精子和卵母细胞质量控制对山羊卵胞质内精子注射（ICSI）受精的影响

ABSTRACT Effects of sperm and oocyte quality control on the efficiency of ICSI of in vitro matured goat oocytes were studied in this paper. The results showed that when injected intracytoplasmically, spermatozoa from caput, corpus and cauda epididymidis resulted in similar rates of fertilization, cleavage and morulae/blastocysts, but when injected subzonally, spermatozoa from caput and corpus gave rise to significantly lower rates of fertilization and embryo development than spermatozoa from the cauda epididymidis and ejaculates. When dead spermatozoa collected from semen that had been preserved in different ways were used for ICSI, those dead from liquid storage at 20 degrees C for 24 h gave rise to the best, but those dead from liquid storage at 5 degrees C for 15 days produced the poorest fertilization and embryo development. When spermatozoa were treated with different concentrations of Triton X-100 before ICSI, significantly higher rates of fertilization, cleavage and morulae/blastocysts were obtained with 0.0005% Triton X-100 than with other concentrations and manual immobilization. Oocytes were classified as of good and poor qualities by treatment in hypertonic sucrose solution, and rates of fertilization and embryo development were significantly higher in the good than in the poor oocytes after ICSI. Post-injection activation of oocytes with either A23187 or ionomycin/6-DMAP significantly increased the rates of fertilization, cleavage and morulae/blastocysts after ICSI. It is therefore concluded that (i) epididymal maturation mainly endowed spermatozoa with the capacity to fuse with the egg plasma membrane; (ii) different methods of semen storage caused different impairment of sperm fertilizing capacity; (iii) pre-injection treatment of spermatozoa with proper concentrations of Triton X-100 might be used to replace manual immobilization for ICSI; (iv) oocyte quality was a major factor influencing the efficiency of ICSI; (v) post-injection activation treatment of oocytes improved fertilization and embryo development after ICSI.     

影响山羊体外受精的因素

以屠宰山羊卵母细胞为材料研究了公羊个体、附睾不同部位精子、成熟培养和受精时卵丘存在与否、卵丘扩展程度及卵龄对山羊体外受精的影响。结果表明 :1)不同公羊精液在受精、卵裂和桑椹 /囊胚率上都有显著差异 ;2 )附睾尾精子和鲜精的受精、卵裂和桑椹 /囊胚率无显著差异 ,但显著高于附睾体和附睾头精子 ;3)成熟培养 2 4和 2 7h卵母细胞的的桑椹胚 /囊胚率显著高于培养 2 1和 30h卵母细胞 ;4 )卵丘扩展 3和 4级卵母细胞受精和桑椹胚 /囊胚率显著高于扩展 0和 1级卵母细胞 ;5 )成熟培养前机械去卵丘严重影响卵母细胞体外受精和桑椹胚 /囊胚率 ;6 )受精前完全去掉卵丘显著影响桑椹胚 /囊胚率     

Effect of mating on sperm distribution in the reproductive tract of the male rat

Extragonadal sperm reserves in male rats were measured in different regions of the genital tract before and subsequent to normal ejaculation. In sexually rested rats, the sperm count (million spermatozoa for the paired organs) in different regions was: distal vas, 18; proximal vas, 9.8; cauda epididymidis, 229; caput + corpus epididymidis, 154. Following mating, the sperm count was reduced in the proximal and distal vas deferens and in the cauda epididymidis. The reproductive tract of mated females was found to contain 29% (no copulatory plug) or 59% (with copulatory plug) of the estimated mean ejaculate, which was estimated from the difference between the sperm counts in the sexually rested rat and following ejaculation. It is concluded that in the rat the immediate source of spermatozoa for ejaculation is the cauda epididymidis, with a smaller contribution arising from the vas deferens.     

Effects of age and frequency of ejaculation on sperm production and extragonadal sperm reserves in stallions.

Extragonadal reserves totalled 89 X 10(9) spermatozoa for 5--16-year-old sexually rested stallions and 60 X 10(9) for 2--4-year-olds. Regardless of age, the cauda epididymidis contained 62% of the total reserves and the vas deferens, including the ampulla, contained 7% of the total reserves of spermatozoa. The caput plus corpus epididymidis from 5--16-year-old stallions (N = 41) contained 14.9 X 10(9) spermatozoa per side as compared (P less than 0.01) to 8.5 X 10(9) for 2--4-year olds (N = 30). Frequency of ejaculation did not influence the number of spermatozoa found in caput plus corpus epididymidis. Across all ages, the number of spermatozoa potentially available for ejaculation from the cauda epididymidis and vas deferens on each side totalled 54 X 10(9). Collection of 5 successive ejaculates from sexually rested stallions removed 40--60% of the available spermatozoa while ejaculation every 2nd day reduced (P less than 0.05) the number of spermatozoa potentially available for ejaculation by 27%. Nevertheless, sufficient spermatozoa are produced daily (6.4 and 4.2 X 10(9) for 5--16-and 2--4-year-olds) to permit use of an average stallion once or twice daily, during spring and summer, providing sexual behaviour is adequate.     

The histochemical localization of prostaglandin synthetase activity in reproductive tract of the male rat.

PG synthetase activity was assessed histochemically in the reproductive tract of male rats. Moderate activity was observed in tails of spermatozoa within the corpus and cauda epididymidis but there was no activity in the caput epididymidis or the seminiferous tubules. The sperm tail activity was maximal for cells within the vas deferens. PG synthetase activity was also observed in individual adipose cells adhering to the testicular capsule, epididymis and vas deferens, and in isolated interstitial cells of the testis and the caput, corpus and cauda epididymidis. Specific cells in the capsules of the testes, epididymis and vas deferens also produced PGs. The activity observed in the interstitial cells of the testis and the caput epididymidis was less than that for the other tissues in terms of the proportion of possible cells. The demonstration of PG synthetase activity paralleled to known loss of arachidonic acid from the phospholipids of the spermatozoa as they pass through the male tract. Endogenous substrate was not limiting in the assay system, even in the testis and caput epididymidis where PG synthesis was not normally observed, indicating that a PG synthesis inhibitor may be present in these two tissues. PG synthetase activity within teased seminiferous tubules was markedly increased by physical trauma. Indomethacin diminished but did not eliminate synthesis.     

Does multivariate analysis of post-thaw sperm characteristics accurately estimate in vitro fertility of boar individual ejaculates?

The objective of this study was to determine if a multivariate pattern analysis of frozen-thawed sperm characteristics of boar semen of unknown fertility, thus identifying groups of ejaculates as "good" or "bad" freezers, would estimate their fertilizing potential in an in vitro embryo production (IVP) system. Frozen-thawed spermatozoa from a single ejaculate collected from 46 boars were evaluated for sperm motility and kinematic patterns, for sperm viability and for early changes in sperm membrane stability. All data generated were used for a multivariate pattern analysis (PATN; CSIRO, Canberra, Australia) which objectively classified all ejaculates within a data set in to one of two groups, categorised as "good" (n = 25) or "bad" (n = 21) according with their freezability. In vitro matured oocytes were exposed to 2000 or 4000 frozen-thawed spermatozoa per oocyte for 6h and then cultured in embryo culture medium for either 6h (assurance of fertilization) or 7 days (to collect data on embryo development). Rates of sperm oocyte penetration and of embryo development significantly (p < 0.05) increased in a sperm:oocyte ratio-dependent manner. A similar pattern was observed when sperm characteristics were grouped. Indeed, ejaculates classified as "good" showed significantly (p < 0.05) higher rates of oocyte penetration, cleavage and of blastocyst formation than those classified as "bad". However, variation was still present among individuals (ejaculates, boars) in their ability to produce blastocysts in vitro. It is therefore concluded that despite the presence of a relationship for ejaculates with good semen quality post-thaw (thus grouped as "good") to higher IVP-results, the presence of individual variation does not allow for an accurate estimation of in vitro fertility based solely on the frozen-thawed semen quality parameters of a single ejaculate from a given boar.     

Activation, pronuclear formation, and development in vitro of pig oocytes following intracytoplasmic injection of freeze-dried spermatozoa

The fertilization of pig oocytes following intracytoplasmic injection of freeze-dried spermatozoa was evaluated. Activation and male pronuclear (MPN) formation were better in oocytes injected with isolated freeze-dried sperm heads than whole freeze-dried spermatozoa, but cleaved embryos were generally difficult to develop to the morula or blastocyst stage. When spermatozoa were freeze-dried for 24 h, oocyte activation and MPN formation in activated oocytes after sperm head injection were inhibited. Embryo development to the blastocyst stage was only obtained after injecting sperm heads isolated from spermatozoa freeze-dried for 4 h and stored at 4 degrees C. The proportion of embryos that developed to the blastocyst stage was not increased by the treatment of injected oocytes with Ca ionophore (5-10 microM). Increasing the sperm storage time did not affect oocyte activation or MPN formation, but blastocyst development was observed only after 1 mo of storage. These results demonstrate that pig oocytes can be fertilized with appropriately freeze-dried spermatozoa and that the fertilized oocytes can develop to the blastocyst stage.     

Leptin accelerates pronuclear formation following intracytoplasmic sperm injection of porcine oocytes: Possible role for MAP kinase inactivation

Leptin, a multifunctional hormone, is present in mammalian oocytes and follicular fluids and cumulus cells. While leptin modulates oocyte maturation in vitro which seems to result in enhancement of embryo development, it is unclear whether leptin treatment of oocytes affects cytoplasmic maturation and fertilization processes. In order to gain a better understanding of the role of leptin during oocyte maturation, we examined microtubule and microfilament assembly following oocyte maturation and blastocyst formation, mitogen-activated protein kinase (MAPK) activity, and pronuclear formation following parthenogenetic stimuli or intracytoplasmic sperm injection (ICSI) in leptin-treated oocytes. Addition of 10 or 100 ng/ml leptin during oocyte maturation did not increase the proportion of metaphase II oocytes, but enhanced development to blastocyst stage by day 7 (P < 0.01) after parthenogenetic activation (PA), accompanied by increased cell number. However there was no effect on the number of apoptotic cells in blastocysts. Following maturation in the presence of leptin, there were more oocytes with normal spindle formation. MAPK activity decreased more rapidly, and pronuclear formation was accelerated after parthenogenetic activation or ICSI of leptin-treated oocytes. These results suggested that exogeneous leptin enhanced spindle assembly and accelerated pronuclear formation following fertilization, possibly via the MAPK pathway.     

Buffalo and cattle hybrid embryo development is decreased by caffeine treatment during in vitro fertilization

Water buffalo are renowned for difficulties in the implementation of assisted reproductive technologies, with both males and females being problematic. In this study, we used cattle oocytes to assess the effect of treatments with heparin and caffeine on buffalo spermatozoa and subsequent fertilization and embryo development in vitro. There was no significant difference between buffalo and bovine spermatozoa in the events associated with fertilization. Fertilization of cattle oocytes with buffalo spermatozoa resulted in 7.8% of oocytes developing into hybrid embryos. A difference in the developmental capability of hybrid embryos compared with the cattle control was observed. This has not been previously reported. The subsequent transfer of a limited number of hybrid embryos did not produce a viable pregnancy. However, control treatments in this experiment also failed to achieve pregnancy, so objective data is not available to provide conclusions about the developmental competence of the buffalo and cattle hybrid embryos. Optimal spermatozoa capacitation treatments achieved 61% fertilization and 21% zygote cleavage into two cell embryos. There was no significant difference in fertilization or development due to heparin or spermatozoa concentrations. However, treatment of buffalo and cattle spermatozoa with caffeine significantly decreased embryo cleavage but also tended to decrease embryo development to the blastocyst stage. These studies suggest that problems with reproduction in buffalo may reside with biological mechanisms associated with the oocyte that are often complicated by poor male reproductive performance. Selection for bull fertility would prevent some of these complications.     

Effects of oviductal and cumulus cells on in vitro fertilization and embryo development of porcine oocytes fertilized with epididymal spermatozoa

This study was designed to evaluate the effects of adding porcine oviductal epithelial cell (POEC) monolayers before or during the fertilization of denuded or cumulus-enclosed oocytes, in terms of fertilization results and subsequent embryo development. The variables determined were: penetration rate, mean number of spermatozoa per oocyte, male pronucleus formation rate, monospermy rate, cleavage rate after 48 h of fertilization, blastocyst rate, and mean number of nuclei per blastocyst. We used cumulus-free and cumulus-enclosed oocytes preincubated or fertilized in the presence of POEC, once the purity in epithelial cells of these cultures had been assessed. All the experiments involved the use of frozen-thawed epididymal spermatozoa to avoid replicate variability. The POEC cultures prepared showed a high proportion of epithelial cells (over 95%). Preincubation of oocytes with POEC before fertilization showed no effects on the fertilization variables determined. In contrast, during IVF under our experimental conditions, these cells attached to the cumulus cells and their interaction had a significant effect on some of the fertilization variables analyzed. The presence of POEC and cumulus cells during IVF increased oocyte penetrability. Moreover, in the absence of POEC, cumulus cells resulted in a reduced monospermy rate. On subsequent embryo culture, a lower cleavage and blastocyst formation rate were recorded when the oocytes had been preincubated with POEC before IVF.     

Influence of sperm:oocyte ratio during in vitro fertilization of in vitro matured cumulus-intact pig oocytes on fertilization parameters and embryo development

The present study was conducted to evaluate the influence of sperm:oocyte ratio during in vitro fertilization (IVF) of in vitro matured cumulus-intact oocytes on fertilization parameters and embryo development in pigs. In vitro matured oocytes surrounded by intact cumulus cells (COC) were inseminated with frozen-thawed spermatozoa at different sperm:oocyte ratios (2000:1, 3000:1, 4000:1, 6000:1, and 8000:1). Denuded oocytes inseminated with 2000 frozen-thawed spermatozoa:oocyte were the control group. A total of 2546 oocytes in five replicates were exposed to spermatozoa for 6 h and then cultured in embryo culture (EC) medium for 6 h (pronuclear formation) or 7 days (blastocyst formation: BF). The penetration rate increased in the COC groups with the sperm:oocyte ratio, reaching the highest rates with 8000:1 spermatozoa:oocyte (72.1 +/- 6.5%), similar to the control (73.5 +/- 3.5%). However, the monospermy was highest with the lower spermatozoa:oocyte rates (82.6-94.8%) and decreased drastically (P<0.05) in the COC group fertilized with 8000 sperm:oocyte (36%). The efficiency of fertilization (number of monospermic oocytes/total number of inseminated oocytes) showed no difference among the COC groups (20-30%) but they were significantly lower (P<0.007) than those obtained by the control group (43.7 +/- 2%). Embryo development was highest in the control group (58% for cleavage and 23% for BF) but not significantly different with the 6000 and 8000 sperm:oocyte COC groups (47 and 50% for cleavage and 19 and 17% for BF, respectively). These results indicate that the use of COC for IVF involves a drop in the efficiency of the fertilization and the necessity to increase the frozen-thawed sperm:oocyte ratio three to four times more to obtain similar embryo development to denuded oocytes.     

Viability of ICSI oocytes after caffeine treatment and sperm membrane removal with Triton X-100 in pigs

Non-adequate decondensation of injected sperm nucleus is one the main problems of intracytoplasmic sperm injection (ICSI) in porcine. With the aim of improving pronuclear formation, the effects on activation and embryo development rates of 0.1% Triton X-100 (TX) sperm pre-treatment for membrane removal and/or 5 mM Caffeine (CAF) addition in oocyte manipulating and culture medium for 2 h after ICSI or artificial activation were studied. The effects of 4 different Ca²⁺ concentrations contained in the injection medium on embryo development after sham injection were also analysed. In Experiment 1, no significant effect on cleavage or blastocyst rate was detected independently of Ca²⁺ concentration contained in the injection medium. In Experiment 2, oocytes injected with TX pre-treated sperm showed a significant higher rate of male pronuclear formation in comparison with oocytes from control group (2PN; 54.1 vs 36.6%). However, no differences on in vitro embryo development, cleavage or blastocyst rates were observed. In Experiment 3, oocytes treated with CAF during and after micromanipulation and injected with sperm pre-treated with TX had a significantly lower oocyte activation rate than any other experimental groups (25.7 vs 56.3–66.3%). No differences were observed in cleavage rates among different experimental groups. However, the CAF group showed a higher blastocyst rate significantly different from TX+CAF group (12.0 vs 1.9%, respectively). In a second approach, the effect of electric field strengths and CAF treatments on oocyte activation was studied. In Experiment 4, oocytes submitted to 0.6 kV/cm showed significant higher activation rates than 1.2 kV/cm ones regardless of the caffeine treatment (83.7 vs 55.9% and 75.7 vs 44.3%; in control and caffeine groups, respectively). No effect of caffeine treatment was observed in any experimental group. In conclusion, TX sperm treatment before ICSI without an additional activation procedure improved male pronuclear formation, but did not improve embryo development until blastocyst stage. No significant effect of caffeine was found when sperm was not treated with TX, although in membrane absence caffeine avoided oocyte activation and embryo development. Finally, caffeine had no effect on female pronuclear formation regardless of electric field strengths applied to the parthenogenetic activation.     

Sexual characteristics and spermatogenesis in males of the parthenogenetic gecko Lepidodactylus lugubris (Reptilia, Gekkonidae)

Obligately parthenogenetic lizards usually are all-female populations of hybrids producing diploid oocytes by premeiotic endomitosis and quasi-normal meiosis. In an all-female strain of the gekkonid lizard Lepidodactylus lugubris several phenotypic males arose spontaneously. The sexual characteristics of these males were studied using light and electron microscopy and compared with normal males of the bisexual genus Lygodactylus. Emphasis was layed on morphology of seminiferous tubules, occurrence of spermatogenic stages and ultrastructure of spermatozoa. The phenotypic males possessed preanal pores filled with secretions and a sexual nephric segment which were exactly the same as in normal, reproductively active males. In the testes, density and morphology of non-spermatogenic cell types, the Leydig and Sertoli cells, indicate a normal production of testicular testosterone and a normal function of the blood-testis barrier, respectively. Both in the normal and the phenotypic males, all meiotic cell types of spermatogenesis can be recognised in the seminiferous tubules and are apparently identical, indicating a normal meiosis without impairment in the phenotypic males. In contrast, the differentiation process of spermatids is markedly disturbed in the phenotypic males of L. lugubris. In the normal male, spermiogenesis results in mature spermatids and spermatozoa with small elongated nuclei, an acrosomal complex, and a flagellar tail possessing one axoneme. Spermatozoa fill both the lumen of most seminiferous tubules and the lumina of ductus epididymidis and ductus deferens. In the phenotypic male, spermiogenesis results in seemingly normal spermatids and in spermatozoa with large, non-elongated, deformed nuclei and/or irregular tails possessing more than one axoneme. Both the lumen of most seminiferous tubules and the lumina of the ductus epididymidis and the ductus deferens contain relatively few spermatozoa. We suggest that the phenotypic males inherited the ability for a premeiotic endomitosis from their all-female ancestral lineage. While in females this leads to quasi-normal meiosis and diploid oocytes capable of development, the small nuclei of the spermatozoa are unable to contain a diploid set of chromosomes. Because of the high amount of deformed spermatozoa and possibly uncontrolled loss of genetic material in structurally normal, but aneuploid spermatozoa we conclude that these otherwise perfect males are infertile, thus constituting another example of gametic sterility.     

Viability of ICSI oocytes after caffeine treatment and sperm membrane removal with Triton X-100 in pigs

Non-adequate decondensation of injected sperm nucleus is one the main problems of intracytoplasmic sperm injection (ICSI) in porcine. With the aim of improving pronuclear formation, the effects on activation and embryo development rates of 0.1% Triton X-100 (TX) sperm pre-treatment for membrane removal and/or 5 mM Caffeine (CAF) addition in oocyte manipulating and culture medium for 2 h after ICSI or artificial activation were studied. The effects of 4 different Ca²⁺ concentrations contained in the injection medium on embryo development after sham injection were also analysed. In Experiment 1, no significant effect on cleavage or blastocyst rate was detected independently of Ca²⁺ concentration contained in the injection medium. In Experiment 2, oocytes injected with TX pre-treated sperm showed a significant higher rate of male pronuclear formation in comparison with oocytes from control group (2PN; 54.1 vs 36.6%). However, no differences on in vitro embryo development, cleavage or blastocyst rates were observed. In Experiment 3, oocytes treated with CAF during and after micromanipulation and injected with sperm pre-treated with TX had a significantly lower oocyte activation rate than any other experimental groups (25.7 vs 56.3-66.3%). No differences were observed in cleavage rates among different experimental groups. However, the CAF group showed a higher blastocyst rate significantly different from TX+CAF group (12.0 vs 1.9%, respectively). In a second approach, the effect of electric field strengths and CAF treatments on oocyte activation was studied. In Experiment 4, oocytes submitted to 0.6 kV/cm showed significant higher activation rates than 1.2 kV/cm ones regardless of the caffeine treatment (83.7 vs 55.9% and 75.7 vs 44.3%; in control and caffeine groups, respectively). No effect of caffeine treatment was observed in any experimental group. In conclusion, TX sperm treatment before ICSI without an additional activation procedure improved male pronuclear formation, but did not improve embryo development until blastocyst stage. No significant effect of caffeine was found when sperm was not treated with TX, although in membrane absence caffeine avoided oocyte activation and embryo development. Finally, caffeine had no effect on female pronuclear formation regardless of electric field strengths applied to the parthenogenetic activation.     

Developmental potential of aged oocyte rescued by nuclear transfer following parthenogenetic activation and in vitro fertilization

Mouse oocyte aged in vitro cannot develop normally following activation. To investigate the roles of nucleus or cytoplasm elements in oocyte aged in vitro process and their subsequent development capability following activation, we reconstructed oocytes with MII chromosome spindle and cytoplasm from aged and fresh oocytes by nuclear transfer. The subsequent developmental potential after parthenogenetic activation (PA) or in vitro fertilization (IVF) was evaluated. After nuclear transfer, more than 75.6% of karyoplast and cytoplast pairs can be fused and reconstructed oocytes have a normal haploid karyotype. Following PA, aged oocytes cannot develop beyond four-cell stage, reconstructed oocytes from fresh nucleus and aged cytoplasm developed to blastocyst with a low percentage (9.1%). Instead, blastocyst formation rate of reconstructed oocyte from aged nucleus and fresh cytoplasm was higher (60.0%). Following IVF, zygote with diploid karyotype can be formed from zona pellucida (ZP)-free oocyte. After cultured in vitro, aged oocytes cannot develop beyond two-cell; reconstructed oocytes from fresh nucleus and aged cytoplasm developed to blastocyst with low percentage (15.0%). However, high blastocyst formation rate (86.2%) can be obtained from reconstructed oocytes from aged nucleus and fresh cytoplasm. Furthermore, after embryo transfer, three viable pups have been obtained, although the efficiency is very low. These observation demonstrated that cytoplasm is more crucial than nucleus to aging process. Fresh cytoplasm could partly rescue nucleus susceptibility to apoptosis from aging in vitro.     

Plasminogen activator and mouse spermatozoa: urokinase synthesis in the male genital tract and binding of the enzyme to the sperm cell surface

When ejaculated mouse spermatozoa were embedded in a plasminogen- containing insoluble protein substrate, a zone of proteolysis developed progressively, centered around the sperm head region. Lysis did not occur in absence of plasminogen or in presence of antibodies against the urokinase-type plasminogen activator (u-PA). Zymographic and immunological analyses confirmed the presence of u-PA in extracts of ejaculated mouse spermatozoa. In contrast, the u-PA activity of sperm cells obtained from testis or from vas deferens was low, although these cells were able to bind added murine u-PA. The sites of u-PA synthesis were identified by measuring u-PA activity and u-PA mRNA content in protein extracts and in total RNA preparations of various portions of the male genital tract. The highest levels of u-PA activity and of u-PA mRNA were found in vas deferens and seminal vesicles. The cells that synthesize u-PA were localized by hybridizing frozen sections of various portions of the genital tract to a u-PA cRNA probe. In all tissues examined, u-PA mRNA was predominantly located in the epithelial layer, and the strongest signal was observed over that of the vas deferens. Hence, the u-PA associated with ejaculated sperm cells is probably acquired from genital tract secretions. Sperm-bound u-PA may participate in the proteolytic events that accompany capacitation and fertilization.     

Concentration of acrosome stabilizing factor (ASF) in rabbit epididymal fluid and species specificity of anti-ASF antibodies

Rabbit Acrosome Stabilizing Factor (ASF) concentrations were measured by immunoradiometric assay (IRA) in lumenal fluids obtained by micropuncture from the caput epididymidis, corpus epididymidis, cauda epididymidis, and the vas deferens of the rabbit. ASF was below the limit of detection in caput epididymidal fluids. Average ASF concentrations (3 bucks) in the corpus epididymidis, cauda epididymidis, and vas deferens were 880, 3363, and 3236 micrograms/ml, respectively. The average level of ASF in the cauda epididymidal fluid (CEF) represents from 10 to 23% of the total protein and is at least tenfold more than the amount previously determined to effect complete decapacitation of rabbit sperm by an in vivo assay. The average ASF concentration in seminal plasma from two vasectomized males was 0.155 micrograms/ml, approximately 100,000-fold less than is present in CEF and 2000-fold less than is present in normal seminal plasma. CEFs or seminal plasma from 11 different species were screened by Western blotting using high titer anti-ASF polyclonal antibodies to detect ASF-like molecules in other species. Only rabbit ASF was recognized.     

Cryopreservation of epididymal dog sperm.

A method of cryopreservation was developed for sperm salvaged from the cauda epididymis and vas deferens of domestic dog testes. Four modifications of the glycerol concentration of a buffer used for cryopreservation of dog ejaculates and two freezing rates were assessed for their effect upon post-thaw spermatozoal motility and morphology. There was no statistical difference between the four glycerol concentrations or the two freezing rates and the buffer containing 6% glycerol and the freezing rate provided by 0.5 ml straws was chosen for further study. This method resulted in a significant reduction in the percentage of live spermatozoa detected with Hoechst staining and a reduction in the percentage of capacitated spermatozoa after freeze-thawing. However, there was no difference in the ability of frozen-thawed spermatozoa to penetrate homologous oocytes.This study demonstrates that cryopreservation of epididymal canine sperm can be performed using methods similar to those established for ejaculates of the same species, and that despite some damage, spermatozoa retain their functional ability.     

Morphology of spermatozoa in the cauda epididymidis before and after electroejaculation and a comparison with ejaculated spermatozoa in the domestic cat

When 2 ejaculates are collected by electroejaculation from the domestic cat within a period of 10 min the first ejaculate has a higher proportion of abnormal spermatozoa than the second. The reason for this difference is not known for the domestic cat, but in other species long-term epididymal storage results in a higher proportion of abnormal spermatozoa. The aims of this study were to determine the proportions of abnormal spermatozoa in the cauda epididymidis and to ascertain if electroejaculation affects this proportion. Therefore the proportions of spermatozoa in the cauda epididymidis with different morphological abnormalities were compared before and after ejaculation. In addition, the proportion of morphologically abnormal spermatozoa in the epididymis was compared with that in the ejaculate. Nine privately-owned domestic cats were anesthetized, and one testicle was surgically removed. An ejaculate was collected by electroejaculation, after which the remaining testicle was ectomized. There were no significant differences in the proportions of different sperm abnormalities between the cauda epididymidis removed before ejaculation and the one removed after ejaculation. A significantly (P = 0.009) higher proportion of spermatozoa with tail abnormalities was found in the ejaculates compared with the cauda epididymides (11.1 and 1.6%, respectively), while, as expected, there was a lower proportion of spermatozoa with distal droplets in the ejaculates than in the cauda epididymides (35.1 and 75.9%, respectively). This new information contributes to the understanding of the etiology of sperm defects in the domestic cat, and is of importance when evaluating a semen sample in this species.     

Haploidy but not parthenogenetic activation leads to increased incidence of apoptosis in mouse embryos.

Aneuploidy underlies failed development and possibly apoptosis of some preimplantation embryos. We employed a haploid model in the mouse to study the effects of aneuploidy on apoptosis in preimplantation embryos. Mouse metaphase II oocytes that were activated with strontium formed haploid parthenogenetic embryos with 1 pronucleus, whereas activation of oocytes with strontium plus cytochalasin D produced diploid parthenogenetic embryo controls with 2 pronuclei. Strontium induced calcium transients that mimic sperm-induced calcium oscillations, and ploidy was confirmed by chromosomal analysis. Rates of development and apoptosis were compared between haploid and diploid parthenogenetic embryos (parthenotes) and control embryos derived from in vitro fertilization (IVF). Haploid mouse parthenotes cleaved at a slower rate, and most arrested before the blastocyst stage, in contrast to diploid parthenotes or IVF embryos. Developmentally retarded haploid parthenotes exhibited apoptosis at a significantly higher frequency than did diploid parthenotes or IVF embryos. However, diploid parthenotes exhibited rates of preimplantation development and apoptosis similar to those of IVF embryos, indicating that parthenogenetic activation itself does not initiate apoptosis during preimplantation development. These results suggest that haploidy can lead to an increased incidence of apoptosis. Moreover, the initiation of apoptosis during preimplantation development does not require the paternal genome.