Utero-placental expression and functional implications of HSD11B1 and HSD11B2 in canine pregnancy†.

Glucocorticoids modulate the feto-maternal interface during the induction of parturition. In the dog, the prepartum rise of cortisol in the maternal circulation appears to be erratic, and information about its contribution to the prepartum luteolytic cascade is scarce. However, the local placental upregulation of glucocorticoid receptor (GR/NR3C1) at term led to the hypothesis that species-specific regulatory mechanisms might apply to the involvement of cortisol in canine parturition. Therefore, here, we assessed the canine uterine/utero-placental spatio-temporal expression of hydroxysteroid 11-beta dehydrogenase 1 (HSD11B1; reduces cortisone to cortisol), and -2 (HSD11B2; oxidizes cortisol to the inactive cortisone). Both enzymes were detectable throughout pregnancy. Their transcriptional levels were elevated following implantation, with a strong increase in HSD11B2 post-implantation (days 18-25 of pregnancy), and in HSD11B1 at mid-gestation (days 35-40) (P < 0.05). Interestingly, when compared pairwise, HSD11B2 transcripts were higher during post-implantation, whereas HSD11B1 dominated during mid-gestation and luteolysis (P < 0.05). A custom-made species-specific antibody generated against HSD11B2 confirmed its decreased expression at prepartum luteolysis. Moreover, in mid-pregnant dogs treated with aglepristone, HSD11B1 was significantly higher than -2 (P < 0.05). HSD11B2 (protein and transcript) was localized mostly in the syncytiotrophoblast, whereas HSD11B1 mRNA was mainly localized in cytotrophoblast cells. Finally, in a functional approach using placental microsomes, a reduced conversion capacity to deactivate cortisol into cortisone was observed during prepartum luteolysis, fitting well with the diminished HSD11B2 levels. In particular, the latter findings support the presence of local increased cortisol availability at term in the dog, contrasting with an enhanced inactivation of cortisol during early pregnancy.

Follicular dynamics during the non-reproductive season in Miranda jennies.

Jennies' follicular wave patterns have not yet been addressed during the non-breeding and transition seasons in anoestrous jennies. Twelve non-pregnant females of the Miranda donkey breed were followed to describe follicular wave characteristics during the non-reproductive season and determine the anoestrous effect in follicular wave patterns. Five jennies enrolled in this study experienced anoestrus during the non-breeding season, but all retained the continuous emergence of follicular waves. The average duration of the waves from emergence to peak was 11.2 ± 0.021 days (3-29 days). The duration of the different type of waves was 9.91 ± 0.034 days for minor waves,12.5 ± 0.232 days for major secondary waves and 12.5 ± 0.057 for major primary waves. The major waves were significantly longer than the minor waves (p < .001). Older jennies presented longer waves (p = .021). In the jennies presenting anoestrus, the wave duration during anoestrus (11.2 ± 0.125 days, n = 31) was not different from the waves detected in the preceding and subsequent ovulatory cycles (11.3 ± 0.084 days, n = 43) (p = .978). The number of follicular waves emerging in each ovulatory cycle (n = 59) was 2.36 ± 0.011, varied from 1 to 4. Only in a small proportion of cycles, one wave (0.8%) was recorded, with 41 cycles (67.2%) presenting two waves; fourteen cycles presenting three waves (24.6%); and three cycles (6.6%) showing 4 waves. It was concluded that follicular waves do occur in anoestrous jennies during the non-breeding season with two-wave cycles being common.

Evaluation of gonadal macroscopic and microscopic morphometry reveals precocious puberty in Bísaro pig.

Bísaro pig (BP), like many other local breeds reared in extensive non-industrial systems, faces many constraints to comply with the EU welfare regulation, particularly regarding the restrictions to surgical castration. In order to adapt an immunocastration protocol to overtake this issue, a puberty timeline is needed. Using gonadal morphometry data from 91 young male BP, this study intended to characterize testicular development, describing the prominent cell types and structures, to ultimately assess the age at puberty in male BP through a mixed prediction model. As expected, the relations between several macro and microscopic parameters and their relation with age were as described within the literature. Post-pubertal animals have larger and heavier gonads, lower Sertoli cell density/tubule, higher Leydig cell density and larger seminiferous tubules. Meiosis was firstly seen in 44-day-old animals, elongated spermatids in 70-day-old animals. Complete spermatogenesis was firstly identified in a 90-day-old animal. Spermatozoa were present in the epididymis of 23 animals, aged from 70 to 240 days old, and in the vas deferens of 14 animals (105 to 240 days old). The prediction model inferred that male Bísaro pigs reach puberty between 14 and 17 weeks (3 and 4 months old) and become sexually capable from 15 to 19 weeks (3.5 and 4.4 months old). These parameters confirm the sexual precocity of this breed.

Co-existing monophasic teratoma and uterine adenocarcinoma in a female dog.

Ovarian teratomas are occasionally reported in dogs; the rarest type is the monophasic teratoma, composed of tissues originating from only one germ layer. Canine endometrial adenocarcinomas are also rare in dogs and mainly affect geriatric females. This report describes the case of co-existing ovarian teratoma and uterine adenocarcinoma in a 10-year-old nulliparous female Boxer presented with lethargy, anorexia and purulent vaginal discharge. Abdominal ultrasonography evidenced pyometra and a mass in the left ovary. This was composed of a uniform whitish tissue with multiple cystic structures. The histology revealed an atrophy of the ovarian parenchyma, compressed by a proliferation of well-differentiated nervous tissue staining positively to vimentin, S100 and neuronal specific enolase (NSE), and negatively to keratin and inhibin. The left uterine horn, whose diameter was markedly increased, showed foci of endometrial cellular atypia, evident nucleoli and mitoses, at light microscopy. To our best knowledge, this is the first report of a co-existing ovarian monophasic teratoma and endometrial adenocarcinoma, two rare reproductive neoplasia in dogs.

An immunohistochemical study on the expression of sex steroid receptors, Ki-67 and cytokeratins 7 and 20 in feline endometrial adenocarcinomas.

BACKGROUND: Endometrial adenocarcinomas are a rare type of tumour in cats. Though different morphologies have been reported, the most frequent histological type of feline endometrial adenocarcinoma (FEA) is the papillary serous. Characterization of molecular markers expression in FEA may contribute to clarify the pathogenesis of these tumours and to assess the differences between normal endometrium and FEA regarding the expression pattern of several proteins. Therefore, this study aimed to evaluate the immunohistochemical profile of a wide panel of antibodies (specific for ER-α, PR, Ki-67, CK7 and CK20) in twenty-four cases of FEA. Comparisons were made between FEA and feline normal cyclic endometrium in follicular (n = 13) and luteal (n = 10) stages. Except for Ki-67, all other molecular markers were assessed independently for the intensity of immunolabeling and for the percentage of cells expressing the protein. RESULTS: This study showed that in FEA a loss of expression occurs for ER-α (P ≤ 0.0001) and less markedly also for PR. The lost in sex steroid receptors concerns a decrease in both the proportion of labelled cells and the intensity of immunolabelling (P = 0.002 and P = 0.024, respectively). Proliferative activity, estimated via Ki-67 immunoreaction, significantly increased in FEA as compared to normal endometrium (P ≤ 0.0001). Feline endometrial adenocarcinomas maintained the CK7+/CK20+ status of normal endometrium. However, FEA showed decreased CK7 intensity of labelling compared to normal endometria (P ≤ 0.0001) and loss of CK20 expression, both in intensity (P ≤ 0.0001) and in percentage of positive cells (P = 0.01), compared to normal tissues. CONCLUSIONS: Data gathered in this study suggest that proliferation in FEA accompanies ER-α down-regulation, possibly following activation of pathways mediated by local growth factors. Moreover, FEA retains combined expression of CK7 and CK20, as evidenced in normal endometrial epithelia, although a decrease in CK7 expression was observed.

Membrane-bound progesterone receptors in the canine uterus and placenta; possible targets in the maintenance of pregnancy.

To date, the biological functions of P4 within the canine placenta have been attributed to maternal stroma-derived decidual cells as the only placental cells expressing the nuclear P4 receptor (PGR). However, P4 can also exert its effects via membrane-bound receptors. To test the hypothesis that membrane-bound P4 receptors are involved in regulating placental function in the dog, the expression of mPRα, -ß, -γ, PGRMC1 and -2 was investigated in the uterine and placental compartments derived from different stages of pregnancy and from prepartum luteolysis. Further, to assess the PGR signaling-mediated effects upon membrane P4 receptors in canine decidual cells, in vitro decidualized dog uterine stromal (DUS) cells were treated with type II antigestagens (aglepristone or mifepristone). The expression of all membrane P4 receptors was detectable in reproductive tissues and in DUS cells. The main findings indicate their distinguishable placental spatio-temporal distribution; PGRMC2 was predominantly found in decidual cells, PGRMC1 was strong in maternal endothelial compartments, and syncytiotrophoblast showed abundant levels of mPRα and mPRß. In vitro decidualization was associated with increased expression of PGRMC1 and -2, while their protein levels were diminished by antigestagen treatment. The involvement of membrane-bound P4 signaling in the regulation of canine placental function is implied, with P4 effects being directly exerted through maternal and fetal cellular compartments. The indirect effects of PGR might involve the modulation of membrane-bound receptors availability in decidual cells, implying a self-regulatory loop of P4 in regulating the availability of its own receptors in the canine placenta.

Effect of Immunocastration on Culled Sows-A Preliminary Study on Reproductive Tract, Carcass Traits, and Meat Quality.

The Bísaro pig is a Portuguese autochthonous breed greatly appreciated for its meat quality and is mainly reared outdoors. Immunocastration could be a solution to avoid undesirable pregnancies and boar taint in cull sows. The present study tested three immunocastration protocols (with Improvac®) according to their reproductive cycle. The first inoculation was performed two weeks after farrowing (IM1, n = 5), at the beginning of estrus (IM2, n = 5), and one week after the end of estrus (IM3, n = 5), followed by a second administration four weeks apart. A control group (C, n = 5) was also included in the same housing conditions. The sample collection included the reproductive tract for morphometric evaluation, neck fat for the quantification of boar taint compounds, and a portion of the Longissimus thoracis et lumborum for meat quality trait assessment. The reproductive tracts from intact sows (C) were significantly heavier compared to the immunocastrated groups (p < 0.05) (1.403 kg C to 0.508 kg IM1, 0.590 kg IM2, and 0.599 kg IM3), suggesting the regression of the reproductive tract to nonstimulated conditions due to immunization against GnRH. The IM1 group exhibited significantly smaller reproductive tract measurements compared to group C for most of the evaluated segments (p < 0.05). No marked differences were observed in the meat quality traits. Therefore, immunocastration can be used in culling sows to avoid ovarian activity, and it is not detrimental to pork quality traits.

Seasonal Variations on Testicular Morphology, Boar Taint, and Meat Quality Traits in Traditional Outdoor Pig Farming.

Traditional outdoor pig farming is renowned for its emphasis on animal welfare and the production of highly valued, quality meat. While seasonality is known to impact certain animals, particularly those raised outdoors, there is a lack of research on Bísaro boars, a native Portuguese breed. This research study was conducted on a total of 20 male entire Bísaro pigs, reared in outdoor pens from 4 to 13 months old, and subsequently slaughtered. The animals were divided into two groups: one slaughtered in winter (Wi, n = 9), and the other in summer (Su, n = 11). The objective was to evaluate testicular morphometry, boar taint compounds, and meat quality traits, including sensory analysis and fatty acid profile. Testicles from the Su group exhibited reduced volume, indicating diminished functionality during that season. While no significant differences were observed in the boar taint compound analysis, panelists could discern a more intense aroma and flavor of boar taint in the Su meat. Other meat quality traits showed no significant variations, but the fatty acid profile displayed higher values in the Wi group. This study reveals that Bísaro boars experience reproductive seasonality, leading to variations in boar taint compounds across the seasons. This information is crucial for farm planning.

Acute Hormonal Responses to Multi-Joint Resistance Exercises with Blood Flow Restriction.

The purpose of this study was to investigate the acute effects of multi-joint resistance exercises (MJRE) with blood flow restriction on hormonal responses. Ten men participated in the study and underwent two experimental protocols in random order: four sets (30, 15, 15, and 15 reps, respectively) of MJRE (half squat and horizontal chest press) were performed with 20% of 1RM and a rest time between sets of 30 s, combined with intermittent blood flow restriction (LI + BFR protocol); and four sets (8, 8, 8, 20 reps, respectively) of the same MJRE performed with 75% of 1RM load (HI protocol), with a 90 s rest between the first three sets and 30 s between the third to the fourth set. Blood samples were collected before (PRE), immediately after (POST), and 15 min after the performance of MJRE (POST15). A time effect was observed for growth hormone (GH) and insulin-like-growth-factor-1-binding-protein-3 (IGFPB-3), but no protocol effects or interactions between protocol and times were observed (p > 0.05). There was no effect of either protocol or time (p > 0.05) on total testosterone, free testosterone, or cortisol concentrations. However, significant (p < 0.05) increases were observed in the GH serum concentrations of 2072.73% and 2278.5%, HI, and LI + BFR protocols, respectively, from the PRE to POST15 test. In addition, there was an increase of 15.30% and 13.29% in the IGFPB-3 concentrations (p < 0.05) from PRE to POST0 times for HI and LI + BFR protocols, respectively. Furthermore, there was a decrease of −6.17% and −11.54%, p = 0.00, between the times POST0 to POST15 in the IGFPB-3 for the HI and LI + BFR protocols, respectively. It is concluded that multi-joint resistance exercises combined with intermittent blood flow restriction seemed to promote acute hormonal responses in a manner similar to traditional exercise with high loads. Future studies may investigate whether chronic use of LI + BFR with MJRE may promote muscle hypertrophy.

Long-Term Immunocastration Protocols Successfully Reduce Testicles' Size in Bísaro Pigs.

This study aimed to find a suitable immunocastration protocol for male Bísaro pigs (BP) due to the breed and production system particularities. Twenty-five male BP were treated with Improvac® according to three protocols: using two (GrpE2 and L2) or three vaccinations (GrpL3) and starting at 9 (GrpE2) or 13 weeks old (GrpL2 and L3). Eleven animals were kept as intact males (GrpC). Scrotal measurements and the morphometry of the testes and epididymides collected at slaughter were used to survey the effectiveness of the immunocastration compared with the age-matched intact controls. Animals in groups E2 and L3 were kept until 57 weeks, after a second vaccination cycle at 49 and 53 weeks of age. Scrotal dimensions decreased to almost initial values in treated animals until 17 (GrpE2) and 21 weeks (GrpL2 and L3), thereafter increasing to post-pubertal values until around 29 or 37 weeks of age for groups E2 and L2, respectively, but only at 41 weeks in group L3. Between 41 and 49 weeks, scrotal dimensions were similar in treated and control animals, decreasing to the predicted pre-puberty size after the second cycle of vaccination. This study suggests the most suited protocol for males slaughtered at older ages includes three administrations of Improvac® starting at 3 months of age, followed by a second vaccination cycle.

Selected Uterine Immune Events Associated With the Establishment of Pregnancy in the Dog.

In the dog, implantation takes place at approximately 17 days of embryonal life and, while exposed to relatively high circulating progesterone concentrations, embryos presence is required for the formation of decidua. Furthermore, a balance between pro- and anti-inflammatory responses in conceptus-maternal communication is crucial for the onset of pregnancy. Strikingly, the understanding of such immune mechanisms in canine reproduction is still elusive. Here, canine uterine samples from pre-implantation (day 10-12, E+) and corresponding non-pregnant controls (E-), implantation (day 17, Imp) and post-implantation (day 18-25, Post-Imp) stages of pregnancy were used to investigate the expression and localization of several immune-related factors. The most important findings indicate increased availability of CD4, MHCII, NCR1, IDO1, AIF1, CD25, CCR7, and IL6 in response to embryo presence (E+), while FoxP3 and CCL3 were more abundant in E- samples. Implantation was characterized by upregulated levels of FoxP3, IL12a, ENG, and CDH1, whereas CD4, CCR7, IL8, and -10 were less represented. Following implantation, decreased transcript levels of TNFR1, MHCII, NCR1, TLR4, CD206, FoxP3, and IL12a were observed concomitantly with the highest expression of IL6 and IL1ß. MHCII, CD86, CD206, CD163, TNFα, IDO1, and AIF1 were immunolocalized in macrophages, CD4 and Nkp46 in lymphocytes, and some signals of IDO1, AIF1, and TNF-receptors could also be identified in endothelial cells and/or uterine glands. Cumulatively, new insights regarding uterine immunity in the peri-implantation period are provided, with apparent moderated pro-inflammatory signals prevailing during pre-implantation, while implantation and early trophoblast invasion appear to be associated with immunomodulatory and rather anti-inflammatory conditions.

Association between polymorphisms in the SOX9 region and canine disorder of sex development (78,XX; SRY-negative) revisited in a multibreed case-control study.

Testicular or ovotesticular disorders of sex development (DSD) in individuals with female karyotype (XX) lacking the SRY gene has been observed in several mammalian species, including dogs. A genetic background for this abnormality has been extensively sought, and the region harboring the SOX9 gene has often been considered key in canine DSD. Three types of polymorphism have been studied in this region to date: a) copy number variation (CNV) in a region about 400 kb upstream of SOX9, named CNVR1; b) duplication of SOX9; and c) insertion of a single G-nucleotide (rs852549625) approximately 2.2 Mb upstream of SOX9. The aim of this study was thus to comprehensively analyze these polymorphisms in a large multibreed case-control cohort containing 45 XX DSD dogs, representing 23 breeds. The control set contained 57 fertile females. Droplet digital PCR (ddPCR) was used to study CNVR1 and the duplication of SOX9. Fluorescent in situ hybridization (FISH) was used to visualize copy numbers on a cellular level. The Sanger sequencing approach was performed to analyze the region harboring the G-insertion. We confirmed that CNVR1 is highly polymorphic and that copy numbers varied between 0 and 7 in the case and control cohorts. Interestingly, the number of copies was significantly higher (P = 0.038) in XX DSD dogs (mean = 2.7) than in the control females (mean = 2.0) but not in all studied breeds. Duplication of the SOX9 gene was noted only in a single XX DSD dog (an American Bully), which had three copies of SOX9. Distribution of the G-nucleotide insertion was similar in the XX DSD (frequency 0.20) and control (frequency 0.14) cohorts. Concluding, our study showed that CNVR1, located upstream of SOX9, is associated with the XX DSD phenotype, though in a breed-specific manner. Duplication of the SOX9 gene is a rare cause of this disorder in dogs. Moreover, we did not observe any association of G-insertion with the DSD phenotype. We assume that the genetic background of XX DSD can be different in certain breeds.

Application of B-mode ultrasonography in the assessment of the dog penis.

With the purpose to characterize the ultrasonographic features of the dog penis, 20 healthy dogs aged from 2 to 11 years old were used. Ultrasonography was performed using a 5-9MHz linear probe and a Philips HD3 scanner. Ultrasonograms were obtained at the penile body (pre- and post-scrotal sections) and at both the bulbus glandis and the pars longa glandis, with the dog placed in dorsal recumbency. The typical anatomy of the dog penis is easily demonstrated in ultrasonographic images, as revealed in this study. On ultrasonograms taken at the body level, the two corpora cavernosa showed a homogeneous, hypoechoic structure; ventrally, the corpus spongiosum appears as a slightly hyperechoic structure surrounding the urethra. In glans ultrasonograms, the most typical feature is the os penis, observed as a strong hyperechoic structure, whose acoustic shadow impairs the screening of adjacent tissues. Lining on its concavity, the corpus spongiosum is observed as a slightly hyperechoic tissue around the urethra. An ovoid, regularly hypoechoic tissue corresponding to the erectile tissue of the pars longa, surrounds these structures. The bulbus glandis is visualized as a regular hypoechoic structure with small anechoic areas, corresponding to sinusoidal spaces. The albuginea is evidenced as a well-defined, hyperechoic external layer. The preputial cavity appears as a thin anechoic layer surrounding the albuginea. The results of this study demonstrate that ultrasonographic assessment of the dog penis is easily performed and accurate enough for penile examination. Furthermore, ultrasonography of the dog penis can become an interesting diagnostic tool to explore in the canine practice, during breeding soundness evaluation and also whenever penile exposition is impossible to achieve.

Ultrasonographic assessment of the feline mammary gland.

The aim of this study is to characterise the feline mammary echotexture using B-mode ultrasonography, which is not routinely used to examine the feline mammary gland. Using a 5-9 MHz linear transducer the ultrasonographic appearance of non-stimulated and stimulated mammary glands was determined in 35 mature intact non-pregnant, pregnant and lactating queens aged from 16 months to 8 years. In intact non-pregnant queens, mammary glands are fairly underdeveloped and on the ultrasonograms they appear with a regular hypoechoic texture and generally show a thickness of less than 2.0mm. The stimulated mammary tissue typically presents a more hyperechoic appearance compared to the non-stimulated gland and a fine granular echotexture. Maximum echogenicity of the mammary gland is reached during lactation. In late pregnancy, the mammary glands reach 6-9 mm in thickness. During lactation, the size of the glands depends on the existence of a suckling stimulus, with the suckled glands reaching about 11 mm in thickness. Ductal structures can only be imaged during late pregnancy and lactation. Ultrasonographic evaluation of the feline mammary gland can become a valuable diagnostic tool to characterise physiological changes and may further contribute to a better characterisation of diseased mammary tissue.

Cardiorespiratory, enzymatic and hormonal responses during and after walking while fasting.

The aim of the present study was to observe whether performing a low intensity endurance exercise following an overnight fasted (FAST) or fed (FED) condition promotes different cardiorespiratory, enzymatic and hormonal responses. Nine male physical active subjects, (age 21.89 ± 2.52 years old, height 175.89 ± 5.16 cm, weight 72.10 ± 4.31 kg, estimated body fat 7.25 ± 2.11%), randomly performed two sessions of 45 minutes' low intensity exercise (individual ventilator threshold) interspersed by seven days, differentiated only in whether they were provided with a standardized meal or not. The oxygen consumption (VO2) and heart rate (HR) were measured continuously at the 30-min rest, the 45-min during and the 30-min post-exercise. The testosterone (T) and cortisol (C) hormones were measured at rest, immediately post-exercise and 15-min post-exercise. The Glucose (GLU), Free fatty acids (FFA) and enzyme lipase activity (ELP) were measured at rest, 15-min and 30-min exercise, immediately, 15-min and 30-min post-exercise. Significantly lower values were observed in FED compared to FAST with: C (nmol/L) from pre (428.87 ± 120.41; 454.62 ± 148.33, respectively) to immediately post-exercise (285.10 ± 85.86; 465.66 ± 137.70, respectively) and 15-min post-exercise (248.00 ± 87.88; 454.31 ± 112.72, respectively) (p<0.05); and GLU at all times, with an exception at 15-min post-exercise. The testosterone/cortisol ratio (T/C) was significantly higher in the FED compared with FAST from pre (0.05 ± 0.02, 0.05 ± 0.01, respectively) to 15-min post-exercise (0.08 ± 0.03, 0.05 ± 0.02, respectively). No other significant differences were observed between conditions. We conclude that fasting prior to low intensity endurance exercise does not seem be advantageous, when it comes to fat loss, compared with the same exercise performed after a meal.

Effects of dietary inclusion level of a mixture of probiotic cultures and enzymes on broiler chickens immunity response.

The current study was conducted to analyse the effect of a mixture of probiotic cultures and enzymes (Probio Enzyme, XVET GmbH, Germany) on the immune response and weight of central lymphoid organs and liver in broilers. A total of 270 male chickens were randomly divided into nine groups, with three replicates of 10 birds each. Treatment groups were fed for either 22 or 42 days with different levels of Probio Enzyme 250, 500, 750 and 1000 g/ton, whereas the control group fed a basal diet without Probio Enzyme. To analyse the effects of dietary supplementation on broilers humoral immune response, the antibodies titres for avian influenza (AI) and Newcastle disease virus (NDV) and to the sheep red blood cells (SRBC) challenge were assayed in birds from each experimental unit, along with the assessment of the weight of the main lymphoid organs and liver. The addition of the Probio Enzyme mixture did not significantly affect the titres of the antibodies against AI and NDV at day 42, despite the wide individual variation observed specially on the antibody titres at day 33. Treatments affected the production of IgG after the second challenge with SRBC (P = 0.003), which was transposed to the correspondent total Ig titres (P = 0.044). Conversely, a lower birds' body weight (BW) was found in the majority of treated groups compared to control (P = 0.031). The spleen was the only lymphoid organ showing differences in the absolute and relative weight (P = 0.003 and P = 0.001, respectively). No differences were found in thymus and Bursa of Fabricius weights. In conclusion, broilers treated with Probio Enzyme showed a satisfactory immune response compared with control, despite the wide variation found after the first vaccine challenge against AI. Moreover, the probiotic mixture dose and duration modulated differently the immune response and the spleen weight, unaffecting the central lymphoid organs weight.