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1.
This study was designed to test the hypothesis that sperm‐bound IgG and IgA decrease binding of bull spermatozoa to oviductal epithelial cells in vitro. Three ejaculates were cryopreserved from each of four antisperm antibody (ASA)‐negative satisfactory breeder bulls. Bulls were then immunized with autologous spermatozoa, and three ASA‐positive ejaculates were cryopreserved from each bull post‐immunization. First, microscopy methods were compared to select the most appropriate assay for evaluation of oviductal binding index (BI). The BI did not differ when the evaluation was performed under fluorescence microscopy (131.1 sperm/mm2; 62.5–251.1 sperm/mm2), phase‐contrast microscopy (160.5 sperm/mm2; 56.8–397.4 mm2) or their combination (116.4 sperm/mm2; 56.8–249.6 sperm/mm2) (Median; IQR). The combination of microscopy methods was selected as it allowed better visualization of cells. Then, BI was compared between ASA‐negative and ASA‐positive ejaculates, and the association between BI and ASA binding was evaluated. The BI was less in ASA‐positive (114.9; 0 to 201.8 sperm/0.1 mm2) than ASA‐negative samples (218.9; 24.7 to 276.8 sperm/0.1 mm2) (P = 0.0002). This reduction in BI was significant in three of the four bulls. Regression analysis identified a negative association between BI and the percentage of IgG‐bound (p = 0.013) but not IgA‐bound spermatozoa. In conclusion, sperm‐bound IgG decreased the ability of bovine spermatozoa to bind to oviductal epithelial cells in vitro.  相似文献   

2.
A study between August 1995 and December 1997 included 343 dairy cattle on 20 farms in the Dar es Salaam region and 2289 zebu cattle on 39 bomas in the Lugoba area (coast region). The aim was to establish the prevalence of bovine tuberculosis (Mycobacterium bovis) and bovine brucellosis (Brucella abortus). In the single intradermal tuberculin test (SIT), 0.9% (3/343) of the animals in Dar es Salaam tested positive and 1.2% (4/343) were doubtful. Positive reactors were found in 10% (2/20) of the farms. In the Lugoba area, 0.6% (14/2206) were positive and 6.8% (149/2206) doubtful, positive cases being found in 21% (8/39) of all bomas. In the slow agglutination test (SAT) for B. abortus, 14.1% (48/341) of the serum samples reacted positively in Dar es Salaam and 2.3% (8/341) were doubtful. Positive SAT reactors were identified on 25% (5/20) of the dairy cattle farms. In the Lugoba area, 12.3% (273/2221) proved to be positive SAT reactors and doubtful reactions were observed in 2.9% (64/2221). SAT-positive animals were detected on 87% (34/39) of all bomas. The prevalence in single herds in Dar es Salaam varied from 4.3% to 5.3% for the SIT and from 2.2% to 50% for the SAT. The prevalence in single herds in Lugoba area was between 1.1% and 2.9% for SIT and from 1.4% up to 62.1% for SAT. The two cattle populations differed significantly (p<0.001) in the prevalence of both bovine tuberculosis and bovine brucellosis. Two cows that were positive reactors were slaughtered and subjected to post-mortem examination, and organ samples were bacteriologically cultured. The occurrence of Mycobacterium tuberculosis was confirmed by polymerase chain reaction (PCR) in both cows.  相似文献   

3.
The effect of melatonin implants administered during non‐breeding season in Rasa Aragonesa rams on sperm motility parameters and other reproductive traits was assessed. In a first experiment, two Rasa Aragonesa rams were implanted (with melatonin group M), remaining other two males as control group (C). Semen of each group was collected from 1 May to 23 June, twice or three times a week, and motility parameters were assessed using a computer‐assisted sperm analysis system. Melatonin increased the percentage of progressive motile spermatozoa, particularly during 46–75 days after melatonin implantation (p < 0.01). In experiment 2, M and C in vitro fertilization ability had been determined by zona‐pellucida binding assays, using spermatozoa from experiment 1, obtained 60–70 days after melatonin was implanted. A significantly higher number of spermatozoa attached per oocyte was observed in frozen‐thawed immature ovine oocytes incubated with sperm from M animals than in those incubated with sperm from the C group (p < 0.01). Finally, a field assay (experiment 3) was performed. In this case, five Rasa Aragonesa rams were implanted with melatonin and three remained as control group. Sperm doses from those animals were used for artificial insemination of 2608 Rasa Aragonesa ewes from 39 different farms at non‐breeding season. Fertility, litter size and fecundity were studied. Semen from melatonin implanted rams seemed to increase both fertility and fecundity in ewes inseminated with spermatozoa obtained 46–60 days after implantation (p < 0.1). Thus, melatonin treatment in rams during non‐breeding season modifies sperm motility parameters and seems to improve the fertilization parameters obtained.  相似文献   

4.
During an experimental reproduction of CBPP, 5 inoculated cows and 5 contacts cows were bled twice a week and antibodies research was performed using complement fixation test (CFT), passive haemagglutination test (PHAT) and slide agglutination test (SAT). In the same period, trachobronchial washings were performed weekly to detect and to count Mycoplasma mycoides subsp. mycoides SC. For four months these tests were used to compare antibodies kinetics with kinetics of mycoplasmas excretion. With titer over 40 as threshold of positivity, PHAT detects antibodies earlier than CFT and SAT at the beginning of infection, but fails to detect chronic carriers. In contacts cows no failure has been observed with CFT and SAT except during prodromic phase of infection. These results are valid for natural infections. However, for inoculated animals, most serious failures to detect infected cows occur.  相似文献   

5.
An interesting pattern of tail-in, head-out sperm agglutination was identified in a Brucella canis seronegative subfertile dog. Centrifuged seminal plasma from this dog could induce a similar pattern of agglutination in six other dogs, but not in ejaculates from a single stallion and two rams. The agglutination pattern was short-lived and appeared to depend on motility of spermatozoa, although intensity of agglutination may have been affected by concentration of agglutinating factor.  相似文献   

6.
应用精子凝集试验测定五个牧场共33头屡配不孕奶牛和86头正常牛的血清抗精子抗体水平,发现30头屡配不孕牛的血清稀释到1∶8时,仍可见明显的凝集反应,而85头正常牛的血清稀释到1∶8时,凝集反应阴性。因此,将1∶8的血清稀释倍数作为诊断母牛是否不孕的指标,阳性和阴性准确率分别可达90.91％和98.84％。此外,用微滴板酶免疫法测定8头屡配不孕牛和30头正常牛的血清孕酮含量分别为9.83±1.85和1.95±0.74n g/ml,两者统计差异显著(P<0.01)。  相似文献   

7.
To examine the efficiency of retrograde sperm transport following intraperitoneal insemination, live and dead spermatozoa were used at different concentrations, and sperm recovery from cervical mucus (0.5 ml) 2, 6, 12 and 24 h following insemination was evaluated. Forty lactating Friesian cows, in their second to fourth lactation period, were used in this experiment. Thirty-six cows received intraperitoneally either live or dead spermatozoa. Each group of six cows received one of three total sperm numbers of 30, 45 and 90 million. Four cows were inseminated with 90 million spermatozoa into the uterus and served as a control group. All cows were inseminated towards the end of oestrus. After intrauterine insemination sperm recovery declined, but motile and/or immotile spermatozoa were recovered from all cows at any time. In cows inseminated intraperitoneally, sperm was recovered from the cervix at 6-24 h when 90 million were inseminated. A greater number of spermatozoa was recovered after dead rather than after live sperm inseminations. Only immotile, intact or broken spermatozoa and tail-less heads were recovered after intraperitioneal insemination using either live or dead spermatozoa. No sperm was recovered for 30 and 45 million inseminations. Our results show that, following intraperitoneal insemination, there is passive sperm transport from the peritoneal cavity to the genital tract close to the time of ovulation, and suggest a higher sperm retention in the genital tract when live as opposed to dead spermatozoa are used.  相似文献   

8.
A mongrel dog, aged 2 years, was found to have only a small number of sperm, immobilization of all sperm, and many sperm agglutinations in its ejaculates, and scrotal palpation revealed a small nodule in the left cauda epididymis. Addition of the dog's seminal plasma or serum to the semen of 2 normal dogs caused immobilization and agglutination of their sperm. Histological examination showed that the nodule was a sperm granuloma. Many lymphocytes were seen in the stroma around the sperm granuloma. Anti-sperm antibodies are presumed to be present in the semen and serum of the asthenozoospermic dog.  相似文献   

9.
The ability to preselect or predetermine the sex of offspring prior to conception is a highly desired technological tool for assisted female breeding programs specifically for milk production, and in males, for meat production and increasing livestock numbers. The current technology is based on the well-known differences in X- and Y-sperm in the amount of DNA. The technology uses modified flow cytometric instrumentation for sorting X- and Y-bearing sperm. The method can be validated on the basis of live births, laboratory reanalysis of sorted sperm for DNA content, and embryo biopsy for sex determination. Currently, the sex of animals has been predetermined with 90 % accuracy by sexing spermatozoa. In the bovine breeding industry, flow cytometric sperm sexing has not fulfilled its original promise. Sexed sperm doses are too expensive for widespread application while the fertility of sexed sperm doses is lower than unsexed ones. Essentially all bovine sexed semen is frozen and then applied through artificial insemination (AI) or in vitro fertilization. There is still a need in the animal breeding industry to develop a technique for sperm sexing that provides sufficient spermatozoa for AI doses, does not compromise sperm fertility, and is widely applicable to a range of species. In this review, we will summarize the current state-of-the-art in sex preselection in domestic animals and some wildlife species using flow cytometric sperm-sorting of X from Y sperm based on DNA differences.  相似文献   

10.
Studies have demonstrated that oviductal fluid (ODF) proteins associate with eggs of numerous species including the bovine. In this study, the association of three ODF proteins, the bovine oestrus‐associated protein, osteopontin (OPN), lipocalin‐type prostaglandin D synthase (L‐PGDS), with the bovine zona pellucida (ZP) was demonstrated by immunohistochemistry and western blot. The biological function of ODF derived egg‐associated OPN and L‐PGDS in sperm binding, fertilization and embryonic development was also explored. In vitro matured bovine oocytes were pre‐incubated with ODF collected by cannula from cows in oestrus, or ODF with antibodies to OPN, L‐PGDS and bovine serum albumin (BSA). Following incubation, oocytes were inseminated with 1 × 105 frozen‐thawed spermatozoa, and they were evaluated for sperm binding, fertilization and embryonic development in vitro. Pre‐treatment of ODF with antibodies to all of proteins reduced sperm binding to the ZP and fertilization in vitro. Cleavage rates were not significantly different among incubations, but rates of embryo development were significantly decreased. We conclude that antibodies to OPN, L‐PGDS and BSA react with oocytes incubated with ODF and inhibit sperm binding, fertilization and embryonic development in vitro, suggesting a potential role of these proteins in these events.  相似文献   

11.
This study was designed to investigate the effects of feeding‐protected conjugated linoleic acid (CLA) on the semen production and sperm freezability in Holstein bulls. Twelve bulls were randomly assigned to two groups (n = 6 per group). Bulls received the normal diet (control group) or the normal diet top‐dressed with 50 g of CLA (treated group) for 10 weeks. The control group received 40 g/day calcium soap of fatty acid. Fresh and post‐thaw semen quality was assessed on ejaculates collected at the 0, 4, 6, 8 and 10 week of supplementation. Semen evaluations including sperm concentration, motion characteristics (subjective and computer‐assisted), viability (Eosin–Nigrosin), membrane integrity (hypo‐osmotic swelling test) and abnormality were conducted. Semen volume, sperm concentration and total sperm output were not affected by dietary treatment (p > .05). The proportion of spermatozoa with abnormal morphology in fresh semen significantly increased (p < .05) in the CLA‐fed group compared to control group. Also, in CLA‐fed group, the proportion of post‐thaw spermatozoa with abnormal morphology at week 10 of trial was significantly higher in CLA than control group (p < .05). Progressive motility tended to be increased in the CLA‐fed group, although dietary supplementation did not affect other CASA parameters or viability in fresh and frozen‐thawed sperm. In this study, CLA supplementation had little positive effect on fresh or post‐thaw sperm quality of Holstein bulls.  相似文献   

12.
Intracytoplasmic sperm injection (ICSI) is an assisted reproduction tool with several applications. Its effectiveness in bovines is lower than that in other species, mainly because of difficulties in the decondensation of the sperm nucleus after injection, and the presence of the acrosome and the plasma membrane which remain intact in this procedure. In this study, we assessed the effect of lysolecithin (LL) and Triton X‐100 (TX), in combination with glutathione (GSH) as sperm pretreatments prior to ICSI. The GSH‐LL and GSH‐TX groups showed 0% of spermatozoa with intact membrane (SYBR 14+/PI), in comparison with the control (63.3%) and GSH (65.7%) groups. The proportions of spermatozoa with damaged acrosome membrane in the GSH‐LL, GSH‐TX, GSH and control groups were 46%, 35.9%, 10.5% and 7.5%, respectively. Sperm chromatin decondensation analysis showed that the groups incubated for 3 hr with GSH presented greater decondensation (p < .05). Although fertilization was improved in all treatment groups evaluated, no differences were observed in the cleavage rate 72 hr after activation in the GSH (73.7%), GSH‐LL (80.2%) and GSH‐TX (77.8%) groups compared to the control (66.3%), neither in the blastocyst rate on day 8 (24.0%, 26.2%, 27.1% and 28.4% for the control, GSH, GSH‐LL and GSH‐TX groups, respectively). No differences were also observed in the total number of cells in all groups. In conclusion, although these sperm treatments promoted nuclear decondensation and induced plasma membrane disruption, these effects were not sufficient to improve bovine embryonic development after ICSI.  相似文献   

13.
Development of new semen cryopreservation techniques improving sperm survival and ensuring availability of viable spermatozoa for a prolonged time‐period after AI is promising tools to reduce sensitivity of timing of AI and enhance overall fertility. The SpermVital® technology utilizes immobilization of bull spermatozoa in a solid network of alginate gel prior to freezing, which will provide a gradual release of spermatozoa after AI. The objective of this study was to compare post‐thaw sperm quality and in vitro sperm survival over time of Norwegian Red bull semen processed by the SpermVital® (SV) technology, the first commercialized production line of SpermVital® (C) and by conventional procedure applying Biladyl® extender (B). Post‐thaw sperm motility was not significantly different between SV, C and B semen (p > .05). However, sperm viability and acrosome intactness were higher for SV than C and B semen (p < .05). Small differences in DNA quality were observed (p < .05). Sperm viability after storage in uterus ex vivo was higher for SV than for C semen (p < .05). Furthermore, sperm survival in vitro over time at physiological temperature was significantly higher for SV semen than C semen as well as B semen during the incubation period of 48 hr (p < .05). In conclusion, the SpermVital® technology is improved and is more efficient in conserving post‐thaw sperm quality and results in higher sperm viability over time in vitro for SV than for C and B semen.  相似文献   

14.
This study assessed the influence of three different anaesthetic protocols on semen quality obtained from the epididymis. Sixty male dogs undergoing to routine sterilization were assigned to three anaesthetic protocols: thiopental group (TG, n = 20), propofol group (PG, n = 20) and ketamine–dexmedetomidine group (KDG, n = 20). Immediately after orchidectomy, the cauda epididymides and vas deferent ducts were isolated and then a retrograde flushing was performed to collect spermatozoa. In experiment 1, after the initial evaluation of the semen (sperm concentration, sperm motility and the percentages of live spermatozoa, abnormal spermatozoa and acrosome membrane integrity), semen samples were diluted in Tris‐glucose‐egg yolk extender and chilled for 48 hr, and the sperm motility was assessed at 6, 24 and 48 hr. In experiment 2, semen samples were diluted in Tris‐glucose‐egg yolk extender and chilled for 24 hr, and then samples were frozen in two extenders with different glycerol concentrations, to reach a final concentration of 50–100 × 106 spermatozoa ml?1, 20% egg yolk, 0.5% Equex and 4% and 5% glycerol, respectively. Mean values of total sperm concentration, sperm viability and the percentages of intact acrosome and abnormal spermatozoa were not significantly different between experimental groups, and therefore, the anaesthetic protocols assessed did not affect sperm parameters mentioned above. However, our study confirmed a detrimental effect of the use of thiopental (TG) over the total sperm motility (p < 0.05) and progressive sperm motility (p < 0.05) of the fresh and chilled epididymal sperm samples. The anaesthetic protocols including the application of propofol or ketamine–dexmedetomidine can be used to recover sperm in domestic canids without significant changes in sperm quality compared when semen is collected routinely and these techniques could be applicable to endangered wild canids.  相似文献   

15.
The aim of the study was to determine the relation between the semen quality, frequency of sperm defects, sperm dimensions and shape, and the ejaculate volume of Large White and Landrace boars. A total of 648 ejaculates collected from 31 Large White and 30 Landrace boars were divided into three groups according to the criterion of the ejaculate volume. In this study Landrace boars produced ejaculates with higher volume, sperm concentration, and total numbers of spermatozoa than Large White boars. Landrace boars also showed a lower frequency of sperm with morphological abnormalities (P < 0.05). Landrace boars sperm had larger heads, which were by 0.15 μm longer, and by a larger perimeter and area (P < 0.05). Landrace boar spermatozoa also had a longer flagellum and were generally larger and by 2.07 μm longer than Large White boar sperm (P < 0.05). Significant differences were also found in the shape of sperm of the two breeds (P < 0.05). Landrace boars sperm had more elongated heads, and the ratio of head size to flagellum length was lower than in Large White boars sperm (P < 0.05). Sperm from ejaculates with low volume had a shorter flagellum and a greater head length/flagellum length ratio than sperm from medium- and high-volume ejaculates (P < 0.05).  相似文献   

16.
This study investigates the effects of iodixanol supplementation in varied concentrations to Tris egg yolk (TEY) extender on the quality and fertilization ability of frozen–thawed sperm of Thai native bulls. Each ejaculate was divided into four different groups, as follows: sperm were treated with TEY extender (control group) and TEY extender supplemented with three different concentrations of iodixanol (1.25%, 2.50% and 5.00%). Semen straws were frozen in liquid nitrogen vapor. After thawing, sperm motility characteristics, viability, plasma membrane integrity and acrosome integrity were determined. Also, frozen–thawed spermatozoa from all groups were used for in vitro fertilization and artificial insemination (AI) in natural estrus Thai native cows. The results showed that the post‐thaw quality of the 2.50% iodixanol group was superior to the other iodixanol groups (P < 0.05). However, iodixanol had no beneficial effect on post‐thaw sperm in vitro fertilization ability and pregnancy rate after AI (P > 0.05). It can be concluded that the supplementation of 2.50% iodixanol extender significantly improves the progressive motility, viability, plasma membrane integrity and acrosome integrity of cryopreserved semen from Thai native bulls, but it has no beneficial effect on in vitro fertilization ability and pregnancy rate after AI.  相似文献   

17.
Seminal plasma (SP) proteins interact with sperm plasma membrane (PM) modulating its functionality. It has been shown that SP proteins can reverse the damage caused by freeze‐thaw; however in these studies, SP has been added to washed sperm (i.e., cells depleted from homologous SP and extender). The aim of the current study was to assess whether the egg yolk‐based extender (EY) modifies SP ability to ameliorate sperm parameters in frozen‐thawed ram spermatozoa. Ejaculates were diluted in EY or soybean lecithin‐based extender (SL) and evaluated before and after freezing to measure the cell damage according to the extender. Even when all classical parameters decreased after freezing, as expected (p < .05), there was no effect of the extender. SP treatment was applied after freeze‐thaw. Sperm were incubated with SP (20% v/v) in the presence of either EY or SL, and sperm parameters were assessed after thawing compared with the same treatments after Percoll sperm selection (washed). Treatments with 20% SP improved sperm total and progressive motility compared with controls regardless of washing and extender (p < .05); however, washed sperm showed higher percentage of total sperm motility compared with those unwashed (p < .05). Moreover, treatment with 20% SP showed significantly higher percentages of PM integrity, sperm with intact acrosomes, integrity of chromatin and non‐capacitated sperm in samples diluted with EY when washed before treatment compared with the other conditions (p < .05). It was concluded that the presence of the extenders and particularly egg yolk alters the SP capacity to reduce the cryodamage.  相似文献   

18.
The objectives of this study were to investigate the effects of polyvinyl alcohol (PVA) as a chemically defined compound in egg yolk (EY)‐free extender by determining the appropriate concentration of PVA and the effect of pH adjustment in EY‐free PVA extenders on dog spermatozoa. Spermatozoa (1 × 108 cells/ml) were frozen with EY‐free extenders supplemented with 0 (control), 0.025, 0.05, 0.1, 0.2 or 0.3 g/100 ml PVA. Sperm progressive motility (PM) was assessed immediately after thawing (IAT) and post‐thaw incubation (PTI), while viability, acrosome integrity and reactive oxygen species (ROS) levels were evaluated after PTI. Additionally, spermatozoa were frozen using EY‐free PVA extenders before pH adjustment (6.45) and after adjustment of pH (6.85). Viability, PM, ROS and gene expression (BCL2 and SMCP) were assessed. Supplementation with 0.05 g/100 ml or more PVA significantly increased PM compared to the control group in the IAT and PTI. Post‐thaw incubation significantly increased sperm motility in all groups. The acrosome integrity in all PVA groups was higher (p < .05) than the control without an effect on ROS and viability. Adjustment of the pH to 6.85 improved (p < .05) sperm PM compared to the non‐adjusted groups without affecting viability, ROS or expression of BCL2 and SMCP. We suggest that PVA supplementation in EY‐free Tris extenders can effectively protect dog spermatozoa during freezing and can maintain higher motility and acrosome integrity. Adjustment of pH in EY‐free PVA extenders can improve post‐thaw sperm motility. Therefore, PVA can be used as a compound in EY‐free extender for the cryopreservation of dog spermatozoa.  相似文献   

19.
Sperm DNA fragmentation is a condition that interferes directly in the reproductive efficiency. Currently, there are several methods for assessing the sperm DNA integrity, such as Alkaline Comet, TUNEL and Sperm Chromatin Structure Assay. However, many of these techniques are laborious and require high‐precision equipment. Thus, the development of new techniques can optimize the evaluation of sperm DNA damage. Therefore, the aim of this study was to standardize the toluidine blue (TB) stain technique for the analysis of DNA fragmentation of dog, cat, bull, stallion and ram spermatozoa. For this purpose, we used six animals of each specie (n = 30), in reproductive age. Sperm was collected by different methods according to the particularities of each species, and such samples were divided into two aliquots: a sperm sample was kept at 5°C (considered as intact sperm DNA), and the remaining samples were submitted to the induction of DNA fragmentation by exposure to ultraviolet light for 4 hr. Samples were then mixed with the intact sample to obtain known and progressive proportions of sperm with fragmented DNA (0%, 25%, 50%, 75% and 100%). Semen smears were performed and subjected to staining with TB. Blue‐stained spermatozoa were considered to have DNA fragmentation. We observed high linear regression coefficients between the expected proportion of damaged DNA and the results of TB for dog, cat, ram, bull and stallion samples. In conclusion, TB stain was considered a fast and effective technique for the study of spermatozoa DNA in several species.  相似文献   

20.
In the period 1996–2006 two specific sperm defects, the knobbed acrosome (KA) defect and the immotile short‐tail sperm (ISTS) defect, showed a strong negative association with fertility in Finnish breeding boars. In this study, we examined the incidence of these two sperm defects in two pig breeds, their effects on fertility and their associations with sperm morphology and testicular histology. Semen samples from 2048 (1097 Yorkshire, 951 Landrace) boars were collected. None of the Landrace boars revealed either the KA defect or the ISTS defect. Of the Yorkshire boars, 0.8% were afflicted with the KA defect and 7.6% with the ISTS defect. Boars diagnosed with the ISTS defect produced no litters. Fertility data were available from two artificially inseminated (AI) boars and six farm breeding boars affected with the KA defect. Breeding boars with 45–81% knobbed spermatozoa (n = 6) did not produce any litters out of 71 sows bred. AI boars with 25–30% knobbed spermatozoa had a poor non‐return rate (on average 47% compared with 85% for normal control boars) and produced small litters, on average 2.5 piglets less than other boars of the same breed. Morphometry of testicular tissue and distribution of different cells in the seminiferous tubules were examined in nine boars. Boars with the KA defect had a smaller diameter of the seminiferous tubules (p < 0.05) and a lower number of Sertoli cells (p < 0.05) than controls. ISTS boars, in turn, had a significantly lower number of elongated spermatids (p < 0.05), and they also produced on average only 12% of the spermatozoa of normal boars. The ISTS defect is a manifestation of an autosomal recessive disease caused by an insertion in the KPL2 gene in porcine chromosome 16. Although we tried to map the KA defect, its aetiology remains unclear.  相似文献   

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