Repeatability and heritability of response to superovulation in Holstein cows

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

Testicular ultrastructure and morphology of the seminal pathway in Prochilodus scrofa

The seminiferous tubules of Prochilodus scrofa present a coiled morphological arrangement with intertubular anastomoses and unrestricted spermatogonial distribution. The structural pattern of the seminiferous tubules is cystic, with cysts formed by cytoplasmic prolongations of Sertoli cells. Inside the cysts are observed different types of germ cells. The seminiferous tubules open individually on the ventral surface of the main testicular duct present in each testis. Each main testicular duct prolongs as a spermatic duct, fusing with the spermatic duct of the opposite side to form the common spermatic duct which opens into the urogenital papilla. The mature sperm cysts break and extravasate their content into the lumen of the seminiferous tubules from which the seminal fluid and the spermatozoa penetrate the main testicular duct, the spermatic duct and the common spermatic duct for semen ejaculation.

Efeitos do tabagismo nos parâmetros seminais

Objectives: We performed a study to evaluate the adverse effects of smoker patient on semen parameters. Material and Methods: We studied retrospective 238 semen specimen, 115 from men smoking and 123 from men nosmoking. Sperm concentration, motility, morphology and vitality were confront in statistic test. Results: In 31% smoke between 1 and 10 cigarettes per day, 26% smoked 10 and 19 per day, 42% smoke more than 20 per day. The distribution of heavy smokes and light smokes did not differ statistically between the groups. Only a significant difference found between smoker and controls, there was about motility, morphology. There is difference in vitality and concentration per ml. Conclusions: This study indicates only a minor effect of smoker patient on male subfertility. Although smokers as a group may not experience reduced fertility, men with marginal semen quality may benefit from quitting smoking.

Superovulation in Cycling Mares Using Equine Follicle Stimulating Hormone (eFSH)

Superovulation would potentially increase the efficiency and decrease the cost of embryo transfer by increasing embryo collection rates. Other potential clinical applications include improving pregnancy rates from frozen semen, treatment of subfertility in stallions and mares, and induction of ovulation in transitional mares. The objective of this study was to evaluate the efficacy of purified equine follicle stimulating hormone (eFSH; Bioniche Animal Health USA, Inc., Athens, GA) in inducing superovulation in cycling mares. In the first experiment, 49 normal, cycling mares were used in a study at Colorado State University. Mares were assigned to 1 of 3 groups: group 1, controls (n = 29) and groups 2 and 3, eFSH-treated (n = 10/group). Treated mares were administered 25 mg of eFSH twice daily beginning 5 or 6 days after ovulation (group 2). Mares received 250 (of cloprostenol on the second day of eFSH treatment. Administration of eFSH continued until the majority of follicles reached a diameter of 35 mm, at which time a deslorelin implant was administered. Group 3 mares (n = 10) received 12 mg of eFSH twice daily starting on day 5 or 6. The treatment regimen was identical to that of group 2. Mares in all 3 groups were bred with semen from 1 of 4 stallions. Pregnancy status was determined at 14 to 16 days after ovulation. In experiment 2, 16 light-horse mares were used during the physiologic breeding season in Brazil. On the first cycle, mares served as controls, and on the second cycle, mares were administered 12 mg of eFSH twice daily until a majority of follicles were 35 mm in diameter, at which time human chorionic gonadotropin (hCG) was administered. Mares were inseminated on both cycles, and embryo collection attempts were performed 7 or 8 days after ovulation. Mares treated with 25 mg of eFSH developed a greater number of follicles (35 mm) and ovulated a greater number of follicles than control mares. However, the number of pregnancies obtained per mare was not different between control mares and those receiving 25 mg of eFSH twice daily. Mares treated with 12 mg of eFSH and administered either hCG or deslorelin also developed more follicles than untreated controls. Mares receiving eFSH followed by hCG ovulated a greater number of follicles than control mares, whereas the number of ovulations from mares receiving eFSH followed by deslorelin was similar to that of control mares. Pregnancy rate for mares induced to ovulate with hCG was higher than that of control mares, whereas the pregnancy rate for eFSH-treated mares induced to ovulate with deslorelin did not differ from that of the controls. Overall, 80% of mares administered eFSH had multiple ovulations compared with 10.3% of the control mares. In experiment 2, the number of large follicles was greater in the eFSH-treated cycle than the previous untreated cycle. In addition, the number of ovulations during the cycle in which mares were treated with eFSH was greater (3.6) than for the control cycle (1.0). The average number of embryos recovered per mare for the eFSH cycle (1.9 ± 0.3) was greater than the embryo recovery rate for the control cycle (0.5 ± 0.3). In summary, the highest ovulation and the highest pregnancy and embryo recovery rates were obtained after administration of 12 mg of eFSH twice daily followed by 2500 IU of hCG. Superovulation with eFSH increased pregnancy rate and embryo recovery rate and, thus, the efficiency of the embryo transfer program.

Variance heterogeneity for milk yield in Brazilian and Colombian Holstein herds

Heterogeneity of variances for milk yield (MY) was determined for Brazilian and Colombian herds. The herds were grouped as high or low variability within each country, using as criterion the phenotypic standard deviation (PSD) of MY in the contemporary groups of cows, from the first to the sixth calving. Brazilian and Colombian herds with PSD greater than 1,168 kg or 1,012 kg, respectively, were classified as high variability while the herd groups with values lower than those were classified as low variability. The genetic parameters for MY within each herd group were estimated using bivariate analysis in an animal model and the restricted maximum likelihood method with a derivative free algorithm, using 72,280 first lactations of cows, daughters of 1,880 sires. Heterogeneous variances were found, and Brazilian herds with high PSD had the greatest additive and residual genetic variances and heritability coefficients for MY. MY genetic correlation coefficients between herds of high and low variability within each country were 0.96 and 0.93 while between countries they varied from 0.72 to 0.81, suggesting that there was a reclassification of animals in the two countries and also heterogeneity of variances. This phenomenon leads to the questioning of the strategy of imported semen usage and the need to do genetic evaluations to identify sires with greater genetic potential for (sub) tropical environmental conditions.

Factors that cause genotype by environment interaction and use of a multiple-trait herd-cluster model for milk yield of Holstein cattle from Brazil and Colombia

Descriptive herd variables (DVHE) were used to explain genotype by environment interactions (G x E) for milk yield (MY) in Brazilian and Colombian production environments and to develop a herd-cluster model to estimate covariance components and genetic parameters for each herd environment group. Data consisted of 180,522 lactation records of 94,558 Holstein cows from 937 Brazilian and 400 Colombian herds. Herds in both countries were jointly grouped in thirds according to 8 DVHE: production level, phenotypic variability, age at first calving, calving interval, percentage of imported semen, lactation length, and herd size. For each DVHE, REML bivariate animal model analyses were used to estimate genetic correlations for MY between upper and lower thirds of the data. Based on estimates of genetic correlations, weights were assigned to each DVHE to group herds in a cluster analysis using the FASTCLUS procedure in SAS. Three clusters were defined, and genetic and residual variance components were heterogeneous among herd clusters. Estimates of heritability in clusters 1 and 3 were 0.28 and 0.29, respectively, but the estimate was larger (0.39) in Cluster 2. The genetic correlations of MY from different clusters ranged from 0.89 to 0.97. The herd-cluster model based on DVHE properly takes into account G x E by grouping similar environments accordingly and seems to be an alternative to simply considering country borders to distinguish between environments.

Laser-assisted hatching of cryopreserved-thawed embryos by thinning one quarter of the zona

Laser-assisted hatching is little documented in the literature regarding its efficacy in cryopreserved-thawed (CT) embryo transfer cycles. The aim of the present study was to evaluate in a randomized manner the efficacy of thinning one quarter of the zona pellucida of CT embryos to a depth of 50-80% of the original thickness, via laser treatment (the qLZT-AH procedure), in improving implantation and pregnancy rates. Two populations were studied: population I, patients who had all their supernumerary embryos cryopreserved, regardless of their morphology, and population II, patients at risk of ovarian hyperstimulation syndrome who had all their embryos cryopreserved. Artificial and natural protocols were used for the embryo transfers. A total of 350 laser-thinned CT embryos were compared with 352 intact zona embryos. No difference in implantation or pregnancy rate was found after using qLZT-AH in either population. These findings suggest that qLZT-AH should not be routinely performed in cryopreserved embryo programmes.

Detecção de Leptospira pomona em sêmen bovino por eletroforese capilar fluorescente

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

Tissue inhibitor of metalloproteinase-2 (TIMP-2) location in the ventral, lateral, dorsal and anterior lobes of rat prostate by immunohistochemistry

Matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) play a major role in extracellular matrix component degradation in several normal and abnormal tissue situations; they are also found in human seminal plasma. MMPs have been found in rat prostate secretions and are nearly lobe specific in expression pattern. The aim of this study was to evaluate whether TIMP-2, like other semen components, is expressed differently from different rat prostatic lobes. Immunohistochemical staining was performed in both young and adult rat ventral (VP), lateral (LP), dorsal (DP), and anterior (AP) prostatic lobes and confirmed by western blotting. TIMP-2 expression was found in the epithelial cells in the following sequence: LP > AP > DP > VP, in both young and adult rats. In this study, 100% of adult LP presented histological signs of prostatitis, where TIMP-2 immunostaining was positive in normal epithelium even with intraluminal neutrophils, but was reduced or absent in the epithelium with intraepithelial leukocytes or with periductal stroma disorganization associated with mononuclear cell infiltration. However, TIMP-2 expression in LP was not induced by prostatitis, since younger rat LPs were also strongly TIMP-2 positive. The distal and intermediate VP regions were TIMP-2 negative, but the proximal regions were strongly stained. Western blotting results confirmed the high TIMP-2 expression in the LP lobe. Thus, TIMP-2 is expressed differently between the prostatic lobes and is another nearly lobe-specific protein, which plays a role in the regulation of MMP activity in seminal plasma and glandular homeostasis. TIMP-2 is also another regional ductal variation of VP. Further studies should address whether TIMP-2 expression is related to the highest incidence of rat LP prostatitis and adenocarcinoma. © 2006 International Federation for Cell Biology.

Antisperm antibodies in infertile men and their correlation with seminal parameters

Aim: Antisperm antibodies (ASA) in males cause the autoimmune disease 'immune infertility'. The present study intended to detect the presence of ASA and their incidence in men with unexplained infertility, as well as to evaluate the correlation between the presence of ASA and semen parameter alterations. Methods: Blood and sperm assessment were collected to carry out a direct and indirect mixed antiglobulin reaction (MAR) test and semen analysis in infertile and fertile men from the University Hospital of the Faculty of Medicine, Sao Paulo State University, Sao Paulo. Results: In the MARtest, 18.18% of infertile men were positive for ASA. In fertile men, no positivity was found. A significant correlation between the presence of ASA with an increased white blood cell count plus a decreased hypoosmotic swelling test result was observed. Conclusions: The results indicate that ASA are involved in reduced fertility. It is not ASA detection per.se that provides conclusive information about the occurrence of damage to fertility. The correlation between infertility and altered seminal parameters reinforce the ASA participation in this pathology. © 2007 The Authors Journal compilation. © 2007 Japan Society for Reproductive Medicine.

Decrease of the DNA damage levels after sperm preparation by layering method

The aim of this prospective study was to determine the DNA fragmentation levels before and after sperm preparation by layering method. A total of 78 patients submitted to assisted reproduction technology (ART) for infertility treatment were evaluated. Ejaculated spermatozoa were obtained by masturbation on the day of ART procedure. The evaluation of DNA fragmentation was performed in the fresh semen and after preparation by a layering method, respectively. After washing with PBS, the sperm pellets were smears and then processed for the terminal deoxyribonucleotidyl transferase (TdT)-mediated dUTP nick-end labelling (TUNEL) assay that was performed using a Cell Death Detection Kit with tetramethylrhodamine-labelled dUTP. For quantitative evaluation, 200 spermatozoa in randomly selected areas on microscope slides were evaluated and the percentage of TUNEL positive spermatozoa was determined. If ≥20% of selected sperm were TUNEL positive, the exam was considered abnormal. The mean percentage of DNA sperm fragmentation before sperm preparation was 17±8.3% and after 7.8±6.5% (p<0.0001). The exam was considered normal in 49 patients before preparation and in 73 patients after (p<0.0001). The sperm preparation with a layering method for the ART procedure is effective to select sperm with a significant decrease of the DNA damage.

The effects of male age on sperm DNA damage in an infertile population

The objective was to investigate the influence of age on sperm DNA damage. Semen samples were collected from 508 men in an unselected group of couples attending infertility investigation and treatment. DNA fragmentation in spermatozoa was measured by TdT (terminal deoxynucleotidyl transferase)-mediated dUTP nick-end labelling (TUNEL) assay; at least 200 spermatozoa in randomly selected areas of microscope slides were evaluated using a fluorescent microscope and the percentage of TUNEL positive spermatozoa was determined. The number of cells with red fluorescence (TUNEL positive) was expressed as a percentage of the total sample [DNA fragmentation index (DFI)]. Age was treated as a continuous variable for regression and correlation analysis. The following male age groups were used: Group I: ≤35 years, Group II: 36-39 years, and Group III: ≥40 years. DFI was significantly lower in Group I than in Group II (P = 0.034) or III (P = 0.022). There was no difference in DFI between Groups II and III. In addition, regression analysis demonstrated a significant increase in sperm DFI with age (P = 0.02). TUNEL assay clearly demonstrates an increase in sperm DNA damage with age. © 2007 Published by Reproductive Healthcare Ltd.

What's the signification of nuclear vacuoles in human sperm?

The aim of this study was to determine the extent of DNA fragmentation and the presence of single/denatured or double stranded of DNA in sperm with large nuclear vacuoles (LNV) selected by high-magnification. A total of 30 patients had fresh semen samples prepared by discontinuous concentration gradient. Sperm with normal nucleus (NN) and LNV were selected at 8400x magnification and placed in different slides. DNA fragmentation was determined by TUNEL assay. Denatured and double stranded DNA was identified by acridine orange fluorescence method. The percentage of DNA fragmentation in LNV sperm (29%) was significantly higher (P<0.001) than NN sperm (15.8%). Therefore, cleavage of genomic DNA in low molecular weight DNA fragments (mono and oligonucleosomes), and single strand breaks (nicks) in high molecular weight DNA occur more frequently in LNV. Identically, the percentage denatured stranded DNA in sperm with LNV (67.9%) was significantly higher (P <0.0001) than NN sperm (33%). The high level of denatured DNA in sperm with LNV suggests precocious decondensation and disaggregation of sperm chromatin fibers. Our results support an association between LNV sperm and DNA damage, and the routine selection and injection of morphological motile sperm at high magnification for ICSI. The adverse effect (DNA fragmentation or denaturation) leads to concern particularly about the possibility of iatrogenic transmission of genetic abnormalities. Copyright - SBRA - Sociedade Brasileira de Reprodução Assistida.

Análise morfológica dos folículos primordiais de ovários de ovino criopreservados em EG, DMSO e sua associaçã o

Objective: Compare the cryoprotectants Dimethyl Sulphoxide (DMSO), Ethylene Glycol (EG) and their association for cryopreservation of sheep ovarian cortex. Methodology: Fragments collected from ovaries were divided into 3 parts. 1. One part from sample was destined for analysis of fresh material. 2. The second part was incubated with solution of freezing having 1,5M EG or 1,5M DMSO or 1,5MEG + 1,5M DMSO and washed for dilution of the cryoprotectants. 3. The third part was submitted to cryopreservation using the same cryoprotectans (EG 1,5M; DMSO 1,5M and EG + DMSO 1,5M) and cryopreserved. In all groups, one part of sample was submitted to pre-antral follicles isolation and the remainder was destined to ultra-structural analysis. Results: After isolation of fresh primordial follicles (control), the percentage of viable follicles was 78,9%. The percentage of viable follicles only exposed to cryoprotectants 1,5M EG, 1,5M DMSO and 1,5M EG + 1,5M DMSO was 77,1%, 68,4% and 60,7% respectively. After cryopreservation were 75%, 60% and 55,6% respectively. Ultra-structural analysis of the primordial follicles derived from fresh ovarian fragments or from fragments just exposed to the cryoprotectants showed similar morphology. However, in frozen samples, alterations of mitochondria were observed in all groups. Despite this, the integrity of the remained organelles was preserved in follicles cryopreserved with EG, while that in others groups (DMSO and association) an excess of vacuolizaton in cytoplasm of oocytes and swelling of nuclear membrane was observed indicating degeneration. Conclusion: The Ehilene Glycol seems to be the cryoprotector more adequated for cryopreservation of sheep ovarian tissue.