs, gagging in 29% and excessive licking of surfaces in 17%. The univariate logistic regression  apparently vomited less often than those fed more often or ad libitum 82%), but significance was unproven when “once a day” was compared to “three times a day or more often” (P = 0.06)  Figs. , 3. The  Figs. , Based on the responses to the survey, 79 dogs younger than 5 years of age were initially excluded as the disease is known to affect older dogs. Thirty-five owners of BSDs with age >5 years volunteered to bring their dogs for assessment. However, five dogs were Malinois and were not included in the study. Two male Groenendaels were excluded due to concurrent chronic disease such as spondylosis, recurrent urinary tract infections and borreliosis diagnosed one year earlier. An asymptomatic, severely obese female Tervuren was excluded on account of increased anaesthetic risk.A total of 27 BSD  with a mean age of 9.4 ± 1.7 years underwent gastroscopy. Age followed a normal distribution both considering all dogs included and each of the study Groups A and B . Mean age was 9.1 ± 1.8 years. Mean CCECAI score was 3.1 ± 2.2. All 16 dogs were vomiting once a month or more often and six of them also showed signs of GER. CCECAI score followed a normal distribution within Group A. There was no significant difference between males (mean CCECAI = 4.2 ± 2.8) and females (mean CCECAI = 2.6 ± 1.8) (P = 0.3).Group B included 11 BSD without signs of gastric disease . Mean age was 9.8 ± 1.4 years. All of these dogs had CCECAI score = 0 and no signs referable to GER.All BSD in the endoscopic study were reported to eat grass, except for one .9/L (range 3.8–14.8) and in Group B, 5.6 × 109/L (range 4–9.7); the difference was non-significant (P = 0.77). WBC followed a normal distribution within Group B (mean WBC = 6.2 ± 1.6), but not in Group A. WBC did not differ between males and females (P = 0.13) with or without regard to grouping. Without regard to grouping, median WBC for all dogs was 5.6 × 109/L (range 3.8–14.8) and seven (25.9%) of the 27 dogs  had WBC below the lower limit of reference range (5.4–17.4 × 109/L). Two dogs in each group (12.9%) had absolute neutropenia , one of which had also lymphopenia .Median WBC in Group A was 5.9 × 10Mean serum CRP levels did not differ between Group A  and Group B  (P = 0.08), nor between males and females (P = 0.63). Serum CRP was normally distributed only within Group B (mean CRP = 9.6 ± 2.3 mg/L). The fixed effect of CRP on CCECAI score was nearly significant at Pr > F = 0.06.At endoscopy, 5/27 dogs  had no distinct focal changes, hence the standard sampling protocol was applied Fig. . VisibleThe histopathological MSS did not differ significantly between Group A (1.6 ± 0.8) and Group B (1.3 ± 0.5) (P = 0.08), nor between males (1.2 ± 0.8) and females (1.6 ± 0.5) (P = 0.21). The MSS followed a normal distribution within both groups. Mean MSS was significantly higher in dogs vomiting once a month or more often (1.6 ± 0.7), than in those for which vomiting was reported as less frequent or absent (1.3 ± 0.6) (Pr > F = 0.028). Mean MSS tended to be higher in dogs diagnosed with glandular dysplasia (1.7 ± 0.6) than in those without dysplastic changes (1.2 ± 0.6) (Pr > F = 0.078). Moreover, an increased odds ratio for vomiting [OR = 9.9 (1.5–63.7)] was confirmed for dogs diagnosed with glandular dysplasia (P = 0.016) but not with mucosal atrophy, mucous metaplasia or GC.Logistic regression analysis showed no statistical difference between Groups A and B concerning mucosal atrophy , mucous metaplasia , glandular dysplasia  nor GC . Regardless of grouping, most BSD had gastric mucosal atrophy (20/27), followed by glandular dysplasia (13/27), mucous metaplasia (6/27), and GC (6/27). Factors such as CCECAI score, GER and laboratory tests showed no significant associations with histopathology. Individual findings are summarized in Table Six dogs were diagnosed with GC Tables , 2. All Comparison of the CRP levels of all dogs in the study with histopathological findings including presence of GC (P = 0.44) failed significance. Notably, serum CRP of dog 686 was 45.1 mg/L, well over two standard deviations above the mean and therefore considered an outlier. When the subgroup of four dogs with ulcerated GC were compared to the other 20 dogs , the odds ratio for an increased CRP with ulcerated GC was nearly significant (P = 0.06), OR = 1.2 (0.99–1.48).Neoplastic lesions were also accompanied by moderate (4/6) to severe (2/6) lymphoplasmacytic infiltration, and mild (1/6) to moderate  eosinophilic infiltration. The GC patient with the most severe clinical disease (CCECAI = 8) was both the youngest dog with GC (7.2 years) and the only one with a mucinous adenocarcinoma (dog 658).In one case , endoscopy revealed a marked fold of 4 × 7 cm in the distal greater curvature protruding into the lumen . Eating grass failed to show significant association with vomiting. Plant-eating has been hypothesized to serve various purposes such as inducing vomiting or controlling gastrointestinal parasite burden , 23. AboVomiting was not applied as the single criterion for patient inclusion to Group A (with GI signs) or B (no GI signs). In the absence of a standardized, objective clinical measure of gastric disease, the Canine Chronic Enteropathy Clinical Activity Index was applied. The CCECAI is used to score signs such as inactivity, inappetence, weight loss and vomiting, which are recognized with upper gastrointestinal disorders, including GC , 18. NevWBC below the reference range occurred in dogs of both groups , with and without GC. Low WBC in itself does not entail an increased risk for anaesthesia or endoscopic procedures and was therefore not regarded as a reason for exclusion. Leukopenia has been proposed as common and possibly even physiological for Belgian Tervuren in a study in North America , but theSerum CRP has been reported as increased in dogs diagnosed with GC as compared to dogs with chronic gastritis and control dogs . In humaPrevious reports have shown higher incidence of GC in male dogs . Our onlNone of the BSD in our study had intestinal metaplasia diagnosed from endoscopic biopsies. This suggests that, at least in this population, the histological pathway towards GC does not follow the human linear cascade proposed by Correa . The mosExtensive efforts to standardize inflammatory changes in canine gastrointestinal pathology have contributed to diagnostic capabilities in this field , 34, 41.Relevant histopathological changes, including GC, were diagnosed in both groups A and B. Our approach to group allocation involved an array of gastrointestinal signs, which were analysed as presence/absence of signs of GER and individual scores for CCECAI. Ultimately, vomiting was the only sign showing an important correlation with MSS and notably with glandular dysplasia (OR = 9.9). There was a trend for increased MSS along with glandular dysplasia . That may suggest a possible pathophysiological association of chronic gastritis with carcinogenesis also in BSD, warranting studies with higher statistical power. Both mucous metaplasia and especially glandular dysplasia correlated with GC, which is also the case in the classic human model of gastric carcinogenesis , 44.Vomiting is regarded as the cardinal sign for gastric localization and consistently mentioned in previous studies on canine GC –10, 18. Early detection of GC can potentially improve patient outcome, enabling surgical removal before such tumours have developed in size and clinical severity, as illustrated by the follow-up of dog 680 (group B) as well as another study showing prolonged survival time for a few dogs treated having only minimally invasive GC . Timely Alongside non-predisposed breeds, a great challenge remains in diagnosing the asymptomatic GC cases. Extensive endoscopic screening of healthy BSD to diagnose occult GC may be unwarranted and thus ethically questionable. As opposed to veterinary medicine, several standard laboratory tests and tumour markers are useful in screening and prognosing human GC –52. As sThis study on ageing Tervuren and Groenendael BSD showed a high proportion of dogs with clinical signs of gastric disease regardless of the presence of GC. Gastric mucosal pathology included atrophy, mucous metaplasia, glandular dysplasia and GC, which were found irrespective of clinical signs.Recurrent vomiting correlated with the severity of mucosal inflammation and presence of dysplasia and should be regarded as an indication for endoscopy and gastric mucosal sampling in BSD. Moreover, the significant association of GC with metaplasia and dysplasia, which are regarded as premalignant in humans, supports the indication for endoscopic surveillance in BSD. However, extensive endoscopic screening due to the occurrence of occult gastric disease remains unrealistic. Therefore, sensitive and specific indicators of GC such as serum or faecal biomarkers are warranted to promote early diagnosis of canine GC.


FPED) and genomic measures using segment based (FROH) and marker-by-marker (FGRM) based approaches.Phenotypic performances of livestock animals decline with increasing levels of inbreeding, however, the noticeable decline known as inbreeding depression, may not be due only to the total level of inbreeding, but rather could be distinctly associated with more recent or more ancient inbreeding. Therefore, splitting inbreeding into different age classes could help in assessing detrimental effects of different ages of inbreeding. Hence, this study sought to investigate the effect of recent and ancient inbreeding on production and fertility traits in Canadian Holstein cattle with both pedigree and genomic records. Furthermore, inbreeding coefficients were estimated using traditional pedigree measure , respectively. Similarly, an extension in first service to conception (FSTC) of 0.29, 0.24 and 0.31 day in heifers was found for every 1% increase in FPED, FROH and FGRM, respectively. Fertility traits that did not show significant depression were observed to move in an unfavorable direction over time. Splitting both pedigree and genomic inbreeding into age classes resulted in recent age classes showing more detrimental inbreeding effects, while more distant age classes caused more favorable effects. For example, a − 1.56 kg loss in 305-day protein yield (PY) was observed for every 1% increase in the most recent pedigree age class, whereas a 1.33 kg gain was found per 1% increase in the most distant pedigree age class.Inbreeding depression was found for all production and most fertility traits, for example, every 1% increase in Inbreeding depression was observed for production and fertility traits. In general, recent inbreeding had unfavorable effects, while ancestral inbreeding had favorable effects. Given that more negative effects were estimated from recent inbreeding when compared to ancient inbreeding suggests that recent inbreeding should be the primary focus of selection programs. Also, further work to identify specific recent homozygous regions negatively associated with phenotypic traits could be investigated. Over the past decade, Canadian Holstein cattle populations have experienced an increase in the annual rate of inbreeding from 0.08 to 0.23%, which were observed from 2000 to 2010 and 2010–2018, respectively . In the Before the availability and popularity of genomic data, estimation of inbreeding depression was predominantly done by calculating inbreeding coefficients from pedigree data and regressing any trait of economic interest on the inbreeding coefficients , 10. MorIdeally, the aim of the genetic selection being practised in livestock species is to increase the frequency of favorable alleles, thus, increasing the level of homozygosity. In essence, inbreeding could result in the depression or enhancement of any trait of economic interest, therefore not all inbreeding is detrimental. Inbreeding increases the expression of deleterious recessive alleles, which are naturally or artificially selected against in a process called genetic purging , 20. Witg centiMorgans (cM), where g is the number of generations to a common ancestor [The partitioning of inbreeding into recent and ancient inbreeding can be examined with pedigree and genomic data. For pedigree data, recent inbreeding can be estimated by tracing the pedigree back relatively few generations to the common ancestor, while ancient inbreeding traces back the pedigree to a more distant common ancestor . In addiancestor .The objectives of this study were to 1) estimate the effect of inbreeding on production and fertility traits in Canadian Holsteins using pedigree and genomic information; 2) assess the effect of recent and ancient inbreeding on production and fertility traits in Canadian Holsteins.The basic descriptive statistics of the phenotypic data are presented in Table FPED and FROH and 0.61 for FPED and FGRM. A correlation of 0.97 was estimated for FROH and FGRM , respectively, whereas, a correlation with a more distant pedigree age of inbreeding (FPED7 − 6) was equal to − 0.12, − 0.13 and − 0.12, respectively. Similarly, correlations between FPED, FROH and FGRM with FROH > 16 were estimated to be 0.51, 0.77 and 0.76, respectively, in contrast to − 0.10, − 0.06 and − 0.08 for FROH2 − 4, respectively. For the model-based age of genomic inbreeding, the correlations ranged from 0.44 to 0.65 for the most recent age and − 0.01 to 0.00 for the most distant age of genomic inbreeding with the classical inbreeding measures. The movement in different directions of the correlations with the different classes of the age of inbreeding was notable, with correlations ranging from − 0.45 to 0.11 for the pedigree classes, − 0.17 to 0.17 for the ROH classes and − 0.48 to 0.06 for the model-based classes  was observed for all production traits based on FPED, FROH and FGRM  increase of 0.29, 0.24 and 0.31 days in FSTC for every 1% increase in inbreeding coefficients based on FPED, FROH and FGRM, respectively, which represents 1.50, 1.24 and 1.60% of the phenotypic means. Despite the statistically non-significant effects (P < 0.44) for cows, a 1% increase in inbreeding coefficients based on FPED, FROH and FGRM resulted in an extension of 0.16, 0.19 and 0.19 days, respectively, for FSTC. In addition, both heifers and cows showed genomic inbreeding depression for number of services (NS). For instance, a 1% increase in FROH resulted in a 0.78 and 0.83 chance of getting re-inseminated after the first insemination for heifers and cows, respectively. An inbreeding depression of 0.96 was observed in NS (P < 0.01) for a 1% increase in FPED for heifers, while a statistically non-significant (P < 0.51) effect of 0.45 was observed for a 1% increase in FPED for cows. To further support the effect of inbreeding, differences in the phenotypic means of animals with low inbreeding levels (5th percentile) and high inbreeding levels (95th percentile) were estimated and are presented in Table FPED, FROH and FGRM, respectively. In a similar fashion, animals with low inbreeding coefficients had 3.26, 5.04 and 7.10 kg more FY based on FPED, FROH and FGRM, respectively, when compared to animals with high inbreeding coefficients. For fertility traits, heifers with high inbreeding levels had on average 6.40, 6.71 and 5.39 more days to age at first insemination (AFS) based on FPED, FROH and FGRM, respectively. Likewise, 2.83, 4.12 and 3.87 less days for FSTC was estimated based on FPED, FROH and FGRM, respectively, for heifers with low inbreeding compared to heifers with high inbreeding. For cows, a more evident increase in NS of 3.56, 1.04 and 1.82% based on FPED, FROH and FGRM, respectively, was estimated for highly inbred cows in comparison to lowly inbred cows.Statistically significant inbreeding depression , which showed a 0.42 increase in days for a 1% increase in FPED4 − 3 (P < 0.05).Splitting the pedigree inbreeding coefficients into different age (generation) classes showed varying effects on phenotypes. Interestingly, inbreeding occurring within the most recent five generations resulted in unfavorable and statistically significant depressing effects on phenotypic traits, however, more distant generations showed favorable, but a statistically non-significant effects on phenotypic traits Fig. . A 1% inP < 0.05  was not statistically significant for all ROH classes, however, shorter segments (ROH < 4 Mb) showed favorable effects while longer segments (ROH > 4 Mb) had unfavorable effects. For AFS in heifers, unfavorable inbreeding effects for all classes of ROH were observed, but only ROH > 8 Mb showed statistical significance (P < 0.05). Additionally, a statistically significant and unfavorable effect of 0.62 and 0.96 was obtained for a 1% increase in FROH8 − 16 and FROH > 16 for NS, respectively, whereas a statistically non-significant, but favorable effect of − 2.26 and − 0.07 was obtained for, FROH1 − 2 and FROH2 − 4, respectively. For fertility traits in cows, only an unfavorable and statistically significant (P < 0.05) effect on NRR for ROH >16 Mb was observed. In general, cow traits follow a similar pattern with shorter segments tending to have favorable effects, while longer segments tending to be unfavorable.ROH was split into age classes with longer ROH indicating more recent inbreeding and shorter ROH suggesting more remote inbreeding. Although the effect of all ROH classes were unfavorable for production traits, only ROH classes with segments longer than 4 Mb were significant at .05 Fig. . For exaFHBD1, FHBD2 and FHBD3 corresponded to a − 40.79, − 33.76 and − 30.53 kg loss in MY, respectively. In contrast, a 1% increase in FHBD4 and FHBD5 was related to 10.06 and 15.65 kg gain in MY, respectively. For fertility traits, a 1% increase in FHBD1 and FHBD2 in heifers prolonged FSTC by 0.28 and 0.28 days, respectively. Conversely, a 1% increase in FHBD5 reduced FSTC by 0.42 days, although, this was statistically non-significant. In addition, a statistically significant increase of 0.83 in NS for cows with a 1% increase in FHBD1 and a statistically non-significant decrease of 0.22 for every 1% increase in FHBD4 in NS in cows was estimated.The age of inbreeding estimated using the model-based approach provided varying effects on phenotypes. Based on this approach, more recent age of inbreeding had statistically significant and unfavorable effects on production traits and more distant ages had statistically non-significant and favorable inbreeding effects Fig. . A 1% inFk _ NEW and Fk _ ANC  by 0.27 and 0.42 days, respectively [FROH and FGRM, respectively. Using genomic inbreeding, Martikainen et al. [FROH and FGRM, respectively. This is in line with the 301 and 315 kg difference between lowly and highly inbred cows reported by Doekes et al. [FROH and FGRM, respectively. Despite FPED having a higher estimated effect of inbreeding on phenotypes compared to FROH, FROH accounted for a larger difference in phenotypic means between lowly and highly inbred animals. These results are similar to those reported in Doekes et al. [FROH over FPED.As with other studies, a 1% increase in pedigree inbreeding has been shown to have a significantly negative effect on production traits –36, whicectively . This trn et al.  also fous et al.  and 161 s et al.  using FRs et al.  and is mFPED5). In addition, Doekes et al. [FPED7 − 6 on MY, FY, PY, IFL and calving interval (CI). These findings are in line with the favorable, but non-significant effects of FPED7 − 6 and FPED8 − 7 on production traits, AFS and FSTC in the present study. The consistency between these studies suggest that recent inbreeding is more detrimental than ancient inbreeding. Previous researchers have found effects of different ROH length classes on phenotypes [Few studies have investigated the effect of pedigree and genomic inbreeding age classes on phenotypes , 23. Thes et al.  reportedenotypes , 38. In enotypes  found thenotypes . Generalenotypes , 40. In enotypes . In an aenotypes , 42. To enotypes , the modFNEW and − 5.52 to 15.51 per unit increase in FANC for the number of pups in first litter. Thus, indicating that new inbreeding causes more deteriorating effects, whereas old inbreeding causes lesser deteriorating effects and sometimes favorable effects. Using Kalinowski’s [FK _ NEW and old FK _ ANC inbreeding, Mc Parland et al. [FK _ NEW. In addition, they observed a significant, but less unfavorable effect of − 8.8 kg and 0.52 day for MY and AFC, respectively, per 1% increase in FK _ ANC. In Dutch Holstein-Friesian cattle, Doekes et al. [FK _ NEW and a non-significant, but favorable effect of 0.03 kg for 305-day FY per 1% increase in FK _ ANC. Those authors mentioned evidence of purging due to favorable effects found with FK _ ANC. In the present study, no significant effects were observed for production traits, however, estimates for FK _ ANC were favorable for MY and PY, whereas FK _ NEW showed unfavorable effects for MY and PY. Conversely, a favorable effect was detected for FY when using estimates from FK _ NEW and FK _ ANC. For fertility traits, significant effects were found only for AFS and CTFS. A favorable effect of − 0.77 and − 0.94 days for AFS and CTFS, respectively, per 1% increase in FK _ ANC, while an unfavorable effect of 1.58 and 1.00 days for AFS and CTFS, respectively, was found for every 1% increase in FK _ NEW. The varying results among these studies could be due to the differences in the populations used, which are subjected to different selection criteria. In the present study, there seems to be no evidence of purging and given the rate at which inbreeding is increasing following the implementation of genomic selection [Some studies have evaluated the effect of new and ancestral inbreeding on phenotypic traits , 43, 44.nowski’s  approachs et al.  found a election , 20. In election , 46. Theelection , 47 and election .A significant and unfavorable effect of classical inbreeding on all production traits and some fertility traits was found. Genomic inbreeding measures seemed to capture more phenotypic differences between lowly and highly inbred animals. Recent inbreeding was found to show more detrimental effects on both fertility and production traits than ancient inbreeding. However, no substantial evidence of purging was uncovered with ancestral inbreeding. The model-based approach of classifying inbreeding into age classes provided similar results to the pedigree age of inbreeding, hence, in the absence of pedigree records, genomic measures could be used. Overall, heterogeneity of inbreeding depression was observed with recent and ancestral inbreeding. In future studies, the molecular architecture of inbreeding could be investigated to identify regions negatively associated with phenotypic traits.Pedigree records for all available animals with genotype and phenotype data were provided by Canadian Dairy Network . The pedigree information consisted of a total of 259,871 individuals that trace back to 1950 as the base year. To ensure that inbreeding estimates were not severely underestimated, pedigree completeness index (PCI) going back five generations and the number of complete generation equivalence (CGE) were estimated using EVA software . AnimalsA total of 50,575 genotyped Holstein cows were available with birth year ranging from 1999 to 2017. Cows were genotyped with the Illumina BovineSNP50 Chip (50 K)  and lower density array panels (10 K - 30 K). Animals with lower density genotypes were imputed to medium density (50 K) using FImpute software . After qPhenotypic records of 46,430 cows with first calving date that ranged from 2008 to 2018 were available for production and fertility traits. For production traits, a total number of 21,194 cows had first lactation records on a 305-day basis for milk yield in kg (MY), fat yield in kg (FY) and protein yield in kg (PY). Fertility traits had a total of 52,948 records and these were split up into heifer (first parity) and cow (second parity) traits. Of these records, 33,610 were for heifers and 19,338 were for cows and all animals with cow records also had heifer records. The following fertility traits were considered in this study: age at first service in days (AFS); days from calving to first service (CTFS); number of services (NS); first service non-return rate to 56 days (NRR); days from first service to conception (FSTC). All traits recorded before and during the first parity are termed heifer traits and traits recorded after the first parity were cow traits. NRR was coded as 1 when no subsequent service took place between 15 and 56 days following the first service and coded 0 if otherwise. NS was coded from 1 to 10 and animals with more than 10 services were assigned as 10. AFS was measured in days and considered to be a heifer trait. CTFS was measured in days and considered to be a cow trait. FSTC was measured in days and considered to be both a heifer and cow trait.Pedigree and genomic data were both used in calculating inbreeding coefficients. With pedigree data, inbreeding measures were divided into three categories: 1) classical pedigree inbreeding measure; 2) pedigree age of inbreeding measure and; 3) ancestral pedigree inbreeding measure. For genomic data, inbreeding measures were divided in two categories namely 1) classical genomic inbreeding measure and 2) genomic age of inbreeding measure. A detailed explanation of these categories follows below.FPED) was estimated for all individuals with phenotypic records by tracing back the pedigree to the founder generation using the algorithm proposed by Meuwissen and Luo [n generation ago to common ancestors, where n represent the specific number of generations to the common ancestors. More specifically, the inbreeding age classes attributable to ancestors from a specific generation is the difference between two successive generations. For example, inbreeding coefficients that occurred due to ancestors in generation seven (FPED7 − 6) can be calculated as the difference between inbreeding coefficients obtained tracing back to seven generations ago FPED7 and coefficients obtained tracing back to six generations ago FPED6 . This procedure was performed to categorize inbreeding into age classes from most recent to ancient inbreeding. The most recent age traced back was three generations ago (FPED3) because the number of inbred animals in generation two were less than 0.02% of the sample size, while the most ancient age was traced back to generation eight (FPED8) due to having similar inbreeding coefficients with older generations. Pedigree age of inbreeding was estimated using the vanrad.f function implemented in PEDIG software [FPED into new inbreeding (Fk _ NEW) and ancient inbreeding (Fk _ ANC), therefore, FPED = Fk _ NEW + Fk _ ANC. The difference between Fk _ NEW and Fk _ ANC is that Fk _ NEW is the probability that alleles in IBD state of a given individual is occurring for the first time in the pedigree of the individual, while Fk _ ANC is the probability that IBD alleles in an individual have occurred previously in at least one ancestor. Kalinowski ancestral pedigree inbreeding was calculated using a gene dropping approach with 106 replications as implemented in GRAIN [Classical inbreeding coefficient ( and Luo  as imple and Luo . Pedigresoftware . Ancestrsoftware  with thesoftware  further software  by splitin GRAIN .FROH) and 2) marker-by-marker based approach (FGRM). Runs of homozygosity were identified with the deterministic sliding window approach implemented in PLINK using the following criteria: a minimum physical length of 1 Mb; a maximum gap of 500 kb between two successive SNP; a minimum of 20 consecutive homozygous SNP and a minimum density of one SNP per 100 kb. The following formula was used for calculating individual segment based genomic inbreeding:ith individual, jth ROH segment in bp, n is the total number of detected ROH and LAUTO is the total length of the autosomes covered by the SNP in bp.Two approaches were used in estimating the classical genomic inbreeding: 1) segment-based approach (runs of homozygosity (ROH); G) following the proposition of VanRaden [ith individual and Gii is the diagonal element of the genomic relationship matrix.Inbreeding in the marker-by-marker based approach was calculated by subtracting one from the diagonal of the genomic relationship matrix  1–2 Mb; 2) 2–4 Mb; 3) 4–8 Mb; 4) 8–16 Mb; and 5) > 16 Mb length classes. These length classes indicate inbreeding resulting from ancient to most recent ancestors. Additionally, genomic age of inbreeding was estimated using the model-based method that uses a Hidden Markov Model (HMM) approach to identify homozygous by descent (HBD) segments [Rk is the rate specific to the kth class. Thus, the length of an HBD segment of any kth class is exponentially distributed with rate Rk. In the current study, a model with 5 HBD classes was defined following predefined default rates as implemented in the R statistical package “RZooRoH” [The genomic age of inbreeding was estimated by classifying already identified ROH length into five different length classes to specify the approximate age or generation in which they occur. As mentioned earlier, deducing the age of inbreeding from ROH length is an expectation that follows an exponential distribution with a mean of 100/2 segments . With thRZooRoH” , 42.a); and error term (e). Inbreeding depression was estimated separately for each trait using the following linear mixed model:y is a vector of phenotypic measurement for MY, FY, PY, AFS, CTFS, NS, NRR and FSTC, b is a vector of systematic effects, β is the coefficient of the linear regression on F, F is a vector of inbreeding coefficients from pedigree or genomic data , a is a vector of random additive genetic effects, cj is a vector of jth non-genetic random effects  and e is a vector of random residual effects, n is the number of non-genetic random effects, X, Z and Wj are incidence matrices that link the fixed effects, random additive genetic effects and jth non-genetic random effects to the phenotypes, respectively. The assumptions for the random effects include: A is the numerator relationship matrix and I is an identity matrix. For age of inbreeding and ancestral inbreeding, inbreeding depression was estimated using the following linear mixed model:βk is the coefficient of the linear regression on inbreeding coefficients within the kth class of inbreeding (Fk), m is the number of inbreeding classes, and all other parameters are the same as mentioned in model I.To estimate the effect of inbreeding on phenotypes, the same models used in the national genetic evaluation for Canadian Holsteins were adapted from Jamrozik et al. , with thAll linear models in this study were fitted using the restricted maximum likelihood procedure implemented in ASReml 4.1 .