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1.
The study was conducted to investigate the effect of relaxin on motility, acrosome reaction (AR), viability and utilization of glucose in fresh and frozen‐thawed bovine spermatozoa. Both semen samples were washed twice through centrifugation (5 min at 600 g), and preincubated for 1 h at 39°C for swim up. The swim‐up separated spermatozoa were resuspended in a sperm Tyrode's albumin lactate pyruvate (Sp‐TALP) medium containing 0 (control) and 40 ng/mL porcine relaxin and incubated for 0–6 h. Sperm motility was determined on the basis of movement quality examined by a phase contrast microscope. Sperm viability and AR were evaluated by using the triple staining technique. The incorporation and oxidation of 14C‐glucose was assessed by a liquid scintillation counter. Motility was improved (P < 0.05) in both fresh and frozen‐thawed spermatozoa by the addition of relaxin to the Sp‐TALP medium, whereas relaxin showed no significant effect on viability in either fresh or frozen‐thawed spermatozoa. The percentage of AR increased (P < 0.05) when fresh or frozen‐thawed spermatozoa were incubated with relaxin. In contrast, the incorporation and oxidation of 14C‐glucose increased (P < 0.05) in both kinds of spermatozoa incubated with relaxin. Thus the results demonstrated that the addition of relaxin to the Sp‐TALP medium increased the motility, AR and utilization of glucose in fresh and frozen‐thawed bovine spermatozoa.  相似文献   

2.
In this study, the relations between fertility (56‐day non‐return rates, 56‐day NRR) after artificial insemination (AI) and bull sperm characteristics post‐thaw, after swim‐up and after co‐incubation with heparin (Hep) and hyaluronan (HA), respectively, were determined, attempting to determine if such a procedure could be of value to evaluate the potential fertilizing ability of frozen‐thawed AI bull spermatozoa. Spermatozoa from 20 semen batches derived from 20 Swedish Red and White AI bulls ranging widely in their field fertility after AI (55–79% 56‐day NRRs) were evaluated with regards to post‐thaw motility, membrane integrity, and migration through a simple swim‐up procedure. Sperm viability and capacitation status were evaluated by two different vital staining procedures and chlortetracycline hydrochloride staining. Sperm motility and membrane integrity post‐thaw (e.g. indicators of sperm viability) were significantly correlated (r = 0.53, p < 0.05 and r = 0.59, p < 0.01, respectively) with fertility. Heparin (5 µg/ml) significantly (p lt; 0.001) increased the frequencies of capacitation and acrosome‐reaction (AR) among swim‐up separated spermatozoa, whereas HA at a concentration of 50 ng/ml did not have any significant capacitating effect. The incidences of capacitated or AR‐spermatozoa following Hep‐treatment were not correlated with fertility. On the other hand, the percentage of viable spermatozoa was significantly (p < 0.001) lower in Hep‐treated samples than in control and HA‐treated samples and was significantly (r = 0.49, p < 0.05) correlated with fertility after AI (56‐day NRR). The results indicate that the percentage of viable spermatozoa after swim‐up separation and heparin‐exposure from a selected population of AI bulls were significantly and positively related to the AI fertility of the donors and thus could be used as a parameter to determine the fertilizing ability of frozen—thawed AI bull spermatozoa.  相似文献   

3.
Oxytocin (OXT) contained in boar semen is known to produce uterine contraction; therefore, we hypothesized that the co‐injection of OXT with sperm would improve artificial insemination (AI) using liquid or frozen‐thawed boar sperm. We initially examined whether OXT added to semen extender improved sperm transport to the oviduct. Although the addition of OXT did not affect the fresh or frozen‐thawed sperm motility or acrosomal integrity, it significantly increased the number of sperm in the oviduct at 6 h after AI injection with OXT, as compared with the control (P < 0.05). Moreover, some sperm were observed in the sperm reservoir of the isthmus in the OXT treatment group, whereas few sperm were observed in the control. When OXT was added to the semen extender immediately prior to AI, the conception rates were significantly higher in both fresh semen and frozen‐thawed semen than in the control group (P < 0.05: liquid, 87.5% vs. 70.5%; frozen‐thawed, 89.8% vs. 75.0%). From these results, we concluded that the addition of OXT to the semen extender assisted in sperm transportation from the uterus to the oviduct, which resulted in improved reproductive performance.  相似文献   

4.
In this study, we tested the hypothesis whether the neutral Comet assay (NCA) and the Sperm‐Sus‐Halomax (SSH) test kit could provide similar measurements of post‐thaw DNA fragmentation of boar spermatozoa. Whole ejaculates or sperm‐rich fractions of boar semen were frozen in an extender containing lactose, lipoprotein fractions isolated from ostrich egg yolk (LPFo), glycerol (lactose‐LPFo‐G) or in a standard boar semen extender (K3), without the addition of cryoprotective substances. In all boars, both the NCA and SSH test showed similar levels of post‐thaw sperm DNA fragmentation in samples of the same ejaculates, regardless of the ejaculate collection procedure and extender. Yet, the levels of post‐thaw sperm DNA damage, detected by the NCA and SSH test, were more accentuated in spermatozoa frozen in the absence of cryoprotective substances. Both the NCA and SSH detected variations among individual boars in terms of post‐thaw sperm DNA fragmentation. Agreement between the measurements of the NCA and SSH was confirmed by scatter plots of differences, suggesting that the DNA integrity tests could detect the same sperm populations, which were susceptible to cryo‐induced DNA damage. The findings of this study indicate that the NCA and the SSH test are effective in detecting similar levels of sperm DNA fragmentation and reinforce their importance in the assessment of frozen‐thawed boar semen quality.  相似文献   

5.
Our previous report indicated that addition of Orvus ES Paste (OEP) to the extender of frozen canine semen protected acrosomes and maintained sperm motility after thawing. In this study, artificial insemination (AI) using the frozen semen was carried out. The frozen semen was prepared using egg yolk Tris-fructose citrate, and the final concentrations of glycerol and OEP were 7% (v/v) and 0.75% (v/v), respectively. AI was performed during the optimal mating period predicted from the peripheral plasma progesterone level. In intrauterine insemination (IUI), the bitches were laparotomized and 1 x 10(8) spermatozoa were infused into one of the uterine horns. In insemination of non-OEP supplemented semen, 3 x 10(8) spermatozoa were inseminated. In intravaginal insemination (IVI), 10-40 x 10(8) spermatozoa were inseminated. Conception was obtained in nine of 10 bitches (90.0%) that underwent IUI. The number of newborns was from 1 to 7 (mean 3.6 +/- 0.9). The mean ratio of the number of puppies to the number of ovulations in the inseminated uterine horn was 71.8%. The number of puppies did not exceed the number of ovulation in the inseminated uterine horn. Conception using non-OEP supplemented frozen semen was unsuccessful in all four bitches. In IVI, conception was not obtained in any of the six bitches that received insemination of 10 x 10(8) or 40 x 10(8) spermatozoa, but two of three bitches that received insemination of 20 x 10(8) spermatozoa were fertilized. It was shown that a high conception rate can be obtained by IUI using OEP-supplemented frozen canine semen. Developmenmt of a non-surgical method of IUI and a method of freezing canine sperm applicable to IVI is necessary.  相似文献   

6.
This study assessed the effect of different semen storage temperatures and the influence of semen pooling in semen viability. In experiment 1, semen samples (n = 30) of five Majorera bucks were individually processed [Individual semen (IS)] and after the first dilution (Tris‐yolk extender), semen‐diluted aliquots from each male were pooled semen (PS). Thereafter, semen samples (IS and PS) were preserved as fresh semen (37 and 20°C), chilled semen (4°C) and frozen semen. Sperm motility and the percentage of abnormal sperm cells and intact membrane acrosomes were defined. Semen preservation at 20 and 4°C did not modify the quality of spermatozoa for the first 24 h, but the conservation at 37°C caused a dramatic fall in the semen motility from 12 h onwards. Furthermore, the longevity of frozen‐thawed semen was limited to 4–6 h. No differences were observed in semen parameters when PS was compared with semen from individual males in any of the preservation protocols assessed. In experiment 2, 120 goats were distributed in four experimental groups: in group fresh individual semen (FIS, n = 30) and group frozen‐thawed individual semen (FTIS, n = 30), does were transcervically inseminated with fresh semen and frozen‐thawed semen from each individual male, respectively, and in group fresh pooled semen (FPS, n = 30) and group frozen‐thawed pooled semen (FTPS, n = 30), goats were transcervically inseminated with FPS and FTPS, respectively. The kidding rate was very close in the FIS and FPS groups (70.0% and 73.7%, respectively), and no significant differences were observed in the fertility rate between FTIS and FTPS. The results of this study confirmed that semen samples may be preserved satisfactorily for 24 h both at 20 and 4°C. In addition, the mixture of semen of different bucks did not significantly modify the semen parameters when compared with semen from individual males.  相似文献   

7.
Sperm DNA fragmentation is one of the major causes of infertility; the sperm chromatin dispersion test (SCDt) evaluates this parameter and offers the advantage of species‐specific validated protocol and ease of use under field conditions. The main purpose of this study was to evaluate sperm DNA fragmentation dynamics in both fresh and post‐thaw bottlenose dolphin sperm using the SCDt following different cryopreservation protocols to gain new information about the post‐thaw differential sperm DNA longevity in this species. Fresh and cryopreserved semen samples from five bottlenose dolphins were examined for sperm DNA fragmentation dynamics using the SCDt (Halomax®). Sperm DNA fragmentation was assessed immediately at collection and following cryopreservation (T0) and then after 0.5, 1, 4, 8, 24, 48 and 72 h incubation at 37°C. Serially collected ejaculates from four dolphins were frozen using different cryopreservation protocols in a TES‐TRIS‐fructose buffer (TTF), an egg‐yolk‐free vegetable lipid LP1 buffer (LP1) and human sperm preservation medium (HSPM). Fresh ejaculated spermatozoa initially showed low levels of DNA fragmentation for up to 48 h. Lower Sperm DNA fragmentation (SDF) was found in the second fresh ejaculate compared to the first when more than one sample was collected on the same day (p < 0.05); this difference was not apparent in any other seminal characteristic. While there was no difference observed in SDF between fresh and frozen–thawed sperm using the different cryopreservation protocols immediately after thawing (T0), frozen–thawed spermatozoa incubated at 37°C showed an increase in the rate of SDF after 24 h. Sperm frozen in the LP1? buffer had higher levels (p < 0.05) of DNA fragmentation after 24‐ and 48‐h incubation than those frozen in TTF or HSPM. No correlation was found between any seminal characteristic and DNA fragmentation in either fresh and/or frozen–thawed samples.  相似文献   

8.
Melatonin is known to protect sperm against freezing-inflicted damage in different domestic species. The aim of the study was to evaluate the effect of supplementation of semen extender with melatonin on the quality and DNA integrity of cooled and frozen/thawed rabbit spermatozoa. We also investigated whether the addition of melatonin to the semen extender could improve the fertility of rabbit does artificially inseminated with frozen/thawed semen. Semen samples collected from eight rabbit bucks were pooled and then diluted in INRA-82 supplemented either with (0.5, 1.0 or 1.5 mM) or without (0.0 mM) melatonin. Diluted semen was cooled at 5°C for 24 hr. For cryopreservation and based on the first experiment's best result, semen samples were diluted in INRA-82 in the presence or absence of 1.0 mM melatonin and then frozen in 0.25 ml straws. Following cooling or thawing, sperm quality and DNA integrity were evaluated. Furthermore, the fertility of frozen/thawed semen was investigated after artificial insemination. Supplementation of semen extender with 1.0 mM melatonin improved (p < .05) motility, viability, membrane and acrosome integrities in cooled semen compared with other groups. Sperm quality and DNA integrity were higher (p < .05) in frozen/thawed semen diluted in 1.0 mM melatonin-supplemented extender than in the control group. Conception and birth rates were higher in does inseminated with 1.0 mM melatonin treated semen compared with the controls. In conclusion, supplementation of semen extender with 1.0 mM melatonin improved the quality of cooled and frozen/thawed rabbit spermatozoa. Melatonin can preserve DNA integrity and enhance the fertility of frozen/thawed rabbit spermatozoa.  相似文献   

9.
The present work studied different spermatozoa parameters and the ability of frozen rabbit spermatozoa to fertilize, in vitro, in vivo‐matured oocytes, as a test to predict their in vivo fertility and prolificacy. Semen from rabbit bucks was frozen using two freezing protocols [in a freezer at ?30°C or in liquid nitrogen vapour (LNV)]. For the in vivo trial, females were inseminated with frozen‐thawed spermatozoa. Oocytes used for in vitro testing were recovered 14 h after ovulation induction from donors and co‐incubated with 2 × 106 frozen‐thawed spermatozoa during 4 h at 37°C in Tyrode's medium under an atmosphere of 5% CO2 in air with maximal humidity. After co‐incubation period, presumptive zygotes were cultured in TCM199 supplemented with 20% foetal bovine serum (FBS), under the same conditions described above. Although no statistical differences were observed between freezing protocols in seminal parameters [motility rate: 40 and 35%, VCL: 35 and 46 μm/s, amplitude of lateral head displacement (ALH): 1.7 and 2.4 μm, for semen frozen at ?30°C and in LNV, respectively], significant differences were noted in the fertilizing ability in vivo and in vitro. Semen frozen at ?30°C showed the highest fertilizing ability in vitro (26.7% vs 6.2 and 8.7% for semen frozen at ?30°C, in LNV and fresh semen, respectively) and the lowest fertility rate in vivo (21.7% vs 64.2% and 70.6% for semen frozen at ?30°C, in LNV and fresh semen, respectively). Sperm frozen at ?30°C seemed to be more capacitated.  相似文献   

10.
The main purpose of the present study was to investigate whether boar seminal plasma affects the transport of spermatozoa in the genital tract of oestrous pigs or not, with special reference to the sperm transport into the oviducts. Altogether 17 gilts were used in three experiments.Experiment I. In nine gilts one uterine horn was injected surgically with 1010 spermatozoa suspended in seminal plasma and the other uterine horn with 1010 spermatozoa suspended in TESNaK-glucose buffer solution. The sperm deposition was performed under general anaesthesia. The gilts were slaughtered 1–2 or 4–6 h after insemination. The genital tract was removed and the numbers of spermatozoa determined in oviducts and in uterine horns.Experiment II. The insemination doses were prepared exactly as in Experiment I. Approx. 24 h before insemination Polyvinylchloride cannulas were inserted into the uterine lumen of the horns, drawn via the midventral incision at linea alba subcutaneously to cutaneous incisions ventral to the vulva opening. One cannula was placed in each uterine horn. At standing heat the insemination doses were slowly injected through the cannulas. The gilts were slaughtered 1 h after insemination and the numbers of spermatozoa within the genital tract were counted.Experiment III. In three gilts under general anaesthesia the uterine horns were ligated 10 cm from the uterotubal junction. The semen doses (containing 2 × 109 spermatozoa), prepared as in Experiment I, were deposited into the uterine horns anterior to the ligatures through a cannula. The gilts were slaughtered 1 h after insemination, and the numbers of spermatozoa within the oviducts and ligated part of the uterine horns were counted.In all three experiments more spermatozoa were, on average, recovered in the oviducts connected to uterine horns inseminated with spermatozoa suspended in seminal plasma. In Experiments I andII this was the case for 10 of 14 gilts and in Experiment III for all the three gilts. It is therefore suggested that boar seminal plasma pro¬motes sperm transport into the oviduct of oestrous pigs. The back¬ground mechanism for this is discussed.  相似文献   

11.
Apoptosis in the testis is required to ensure an efficient spermatogenesis. However, sometimes, defective germ cells that are marked for elimination during this process escape elimination in the testes, giving rise to ejaculates with increased percentages of abnormal and apoptotic spermatozoa and a high percentage of apoptotic bodies. Apoptosis markers in the ejaculate have been associated with low fertility, either in animals or humans. Therefore, the goal of this study was to investigate whether fresh equine semen contains apoptotic bodies [initially named Merocyanine 540 (M540) bodies] and to study the relationship between the quantity of these bodies and cell concentration, the volume of ejaculate, viability and motility. Moreover, we also studied whether the presence apoptotic bodies in fresh semen was related to the resistance of the stallion spermatozoa to being incubated at 37°C or being frozen and thawed. Fresh equine semen was stained with fluorescent dyes such as M540 and Annexin‐V. Active Caspase 3 was studied in fresh semen through Western blotting and immunofluorescence with a specific antibody. Sperm kinematics was assessed in fresh, incubated and thawed samples using computer‐assisted semen analysis, and viability was evaluated with the LIVE/DEAD Sperm Viability Kit. Overall, our results demonstrate for the first time the presence of apoptotic bodies in equine semen. The quantity of apoptotic bodies was highly variable among stallions and was positively correlated with Caspase 3 activity in fresh samples and negatively correlated with the viability and motility of stallion spermatozoa after the cryopreservation process.  相似文献   

12.
The purpose of the present study was to compare the number of spermatozoa obtained from different parts of the oviducts and the uterine horns of sows after intrauterine insemination (IUI) and conventional artificial insemination (AI), 24 h after insemination. Twelve crossbred (Landrace x Yorkshire) multiparous sows were used in the experiment. The sows were examined for standing oestrus using a back pressure test and were examined every 4 h after standing oestrus by real-time B-mode ultrasonography to estimate the time of ovulation. The sows were allocated to two groups, group I sows (n = 6) were inseminated by a conventional AI technique with 3 x 10(9) motile spermatozoa in 100 ml of extended semen, and group II sows (n = 6) were inseminated by an IUI technique using 1 x 10(9) motile spermatozoa in 50 ml of extended semen. A single dose of AI or IUI was given using the same boar, 8-10 h before the expected time of ovulation during the second oestrus after weaning. Twenty four hours after insemination, the sows were ovario-hysterectomized. The oviducts and the uterine horns were removed and divided into seven parts, the cranial, middle and caudal uterine horns, the utero-tubal junction (UTJ), the cranial and caudal isthmus, and the ampulla. All parts of the reproductive tract were flushed and the spermatozoa were counted using a haemocytometer. The results revealed that the spermatozoa were found in both the oviducts and the uterine horns in all animals. The number of flushed spermatozoa in the UTJ of groups I and II, was 142,500 and 131,167 (p > 0.05), and in the caudal isthmus was 1411 and 1280 (p > 0.05), respectively. The proportion of spermatozoa in different parts of the reproductive tract in relation to the total number of spermatozoa within the tract was not significantly different between groups I and II (p > 0.05). It could be concluded that IUI, with a three-time reduction in the number of spermatozoa used resulted in the same number of spermatozoa to be deposited in the sperm reservoir around ovulation time.  相似文献   

13.
The objective of this study was to investigate whether butylated hydroxytoluene (BHT) could be used as a suitable supporter or alternative of egg yolk during preservation of goat spermatozoa. Three in vitro experiments and a fertility test were conducted to evaluate the effect of BHT on viability of chilled‐stored semen as well as motility and kidding rate of frozen‐thawed spermatozoa. In the first two experiments, ejaculates (n = 30/experiment) were collected from 10 bucks, split, diluted with egg yolk‐based and egg yolk‐free extenders supplemented with or without 0.3, 0.6, 2, 5 and 8 mm BHT and stored at 5°C for 168 h. In the third experiment, 30 ejaculates were collected from the above‐mentioned bucks, split and diluted with egg yolk‐free extenders supplemented with or without 0.3, 0.6 and 0.9 mm BHT and egg yolk‐based extenders supplemented with or without 5 mm BHT. Diluted semen was cooled to 5°C over a period of 4 h, frozen and thawed in the form of 0.3‐ml pellets. In the fertility test, 75 ejaculates were collected from two proven fertile bucks, split, diluted with egg yolk‐free extenders containing 0.6 mm BHT and egg yolk‐based extenders supplemented with or without 5 mm BHT, frozen and thawed as described above. An insemination volume of 0.6 ml containing 120–140 × 106 progressively motile spermatozoa was used for a single cervical insemination of cloprostenol‐synchronized does (n = 230). The results showed that addition of 5 mm BHT to egg yolk‐deficient (2.5%) extenders significantly improved viability of chilled‐stored semen together with motility (48.5%) and fertility (62.5%) of frozen‐thawed spermatozoa. Replacement of egg yolk in semen extenders by 0.6 mm BHT could sustain not only viability of chilled‐stored semen but also post‐thaw motility (47.5%) and fertility (53.75%) of frozen‐thawed spermatozoa. In conclusion, supplementation of semen diluents with BHT can ameliorate preservability of goat sperm.  相似文献   

14.
This study investigated the effects of long‐term extenders on post‐thaw sperm quality characteristics following different holding times (HT) of boar semen at 17 and 10°C. Sperm‐rich fractions, collected from five boars, were diluted in Androhep® Plus (AHP), Androstar® Plus (ASP), Safecell® Plus and TRIXcell® Plus (TCP) extenders. The extended semen samples were held for 2 hr at 17°C (HT 1) and additionally for 24 hr at 10°C (HT 2), after they were evaluated and frozen. CASA sperm motility and motion patterns, mitochondrial membrane potential (MMP), plasma membrane integrity (PMI) and normal apical ridge (NAR) acrosome integrity were assessed in the pre‐freeze and frozen‐thawed semen. The Vybrant Apoptosis Assay Kit was used to analyse the proportions of viable and plasma membrane apoptotic‐like changes in spermatozoa. Results indicated that boar variability, extender and HT significantly affected the sperm quality characteristics, particularly after freezing‐thawing. Differences in the pre‐freeze semen were more marked in the sperm motion patterns between the HTs. Pre‐freeze semen in HT 2 showed significantly higher VCL and VAP, whereas no marked effects were observed in the sperm membrane integrity and viability (YO‐PRO‐1?/PI?) among the extenders. Post‐thaw sperm TMOT and PMOT were significantly higher in the AHP and ASP extenders of HT 2 group, whereas VSL, VCL and VAP were markedly lower in the TCP extender. Furthermore, spermatozoa from the AHP‐ and ASP‐extended semen of HT 2 group were characterized by higher MMP, PMI and NAR acrosome integrity following freezing‐thawing. In most of the extenders, the incidence of frozen‐thawed spermatozoa with apoptotic‐like changes was greater in HT 1. The findings of this study indicate that holding of boar semen at 10°C for 24 hr in long‐term preservation extenders modulates post‐thaw sperm quality characteristics in an extender‐dependent manner. These results will further contribute to the improvement in the cryopreservation technology of boar semen.  相似文献   

15.
The aim of this study was to evaluate whether the season of ejaculate collection influences seminal quality parameters of pre‐ and post‐freeze–thawing in Xinong Saanen bucks. Ejaculates were collected from eight bucks throughout the four seasons (spring, summer, autumn and winter) in a 12 months’ time period, identified in the Northern Hemisphere. Semen samples were evaluated by the combinations of conventional and Computer‐Assisted Sperm Analysis (CASA) when fresh and after frozen–thawed, respectively. The results clearly demonstrated that season of ejaculate collection influenced (p < 0.05) fresh semen quality. Highest semen quality was observed during autumn. On the contrary, undesirable indices (significantly lower, p < 0.05) were observed in winter as compared with the other remaining seasons. CASA has clearly shown the influences of seasonal variations on semen motility parameters. Furthermore, season of ejaculate collection was also found to influence sperm freezability. Semen characteristics after frozen–thawed followed a similar pattern with that of fresh ejaculate except in spring. The results revealed that sperm quality was higher (p < 0.01) in summer and autumn than in spring and winter. In conclusion, seasonal variation influences semen quality in Xinong Saanen bucks. In addition to summer and autumn, fresh ejaculates in spring can also be successfully used for AI. Sperm from ejaculates collected during summer and autumn are more suitable for cryopreservation. Hence, it is possible to increase the efficiency of goat breeding by manipulating the seasonal variations of semen quality for immediate AI and/or cryopreservation.  相似文献   

16.
Cryopreservation of boar spermatozoa offers an effective means of long‐term storage of important genetic material. Many researchers have investigated how to improve reproductive performance by artificial insemination (AI) using cryopreserved boar spermatozoa. Recently, we and other groups reported that high conception rates (70–80%) can be achieved by AI with frozen‐thawed boar spermatozoa using a modified temperature program during freezing, or a novel cryopreservation extender to improve sperm quality (including sperm survivability, motility, membrane status and fertilization ability) after thawing, or a novel sperm infusion method, deep intra uterine insemination. However, these techniques have not yet been used for commercial pig production. The variation in sperm freezability among boars or among ejaculations in an identical boar is one of the main reasons for this problem. In our previous study, it was revealed that some components of seminal plasma have a negative effect on the freezability of boar sperm. One of these factors is bacteria‐released endotoxin (lipopolysaccharide: LPS). LPS binds to Toll‐like receptor‐4 (TLR‐4) expressed on the sperm surface, resulting in induction of apoptosis. On the other hand, seminal plasma suppresses cryo‐capacitation induced by thawing stress. On the basis of these findings, we designed a novel protocol of AI using frozen‐thawed boar sperm.  相似文献   

17.
An Overview of Low Dose Insemination in the Mare   总被引：1，自引：0，他引：1  
The need for relatively high numbers of spermatozoa for artificial insemination limits our application of recently available technologies such as sex‐sorted semen. The fertility of two different methods of low dose insemination using fresh, frozen and sex‐sorted semen are compared in this overview. Satisfactory conception rates are described using very low doses of spermatozoa inseminated by either hysteroscopic or deep uterine insemination methods, proving the stallion is fully fertile. The hysteroscopic method appears to give higher conception rates when inseminating fewer than 5 × 106 spermatozoa and is therefore, the preferred method of insemination for sex‐sorted spermatozoa. However, hysteroscopic deposition of low numbers of spermatozoa from infertile stallions does not appear to improve their fertility.  相似文献   

18.
Transgenesis constitutes an important tool for pharmacological protein production and livestock improvement. We evaluated the potential of laparoscopic insemination (LI), in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) to produce egfp-expressing ovine embryos, using spermatozoa previously exposed to pCX-EGFP plasmid in two different sperm/DNA incubation treatments: "Long Incubation" (2 h at 17 C) and "Short Incubation" (5 min at 5 C). For LI, Merino sheep were superovulated and inseminated with treated fresh semen from Merino rams. The embryos were recovered by flushing the uterine horns. For IVF and ICSI, slaughterhouse oocytes were fertilized with DNA-treated frozen/thawed sperm. All recovered embryos were exposed to blue light (488 nm) to determine green fluorescent morulae and blastocysts rates. High cleavage and morulae/blastocysts rates accompanied the LI and IVF procedures, but no egfp-expressing embryos resulted. In contrast, regardless of the sperm/plasmid incubation treatment, egfp-expressing morulae and blastocysts were always obtained by ICSI, and the highest transgenesis rate (91.6%) was achieved with Short Incubation. In addition, following the incubation of labeled plasmid DNA, after Long or Short exposure treatments, with fresh or frozen/thawed spermatozoa, only non-motile fresh spermatozoa could maintain an attached plasmid after washing procedures. No amplification product could be detected following PCR treatment of LI embryos whose zonae pellucidae (ZP) had been removed. In order to establish conditions for transgenic ICSI in the ovine, we compared three different activation treatments, and over 60% of the obtained blastocysts expressed the transgene. For ICSI embryos, FISH analysis found possible signals compatible with integration events. In conclusion, our results show that in the ovine, under the conditions studied, ICSI is the only method capable of producing exogenous gene-expressing embryos using spermatozoa as vectors.  相似文献   

19.
The present study was performed to investigate the number of either the spermatozoa or the embryos in the reproductive tracts of sows after unilateral, deep, intra uterine insemination (DIUI). Two experiments were conducted, 10 sows were used in experiment I and eight sows were used in experiment II. Transrectal ultrasonography was used to examine the time when ovulation took place in relation to oestrus behaviour. The sows were inseminated with a single dose of diluted fresh semen 6-8 h prior to expected ovulation, during the second oestrus after weaning. In experimental I, five sows were inseminated by a conventional artificial insemination (AI) technique using 100 ml of diluted fresh semen, containing 3000 x 10(6) motile spermatozoa and five sows were inseminated by the DIUI technique with 5 ml of diluted fresh semen, containing 150 x 10(6) motile spermatozoa. The sows were anesthetized and ovario-hysterectomized approximately 24 h after insemination. The oviducts and the uterine horns on each side of the reproductive tracts were divided into seven segments, namely ampulla, cranial isthmus, caudal isthmus, utero-tubal junction (UTJ), cranial uterine horn, middle uterine horn and caudal uterine horn. Each segment of the reproductive tracts was flushed with Beltsville thawing solution (BTS) through the lumen. The total number of spermatozoa in the flushing from each segment were determined. In experimental II, eight sows were inseminated by the DIUI technique using 5.0 ml diluted fresh semen containing 150 x 10(6) motile spermatozoa. The sows were anesthetized 61.1 +/- 12 h after insemination (48-72 h) and the embryos were flushed from the oviduct through the proximal part of the uterine horn. It was revealed that, in experimental I, the spermatozoa were recovered from both sides of the reproductive tract in the AI-group, and from unilateral side of the reproductive tract in the DIUI-group (three sows from the left and two sows from the right sides). The number of spermatozoa recovered from the reproductive tracts was higher in the AI- than the DIUI-group (p < 0.001). In experiment II, fertilization occurred in five of eight sows (62.5%) after DIUI. The number of ova that ovulated were 16.4 +/- 2.6 per sow and the embryos numbering 11.4 +/- 2.3 per sow were recovered from both sides of the reproductive tract. In conclusion, the spermatozoa given by DIUI could be recovered from only one side of the reproductive tract of sows at approximately 24 h after DIUI via the flushing technique. However, embryos were found in both sides of the oviducts and the proximal part of the uterine horns 48-72 h after insemination, indicating that the fertilization occurred in both sides of the oviducts.  相似文献   

20.
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