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The purpose of the present study was to examine the ability of a tetraparental Chimera in producing IVF embryos. Cumulus oocytes complexes (COCs) were matured in vitro for 22 h. Frozen-thawed sperm of a Chimera (CH), as well as Japanese Black (JB), Limousin (L), Japanese Brown (JBr), Holstein (H) bulls were used for IVF. The chromosome preparations were made from peripheral lymphocytes. Based on chromosome analysis the Chimera had apparently normal chromosomes (29 acrocentric pairs, one large sub metacentric X chromosome and one small sub metacentric Y chromosome). The proportion of acrosome reacted spermatozoa after 1 h incubation was higher (P < 0.01) with the Chimera (CH) than with the Holstein and in Japanese Brown bulls, but did not differ from Japanese Black and Limousin bull sperm (79.0%, 71.2%, 72.5%, 57.8% and 57.0% for CH, JB, L, JBr and H sperm, respectively). Fertilization rates observed after 5 h of sperm-oocyte incubation with Chimera (O-CH) sperm were higher (P < 0.05) than with Japanese Brown (O-JBr) and (P < 0.01) than with Holstein (O-H) sperm, but did not differ from Japanese Black (O-JB) and Limousin (O-L) sperm (36/44, 81.8%; 28/35, 80.0%; 25/36, 69.4%; 19/43, 44.2% and 6/33, 18.2% for O-CH, O-JB, O-L, O-JBr and O-H, respectively). The cleavage rates of IVM oocytes inseminated with Chimera sperm were also higher (P < 0.001) than in Holstein, (P < 0.01) Japanese Brown and (P < 0.05) Limousin, but did not differ from Japanese Black sperm (181/239, 75.7%; 123/171, 71.9%; 108/186, 58.1%; 80/196, 40.8% and 30/186, 16.1% for O-CH, O-JB, O-L, O-JBr and O-H, respectively). The blastocyst rates of IVM oocytes inseminated with sperm were higher (P < 0.05) than in Limousin, Japanese Brown and Holstein, but did not differ from Japanese Black (69/181, 38.1%; 48/123, 39.0%; 27/108, 25.0%; 7/30, 23.3% and 16/80, 17.8% for O-CH, O-JB, O-L, O-JBr and O-H, respectively). Three findings suggested the sperm from this tetraparental Chimeric bull were able to be used for producing bovine IVF embryos.
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PMID:Fertility of sperm from a tetraparental chimeric bull. 923 Dec 45

In aging Brown Norway rats, both spermatogenesis and steroidogenesis decrease. Little is known about changes in the epididymis during aging. However, since the two major components entering the epididymis from the testis change, we hypothesized that epididymal histology would be affected by advancing age. The epididymides of Brown Norway rats ranging in age from 3 to 24 mo were prepared for light and electron microscopy. Striking quantitative and qualitative changes were noted. There was an age-dependent increase in the thickness of the basal membrane and in the number of halo cells. There were also major segment-specific changes in the appearance of cells along the epididymis with age. At 12 mo, basal cells in the initial segment emitted pseudopods into the basement membrane. By 18 mo, in the caput epididymidis, clear cells were filled with lysosomes; these cells frequently showed bulging protrusions into the lumen. In the corpus epididymidis, the cytoplasm of principal cells had numerous large lysosomes both below and above the nucleus; apical cells were usually occupied by one giant membranous lysosome. In the proximal cauda, clear cells became filled with dense lysosomes, and principal cells presented large clear vacuoles; debris from spermatozoa was found in the larger vacuoles. In summary, aging of the epididymis was accompanied by the emergence of characteristic features of aging and activation of the immune system. Furthermore, there were many cell- and segment-specific changes. Finally, these changes were not related to the presence of spermatozoa, often preceding their disappearance, thus indicating that there may be an intrinsic mechanism of aging in epididymal epithelial cells.
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PMID:Segment-specific morphological changes in aging Brown Norway rat epididymis. 947 7

Remarkable changes occur during aging in the testis and epididymis of the Brown Norway rat. A dramatic increase in the number of halo cells, which are present in the epididymal epithelium and originate from the immune system, is found in animals of increasing age. Halo cells have been postulated to be either lymphocytes or monocytes. We hypothesized that halo cells are a mixture of different immune cells and that their relative composition changes with age. To verify this hypothesis, markers for helper T lymphocytes, cytotoxic T lymphocytes, B lymphocytes, and monocytes-macrophages were used to identify the major categories of immune cells in the epididymides of Brown Norway rats ranging in age from 3 to 24 mo. The numbers of immunocompetent cells in the epididymis were determined in relation to age, epididymal segment, and luminal content. We found that monocytes, helper T lymphocytes, and cytotoxic T lymphocytes belong to the population of halo cells. In addition, a segment-specific increase with age in the number of these immune cells was noted. Finally, we report a segment-specific recruitment of cytotoxic T lymphocytes and monocytes-macrophages in the epididymal epithelium of aged rats whose epididymal lumen contained few spermatozoa. We postulate that accumulation of damaged epithelial cells and antigens of germ cell origin, leaking through a dysfunctional blood-epididymis barrier, may contribute to the active recruitment of immune cells with age.
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PMID:Distribution of immune cells in the epididymis of the aging Brown Norway rat is segment-specific and related to the luminal content. 1045 48

The Brown Norway rat provides a useful model to study aging of the male reproductive tract because of the selective age-dependent pathological changes that are found in the testis, epididymis, and prostate. In the testis, there is a clear age-dependent decrease in both steroidogenesis and spermatogenesis. In the epididymis, some striking segment-specific changes occur at the histological and biochemical levels prior to the major loss of spermatogenesis. We hypothesized that formation of spermatozoa in the testis and maturation of spermatozoa in the epididymis (ie, acquisition of motility and loss of the cytoplasmic droplet) may be altered during aging. Changes in the morphology of spermatozoa were assessed by light and electron microscopy. Using computer-assisted sperm analysis, the motility parameters of spermatozoa obtained from the caput and cauda epididymidis of young and old Brown Norway rats were compared. In old animals, we also compared the motility of spermatozoa from epididymides adjacent to regressed testes with those from epididymides adjacent to nonregressed testes. There was a marked increase with age in the number of spermatozoa with abnormal flagellar midpieces; the nature of these defects did not change with age. In caput epididymidis, the percentage of motile sperm was similar in young and old rats. In contrast, the percentage of motile spermatozoa was significantly decreased in cauda epididymidis of old rats; spermatozoa from the regressed testis side had altered motility characteristics. Furthermore, in the cauda epididymidis on the regressed testis side of aged Brown Norway rats, the proportion of spermatozoa that retained their cytoplasmic droplet was markedly elevated. Some of these effects are likely due to changes taking place in spermatozoa during the process of spermatogenesis in the testis (eg, formation of the flagellum), whereas others could occur during sperm maturation in the epididymis (eg, acquisition of motility). The multiple effects of aging on sperm morphology, the acquisition of motility, and the shedding of the cytoplasmic droplet clearly indicate that the quality of spermatozoa is affected by aging.
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PMID:Sperm structural and motility changes during aging in the Brown Norway rat. 1122 97

1. An experiment was conducted to evaluate indices of fertility including the sperm penetration (SP) assay as a technique for the prediction of fertility. Forty-eight males consisting of White Leghorn (WL), New Hampshire (NH), Iraqi Brown (IBr) and Iraqi Barred (IBa) (12 males each) and 64 WL hens were divided at random into 4 groups of 4 replicates of 3 males and 4 females each. 2. At the beginning of each week semen was collected from males and pooled by breed of male. Hens in each breeding group were inseminated once weekly, by breeding group, for 4 consecutive weeks with pooled semen from WL, NH, IBr and IBa males (WLxWL, NHxWL, IBrxWL and IBaxWL). 3. The differences in percentage of dead sperm, acrosomal abnormalities, mass motility, individual motility and spermatocrit between the experimental breeds demonstrated the superiority of WL and NH males in all these quantitative characters of the semen. On the other hand, WL hens inseminated with spermatozoa from NH males had significantly more sperm-egg penetration (SP) holes than WL hens inseminated with spermatozoa from other breeds of males. The breed of males used for insemination affected fertility, hatchability and embryonic mortality. 4. The highest fertility and hatchability and lowest embryonic mortality were observed in eggs laid by hens inseminated with spermatozoa from WL and NH males in comparison with hens inseminated with spermatozoa from Iraqi males. 5. There was a strong positive correlation between SP values and fertility for WLxWL, NHxWL, IBrxWL and IBaxWL. The correlation for all breeds combined was also significant. In addition, SP was also positively correlated with hatchability and negatively correlated with embryonic mortality.
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PMID:Sperm-egg penetration in laying breeder flocks: a technique for the prediction of fertility. 1142 37

In the epididymis a series of complex, sequential events transform immature, spermatozoa into mature, motile sperm with fertilizing ability. These events are not intrinsic to germ cells but rather are a direct result of exposure to, and interaction with, the environment created by the epididymal epithelium. Regional differences along the epididymis are essential in the establishment of the environment required for sperm maturation. Although parts of this process have been identified, the molecular basis for the segment-specific differences and how they contribute to the process of sperm maturation, are not yet resolved. The identification of genes expressed in a region-specific manner will provide valuable insight into the functional differences between the regions. To characterize gene expression in the different regions of the epididymis, microarrays containing 1176 rat cDNAs were used to examine gene expression in the initial segment, caput, corpus, and cauda epididymidis of the adult Brown Norway rat. Overall, the cauda epididymidis expressed the most genes and the corpus epididymidis the fewest. A small percentage of genes (3%) were expressed highly (greater than fivefold the average expression on the array) along the tissue. Segment-specific gene expression for genes expressed at high levels was observed in all epididymal segments except the corpus epididymidis. Of the genes on the array, 36% were expressed in all four epididymal segments; expression changes that were a minimum of twofold in either direction between adjacent segments are discussed. The expression of cathepsins and oxidative stress-related genes was investigated. Six of the eight cathepsins on the array (B, C, E, H, L, and K) were expressed above twofold background and showed different levels of expression along the duct with cathepsin K showing the most dramatic change (i.e., a decrease of 87% between the initial segment and the corpus epididymidis). There was also differential expression along the epididymis of many genes associated with oxidative stress defenses. Using the power of expression array technology, we have identified novel transcripts expressed in a segment-specific manner and been able to assess how the expression of several selected gene families is modulated along the epididymis.
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PMID:Dynamic changes in gene expression along the rat epididymis. 1151 30

For optimizing routine freezing of bull semen, we examined three different cryopreservation methods using either TRIS-egg yolk-citrate extender or Biociphos-Plus. Biociphos-Plus (IMV, France) has been marketed as an extender, in which egg yolk is replaced by a sterile soybean extract to reduce the contamination risk derived from animal borne substances. We used 78 bulls of various breeds (Brown Swiss, Holstein, Simmental) between 12 and 23 months of age, and we produced a total of 800-1000 straws (0.25 ml, 20 x 10(6) spermatozoa) from each bull using three different methods. In method A, we used TRIS-egg yolk as extender and packaged at 4 degrees C. In method B, we also used TRIS-egg yolk but packaged at room temperature (RT) between 18 and 22 degrees C. In method C, Biociphos-Plus served as extender and we packaged at RT. We compared methods A, B and C by using post-thaw motility, viability, morphology and osmotic resistance as semen quality parameters. In addition, we recorded 75-day nonreturn rates (NR75) to detect the effect of extenders on fertility. With the exception of primary defects, all laboratory parameters investigated were significantly (P < 0.05) better in methods A (TRIS-egg yolk, 4 degrees C) and B (TRIS-egg yolk, RT), compared to method C (Biociphos-Plus, RT). We recorded no significant difference between methods A and B. We could not verify the differing laboratory results by fertility data (NR75). However, when we analyzed NR75 for a single breed, significant (P < 0.05) differences existed between methods A and B compared to method C in Simmental and Holstein but not in Brown Swiss. We obtained best results in Simmental using method A (69%, n = 3384), while method C (61.4%, n = 763) was superior to methods A (57.6%, n = 698) and B (57.3%, n = 737) in Holstein. After considering various factors like preparation of extender, cost of materials and ambient working temperature, we concluded from our data that bull semen processing using TRIS-egg yolk extender and RT for packaging (method B) produced the best semen quality and field fertility.
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PMID:Comparison of Biociphos-Plus and TRIS-egg yolk extender for cryopreservation of bull semen. 1204 2

Sexual development and pubertal traits were studied in Holstein Frisian (Ho) and Brown Swiss (BS) bulls born and maintained under tropical conditions. Characteristics evaluated every 2 weeks, from 27 to 63 weeks of age, included live weight, scrotal circumference, testicular diameter, semen quality and sexual behavior. Puberty was defined as the age at which a bull first produced an ejaculate containing at least 50 x 106 spermatozoa, with a minimum of 10% progressive motility. Testicular growth was linear in Ho bulls and quadratic in BS bulls. There was no breed difference in age at puberty (Ho, 333 +/- 15.8 days; BS, 311 +/- 10.5 days). However, at puberty, live weight and scrotal circumference tended to be greater in Ho (276 +/- 16.9 kg and 28.4 +/- 1 cm, respectively) than in BS bulls (233 +/- 11.3 kg and 25.9 +/- 0.7 cm, respectively), and testicular diameter was larger for Ho (5.5 +/- 0.24 cm) than for BS bulls (4.8 +/- 0.16 cm). Pooled data for all bulls for semen characteristics at puberty were: volume, 6.3 +/- 0.6 ml; progressive motility, 26.8 +/- 4.4%; sperm concentration, 58.5 +/- 13.9 x 10(6) spermatozoa/ml, and 351.5 +/- 91.2 x 10(6) spermatozoa/ejaculate. These values improved until at least 18 weeks after puberty. Eighty-five percent of bulls mounted heifers by 206 days of age, but only a few bulls had mounts with ejaculation during the study. It was concluded that reproductive development was similar between Ho and BS bulls, but slower than that reported for dairy bulls in temperate areas. Variation in some characteristics, such as scrotal circumference, was observed among bulls within each breed group, which might be of benefit for genetic selection.
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PMID:Sexual development of dairy bulls in the Mexican tropics. 1221 92

The epididymis is the site for the transport, maturation, and storage of spermatozoa. Regulation of epididymal structure and function is highly dependent on the ipsilateral testis. At the molecular level, however, few studies have been undertaken to determine which genes are expressed in the epididymis under testicular regulation. The goal of this study was to identify genes for which expression is regulated after orchidectomy, both throughout the epididymis and in a segment-specific manner. Microarrays spotted with 474 rat cDNAs were used to examine gene expression changes over the first 7 d post orchidectomy in the initial segment, caput, corpus, and cauda epididymidis of the adult Brown Norway rat. Using k-means cluster analysis, we show that four patterns of gene expression are activated in each epididymal segment over the first week following orchidectomy. Transient up-regulation of gene expression in the epididymis after orchidectomy is described for the first time. Potential androgen-repressed genes, including Gpx-1, show increased expression in the epididymis after orchidectomy. Several glutathione-S-transferases and calcium-binding proteins decline throughout the epididymis after orchidectomy, indicating that these may be novel androgen-regulated epididymal genes. Other genes coding for metabolism-associated proteins, transporters, and alpha-1 acid glycoprotein show segment-specific regulation in the epididymis after orchidectomy. Finally, we describe the expression of the previously uncharacterized heat shock proteins, and apoptosis-associated genes in the epididymis after orchidectomy. Thus, gene expression in the epididymis is differentially affected over time after orchidectomy. These results provide novel insight into androgen-dependent and segment-specific epididymal function.
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PMID:Gene expression is differentially regulated in the epididymis after orchidectomy. 1258 75

We studied expression of protooncogene c-kit receptor in Brown Norway rat Rattus norvegicus testis during different stages of postnatal development. Several regions from within the c-kit gene encompassing different domains were amplified employing reverse transcriptase polymerase chain reaction, and the resultant amplicons were cloned and characterized. Maximum expression of c-kit was observed in the testes during the days 10 to 30, suggesting its involvement in transition of primary spermatocytes towards formation of mature spermatozoa. Multiple novel transcripts originating from the extracellular domain were also identified, though their functions remained unknown. The evolutionary divergence of c-kit cDNA of 10 other vertebrates was studied using their sequences from the GenBank. Analyses of c-kit cDNA and its protein sequences in rat and related genomes showed organizational uniqueness across the species. Construction of phylogenetic tree, based on c-kit cDNA and protein sequences delineated all the species successfully and was found to be in accordance with the established positioning of these animals. The organizational uniqueness of c-kit cDNA sequences from the extracellular domain may be exploited as a useful tool in delineating phylogenetic relationship of different species.
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PMID:Expression of protooncogene c-kit receptor in rat testis and uniqueness of extracellular domain across the species with potential in molecular phylogeny. 1496 71


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