BSA在精子获能中的作用及其替代物研究进展

摘　要:精子获能(Capacitation)是受精过程能否完成的一个关键因素,是生殖机理研究和体外受精(IVF)技术研究中的一个重要方面,但精子获能的机理仍然不甚明确。精子获能孵育液和促获能物质的研究是研究精子获能的基础,牛血清白蛋白(BSA)是经典的精子获能液中不可或缺的添加物,具有促使大多数哺乳动物精子获能、防止精子质膜之间发生黏连等作用。但由于BSA 是生物制剂,化学成分不确定并且作用复杂,给试验研究带来不便。因此,成分和功能相对确定的替代物被研究,像聚乙烯醇(PVA),环化糊精(Cyclodextrin)和合成蛋白等。这方面的研究仍处于起步阶段,有必要继续进行。     

金黄地鼠体外受精的初步研究

实验用ＴＹＨ、Ｔ６两种培养液做为精子获能培养液和受精培养液进行了金黄地鼠的体外受精。用经获能培养的精子分别与具卵丘和去卵丘的卵子进行受精培养。结果在ＴＹＨ中分别得到了２７．５％和３３．３％的受精率。在Ｔ６中得到了４７．２％和２６．３％的受精率；用未经获能培养的精子分别与具卵丘和不具卵丘卵子进行受精培养，结果在ＴＹＨ中得到了４１．８％和７．１％的受精，在Ｔ６中得到了４８．５％和５７．７％的受精率。实验结果说明：（１）适用于小鼠体外受精的培养液Ｔ６和ＴＹＨ并不适合于金黄地鼠，其主要原因是不能使精子有效获能；（２）卵丘细胞的存在有使受精率提高的趋势；（３）来自不同公鼠的精子的受精能力差异很大，进行体外受精时必须严格挑选公鼠     

Effect of Different Media and Protein Source on Equine Gametes: Potential Impact During In Vitro Fertilization

Equine in vitro fertilization (IVF) is still inconsistent. In the present work, we studied how modified Whitten's (MW) medium and Tissue Culture Medium 199 (TCM) added with Foetal Bovine Serum (FBS; 10% v/v) or Bovine Serum Albumin (BSA; 7 mg/ml) affected equine gametes to subsequently run IVF trials. Compact (Cp) and expanded (Ex) cumuli equine oocytes were matured and placed in TCM or MW supplemented with BSA or FBS for 18–20 h (no sperm added). In Ex oocytes, TCM‐199 added with FBS or BSA resulted in higher metaphase II (MII) rates (75.7% and 62.7%, respectively) than MW added with BSA (54%) or FBS (52.2%; p < 0.05); this was not observed for Cp oocytes. Equine sperm were capacitated in the same media at 10 × 10⁶ sperm/ml for 4 h at 37°C; total motility and protein tyrosine phosphorylation (PY) were evaluated. While motility remained unchanged, TCM or MW added with FBS enhanced the number of sperm showing PY‐stained tails (25 ± 4.8% and 31 ± 6.6%; mean ± SEM, respectively) over BSA supplemented media (3 ± 1.2% and 11.7 ± 1.1%) for TCM and MW (p < 0.05). In view of the previous results, sperm were capacitated in TCM + FBS and MW + BSA (control); IVF trials were run in the same media supplemented with 200 ng/ml of progesterone, but no fertilization occurred. Our results show that TCM + FBS enhances Ex equine oocyte's meiotic competence over MW + BSA and TCM or MW added with FBS successfully induce equine PY over media supplemented with BSA.     

In vitro capacitation of stallion spermatozoa assessed by the lysophosphatidylcholine-induced acrosome reaction and the penetration rate into in vitro-matured, zona-free mare oocytes

The purpose of this study was to evaluate the ability of various chemicals to induce capacitation of stallion spermatozoa using 2 different assay systems. In Experiment 1, freshly ejaculated spermatozoa were treated for 0, 3 and 6 h with 10 μ g/ml heparin, 0.5 mM hypotaurine or 5 mM caffeine, or were incubated for 0, 3 and 6 h following 1 min exposure to 0.1 μ M ionophore A23187. The acrosome reaction (AR) in the capacitated spermatozoa was induced by 15 min challenge with 100 μ g/ml lysophosphatidylcholine (LPC). In the BO/BSA-control medium (Brackett and Oliphant medium with 0.3% BSA), mean percentage of AR spermatozoa at 0 h was 30%, and the AR rates increased to 40 and 48% after 3 and 6 h incubation, respectively. There was no significant further increase of the AR rates in the spermatozoa treated with heparin (50% at 6 h) and hypotaurine (58% at 6 h) when compared to the control. Caffeine had a beneficial effect on inducing sperm capacitation after 3 and 6 h incubation (AR rates; 61 and 66%, respectively, P<0.01). Immediately after ionophore A23187 treatment, the AR rate increased to 56%, and reached 68 and 67% after 3 and 6 h incubation, respectively (P<0.01). Spermatozoal motility at any time points did not differ between control and any chemical treatment groups, except one treatment (ionophore; 3 h group).In Experiment 2, frozen-thawed spermatozoa were treated with 4 different chemicals as described above. Aliquot of spermatozoa was added to a microdrop of BO/BSA medium in which 6 to 10 in vitro-matured, zona-free mare oocytes were placed, and the oocytes were fixed and stained 20 h after insemination. The penetration rate by BO/BSA-treated spermatozoa was 76%, which was comparable to the results with heparin (73%), hypotaurine (78%) and caffeine (58%). In contrast, treatment of spermatozoa with ionophore A23187 gave a significantly lower penetration rate (30%) than the control value. Surprisingly these two experiments had different conclusions in assessing capacitation of stallion spermatozoa.     

The effect of porcine follicular fluid on the interaction of boar spermatozoa with zona-free hamster ova.

Boar spermatozoa were cocultured with zona-free hamster ova (eggs) to assess the effects of preovulatory porcine follicular fluid (pFF) in the capacitation medium or gamete coculture (fertilization) medium (pFF; 0, 10 or 40% v/v) on subsequent sperm-egg interaction. Increasing pFF concentrations in the capacitation medium resulted in a progressive decrease in the average numbers of sperm attaching to or penetrating each ovum. When pFF was included in the fertilization medium, but not in the capacitation medium, the average numbers of sperm attaching to or penetrating each ovum and the percentage of ova with sperm attached decreased markedly with increasing pFF concentrations. The percentage of ova with greater than five sperm attached decreased from 84% to 13% and 0% with 0%, 10% and 40% pFF, respectively. Sperm attachment was completely inhibited in approximately 50% of the ova cocultured in 40% pFF. The percentage of ova penetrated by greater than five sperm decreased from 82% to 21% and 7% with 0%, 10% and 40% pFF, respectively. Preincubation of ova in 40% pFF prior to coculture with sperm also resulted in a reduction in sperm attachment and penetration. These results suggest that pFF contains substance(s) that alter the ability of boar spermatozoa to interact with the hamster ovum plasma membrane in vitro.     

Factors required during preculture of rat oocytes soon after sperm penetration for promoting their further development in a chemically defined medium

Blastocyst formation in a chemically defined medium (mR1ECM) of rat oocytes soon after sperm penetration is less frequent than in those undergoing male pronuclear formation. This inhibition is released by preculturing the oocytes for a few hours in modified Krebs-Ringer bicarbonate solution (mKRB). The present study examined the effects of phosphate (Pi), bovine serum albumin (BSA) and osmolarity during preculture of sperm penetrated rat oocytes on their development to blastocysts in mR1ECM in vitro. These are the major factors that differ between mR1ECM and mKRB. When oocytes collected at 0730-0800 h on the day following mating and freed from cumulus cells were precultured for 5 h in mKRB or Pi-free mKRB and then cultured for 127 h in mR1ECM, about 73-74% of oocytes developed to blastocysts. In both media, replacement of BSA with polyvinylalcohol (PVA) or osmolarity of 246 mOsM reduced blastocyst formation compared with media containing BSA or with osmolarity of 304 mOsM; blastocyst formation was greatly inhibited when oocytes were precultured in media with PVA and osmolarity of 246 mOsM. On the other hand, when precultured in mR1ECM or mR1ECM with osmolarity of 304 mOsM or BSA instead of PVA, fewer oocytes developed to blastocysts than those precultured in Pi-free mKRB and mR1ECM with osmolarity of 304 mOsM and BSA. These results indicate that both BSA and osmolarity, but not Pi, are essential factors during preculture of rat oocytes soon after sperm penetration for promoting their further development to blastocysts in a chemically defined medium.     

Defined system for in vitro production of porcine embryos using a single basic medium

We have previously indicated that porcine blastocysts can be produced by in vitro fertilization (IVF) and culture (IVC) in chemically defined porcine gamete medium (PGM) and porcine zygote medium (PZM)-5, respectively, In the present study, the effects of basic media and macromolecular components on in vitro maturation (IVM) were investigated to develop a defined system for in vitro embryo production using a single basic medium through IVM, IVF and IVC. Porcine immature oocytes were matured in porcine oocyte medium (POM) or modified North Carolina State University (mNCSU) 37, which were supplemented with either 10% (v/v) porcine follicular fluid (pFF) or 3 mg/ml polyvinyl alcohol (PVA) as a macromolecular component (designated POM+pFF, POM+PVA, mNCSU37+pFF and mNCSU37+PVA). In the maturation with mNCSU37+PVA, the percentages of oocytes that reached the metaphase II stages were significantly lower than those in the other treatments. Following IVM with the above media, oocytes were treated with an electrical stimulus and cycloheximide for parthenogenetic activation and were cultured in PZM-5 for 5 days. The rates of cleavage and blastocyst formation of parthenogenetic oocytes were significantly lowered for maturation with mNCSU37+PVA compared with the other treatments, while there were no significant differences in the total numbers of cells in blastocysts among the treatments. Following IVF and IVC, the rates of penetration, male pronucleus formation, cleavage and blastocyst formation were significantly lower when oocytes were matured in mNCSU37+PVA than in other maturation media. The normal fertilization rate was significantly higher in POM+PVA compared with the other treatments, although the total number of cells in blastocysts was reduced with the addition of PVA to both POM and mNCSU37 compared with pFF supplementation. These results demonstrate that porcine blastocysts can be produced by the defined system using a single basic medium.     

Live foals produced from sperm-injected oocytes derived from pregnant mares

Recently, in vitro fertilization (IVF) in the horse has met with less than anticipated results. Various problems associated with equine IVF include: (1) the inability to collect large numbers of good quality oocytes, (2) the alteration of the zona pellucida associated with in vitro maturation of equine oocytes, and (3) the improper preparation of equine sperm cells for IVF of these oocytes. Therefore, this study was conducted to achieve fertilization via sperm injection of equine oocytes and to produce live offspring from this IVF procedure. Oocytes were collected by transvaginal ultrasound-guided oocyte retrieval procedures from early pregnant mares of mixed breeds (day 14 to day 70 of pregnancy) and were matured in vitro and subjected to intracytoplasmic sperm injection (ICSI). Injected oocytes were then cultured for 48 hours in either TCM-199 or P-1 medium (glucose and phosphate-free medium) supplemented with 15% fetal bovine serum. Cleavage rates for embryos cultured in the two culture media were different (47% vs. 63% in TCM-199 and P-1, respectively). Also, four Grade 1 embryos were surgically transferred into the oviducts of four recipient mares (one embryo/mare) at 48 hours post-ICSI, with three pregnancies (75%) developing as ultrasonically demonstrated by the presence of an embryonic vesicle in the uterine body by day 16 post-ICSI. On June 23rd one live filly was born after 328 days of gestation and subsequently, a second healthy filly was born after 319 days of gestation. To our knowledge, this is the first report of live foals resulting from in vitro fertilization (via ICSI) of in vitro matured oocytes recovered from pregnant mares using an efficient, repeatable transvaginal ultrasound-guided procedure.     

卵泡大小和颗粒细胞对山羊卵泡卵母细胞体外成熟和体外受精的影响

用切割法采集卵泡液,收集卵丘一卵母细胞复合体（Cumulus oocytes comlexs,COCs）和自然裸卵,将部分COCs去除卵丘细胞获得机械裸卵,COCs放入体外成熟培养液中培养为成熟卵母细胞,加入获能的精子液,进行体外受精。结果表明：卵母细胞的体外成熟率和卵裂率与卵泡直径密切相关,大卵泡（80．95％,P〈0．01）和中等卵泡（75．50％,P〈0．05）的卵母细胞成熟率高于小卵泡（50．27％）;犬卯泡（53．53％）和中等卵泡（47．13％）的卵裂率显著高于小卵泡的32．26％（P〈0．05）。COCs、机械裸卵和自然裸卵的体外成熟率分别为75．0％、54．2％和10．5％,差异极显著（P〈0．01）,卵裂率分别为53．8％、10．8％和0％,差异极显著（P〈0．01）。对照组和1×10^5、1×10^6个／mL颗粒细胞组卵母细胞体外成熟率分别为68．6％、69．6％和67．8％,无显著差异（P〉0．05）,但均显著高于1×10^7个／mL（51．5％,P〈0．05）和1×10^10个／mL（35．5％,P〈0．05）颗粒细胞组,但各组间的体外受精率无显著差异（P〉0．05）。结果提示,大卵泡和中卵泡的卵母细胞的体外成熟率和卵裂率显著高于小卵泡,体外成熟培养液中添加高浓度的颗粒细胞能显著抑制卵母细胞的体外成熟。     

Effect of cumulus cell removal and sperm pre‐incubation with progesterone on in vitro fertilization of equine gametes in the presence of oviductal fluid or cells

In spite of many attempts to establish an in vitro fertilization (IVF) technique in the equine, no efficient conventional IVF technique is available. The presence of oviductal fluid or oviductal cells during IVF helps to improve embryo production in vitro but is not sufficient to reach high fertilization rates. Thus, our aim was to perform equine IVF either after sperm pre‐incubation with oviductal fluid or in the presence of oviductal cells, and to evaluate the effect of cumulus removal from the oocyte or sperm pre‐incubation with progesterone. In experiments 1 and 2, IVF was performed in the presence of porcine oviduct epithelial cells. The removal of cumulus cells from equine oocytes after in vitro maturation tended to increase the percentage of fertilization when fresh sperm was used (1/33 vs. 4/31, p > 0.05) but had no effect when frozen sperm was used (1/32 vs. 1/32). Equine sperm pre‐incubation with progesterone did not significantly influence the fertilization rate when fresh or frozen sperm was used (2/14 vs. 2/18 for fresh, 1/29 vs. 1/25 for frozen). In experiments 3 and 4, IVF was performed after pre‐incubation of sperm with porcine oviductal fluid. The removal of cumulus cells tended to increase the percentage of fertilization when fresh sperm was used (1/24 vs. 3/26, p > 0.05). Sperm pre‐incubation with progesterone did not significantly influence the fertilization rate when fresh or frozen sperm was used (2/39 vs. 2/36 for fresh, 2/37 vs. 1/46 for frozen), but two 3–4 cell stage zygotes were obtained with fresh sperm pre‐incubated with progesterone. This is an encouraging result for the setting up of an efficient IVF procedure in equine.     

Effects of bovine serum albumin on function of cryopreserved stallion spermatozoa during medium culture and uterine tube epithelial cell coculture

OBJECTIVE: To compare function of cultured cryopreserved stallion spermatozoa in a modified Tyrode's medium (TM), with or without bovine serum albumin (BSA), or in uterine tube (oviduct) epithelial cell (OEC) coculture in TM, with or without BSA. SAMPLE POPULATION: Cryopreserved spermatozoa from 6 proven stallions and OEC from bovine reproductive tracts in follicular phase. PROCEDURE: Thawed spermatozoa were cultured in TM, with or without BSA, or cocultured with OEC monolayers in TM, with or without BSA. Percentages of capacitated and acrosome-reacted spermatozoa were measured at 5 hours for TM cultures. Spermatozoal survival and motility characteristics were observed over time for all culture methods. Number of spermatozoa attaching to OEC were compared for cocultures. RESULTS: Use of TM without BSA altered spermatozoal function in cell-free medium culture and OEC coculture. A higher percentage of spermatozoa were acrosome reacted in TM with BSA, although percentages of capacitated spermatozoa did not differ. Spermatozoa survived longer and maintained superior motion in TM culture without BSA and in OEC cocultures. More spermatozoa were able to attach to OEC in TM without BSA. CONCLUSIONS: Incubation of cryopreserved spermatozoa in media with BSA resulted in rapid decrease in percentage of intact, motile spermatozoa and limited their ability to interact with OEC. CLINICAL RELEVANCE: Current culture media used for assisted reproduction techniques in horses do not provide functionally capacitated spermatozoa. Removal of BSA from such media improves spermatozoal quality and survival.     

Addition of D-penicillamine,hypotaurine, and epinephrine (PHE) mixture to IVF medium maintains motility and longevity of bovine sperm and enhances stable production of blastocysts in vitro

The present study aimed to establish an efficient system for bovine embryo production by in vitro fertilization (IVF) that can achieve stable normal fertilization and blastocyst developmental rates in any bull without optimization of the sperm concentration in IVF medium. We examined the effects of a PHE mixture (20 μM D-penicillamine, 10 μM hypotaurine and 1 μM epinephrine), theophylline (2.5 mM), and sperm concentration (1, 2 or 5 × 10⁶ cells/ml) on fertilization and blastocyst developmental rates. High cleavage rates (78.3 to 92.4%) and blastocyst developmental rates (31.9 to 62.0%) at day 7 were obtained in the presence of PHE and theophylline in IVF medium with a sperm concentration of 2 × 10⁶ cells/ml using sperm from 9 bulls. In addition, the synergistic effect of PHE and theophylline on normal fertilization (2 pronuclei) was clarified at 12 h after IVF with a sperm concentration of 1 × 10⁶ cells/ml. Moreover, high linearity, high flagellar beat cross frequency, and low amplitude of lateral head of motile sperm were found by computer-assisted sperm analysis. In conclusion, the combination of the PHE mixture and theophylline synergistically accelerates sperm motility and sperm penetration of bovine oocytes. Theophylline activates sperm motility with increasing intracellular cAMP. However, PHE prevents an excessive increase of cAMP and maintains sperm motility without hyperactivation. When the combination of PHE and theophylline is added to IVF medium at a sperm concentration of 2 × 10⁶ cells/ml, we can achieve stable normal fertilization and blastocyst development in any bull.     

牛成熟卵母细胞体外受精的研究进展

这篇论文论述了牛成熟卵母细胞体外受精的研究进展,主要从精子的制备方法,获能液的组成、精子获能的判定、受精方,法以及卵母细胞受精与否的评定5个方面加以阐述,并展望了体外受精的研究方向。     

Effect of human tubal fluid medium and hyperactivation inducers on stallion sperm capacitation and hyperactivation

Conventional in vitro fertilization has not yet been implemented in the equine species. One of the main reasons has been the inability to develop a culture medium and incubation conditions supporting high levels of stallion sperm capacitation and hyperactivation in vitro. Although different culture media have been used for this purpose, human tubal fluid (HTF) medium, widely used in the manipulation of human and mice gametes, has not been reported so far in stallion sperm culture. The first part of this study aimed to compare HTF and Whitten's media on different stallion sperm quality and capacitation variables. Additionally, the effect of procaine, aminopyridine and caffeine in both media was evaluated on sperm motility parameters at different incubation times. Integrity and destabilization of the plasma membrane were evaluated by merocyanine 540/SYTOX Green (MC540), mitochondrial membrane potential (?Ψm) using tetramethylrhodamine methyl ester perchlorate (TMRM), acrosome membrane integrity by PNA/FITC and tyrosine phosphorylation by P‐tyrosine mouse mAb conjugated to Alexa Fluor® by flow cytometry. Motility parameters were evaluated using the integrated semen analysis system (ISAS®). We found no differences between Whitten's and HTF media and incubation time in terms of sperm viability, uninduced acrosome membrane damage or mitochondrial membrane potential at 30‐ and 120‐min incubation. Membrane fluidity (MC540) increased in both media at 30‐ and 120‐min incubation compared to noncapacitating conditions. Similarly, tyrosine phosphorylation increased in both media in capacitating conditions at 2‐ and 4‐hr incubation compared to noncapacitating conditions. Although procaine showed the best result in terms of sperm hyperactivated motility in both media, aminopyridine also showed parameters consistent with the hyperactivation including an increase in curvilinear velocity and decrease in straightness. In conclusion, HTF medium and aminopyridine equally support capacitation‐related parameters in stallion sperm.     

In vitro development of equine oocytes from preserved ovaries after intracytoplasmic sperm injection

In this study, we evaluated the meiotic competence of equine oocytes from ovaries preserved for one day. We also investigated fertilization, cleavage rate, developmental competence and freezability of equine embryos after intracytoplasmic sperm injection (ICSI). After collection from ovaries, the oocytes were classified into two groups comprised of those having compact cumulus layers (Cp) or those having expanded cumulus layers (Ex). Oocytes with a first polar body were subjected to fertilization by ICSI using frozen-thawed stallion spermatozoa and were then cultured in CR1aa medium. The rates of metaphase II-stage oocytes, normal fertilization and cleavage were not significantly different between the two oocyte categories (38.5, 70.0 and 48.7% for CP and 43.5, 60.0 and 58.8% for Ex, respectively). However, the blastocyst development rate of Ex was significantly (P<0.05) higher than that of Cp (25.5 vs. 7.7%). Three Cp-derived and 12 Ex-derived early blastocysts were cryopreserved using the slow cooling protocol, and all of them developed to hatching blastocysts after thawing. These results suggest that equine oocytes fertilized by ICSI can develop to the preimplantation stage in culture conditions similar to those used in the bovine. Furthermore, the Ex oocytes had higher developmental competence than the Cp oocytes, and the in vitro-produced blastocysts had high viability after freezing and thawing.     

Nuclear and cytoplasmic maturation of bovine oocytes cultured with dbc AMP, FSH and hCG

The present investigation was undertaken to study the effect of addition of dbc AMP on bovine oocyte maturation and fertilization in vitro. The bovine oocytes isolated from 2–8 mm follicles were cultured for 26 h in TCM-199. The maturation rate (71.4 %) did not significantly increase after supplementation of the culture medium with dbc AMP (86.3 %.) or FSH + hCG (86.3 %). The in vitro fertilization rate of oocytes based on sperm penetration and presence of sperm tail in the ooplasm increased significantly in the dbc AMP (34.7 %) and the dbc AMP + FSH + hCG (33.9 %) treated groups when compared with untreated controls (17.9 %). However, dbc AMP treated oocytes were not able to secure the formation of male pronucleus 20 h after in vitro fertilization, while in oocytes matured in dbc AMP free medium both pronuclei were present in approximately 15 % of the penetrated oocytes. Also, the sperm head decondensation was blocked or slowed down by the dbc AMP treatment. It is concluded (1) that dbc AMP may improve the condition for the interaction of oocytes with spermatozoa, and (2) that the ooplasm of such dbc AMP treated oocytes apparently is not able to decandense the sperm head and transform it to the male pronucleus.     

Premature capacitation of frozen-thawed spermatozoa from subfertile Japanese black cattle

Artificial insemination (AI) subfertility is an indication of failure of AI with frozen-thawed sperm classified as normal by conventional semen examination. Recently, 8 AI-subfertile Japanese Black cattle (S1-S8) were identified using the routine AI test or in vivo fertilization test, which included AI with frozen-thawed sperm of superovulated females and subsequent non-surgical recovery of presumptive zygotes. In the present study, we assessed capacitation states and in vitro oocyte penetration of frozen-thawed sperm from these bulls to estimate causal factors of AI subfertility. Frozen-thawed sperm from 8 AI-subfertile (S1-S8) and 9 fertile (F1-F9, control) bulls were washed and then used for a chlortetracycline (CTC) staining assay and in vitro fertilization test. The CTC staining assay revealed that approximately 50% of the sperm from 4 of the AI-subfertile bulls (S5-S8) were prematurely progressing into the capacitation state immediately after washing and resuspension in a CaCl(2)-lacking medium. In contrast, most of the sperm from the fertile bulls and other AI-subfertile bulls (S1-S4) remained uncapacitated. Addition of CaCl(2) to the medium effectively promoted a spontaneous acrosome reaction in the sperm samples from the AI-subfertile bulls (S5-S8). Moreover, the in vitro fertilization test showed that rates of sperm penetration into oocytes were significantly lower in sperm samples from the AI-subfertile bulls (S5-S8) than in the control sperm samples from the fertile bulls (F2-F4 and F7-F9). It has previously been suggested that prematurely capacitated sperm undergo a spontaneous acrosome reaction possibly due to uncontrolled influx of calcium ion, and consequently they possess relatively lower in vitro fertilizing ability. It is therefore possible that premature capacitation of sperm used for AI is a causal factor of subfertility of male Japanese Black cattle and a potentially good marker for identification of subfertile bulls for removal from AI programs.     

Methyl‐β‐Cyclodextrin Improves Sperm Capacitation Status Assessed by Flow Cytometry Analysis and Zona Pellucida‐Binding Ability of Frozen/Thawed Bovine Spermatozoa

Mammalian sperm undergo a series of biochemical transformations in the female reproductive tract that are collectively known as capacitation. Cyclodextrins added to the sperm culture medium have been described to induce in vitro sperm capacitation, enabling its use in protein‐free media. However, the additive capacitating effect of methyl‐β‐cyclodextrin (MβCD) in the medium containing bovine serum albumin (BSA) is unknown in the bovine species. In this study, we evaluated the effects of incubating frozen–thawed bovine spermatozoa in a BSA‐containing medium supplemented with MβCD on different sperm quality and functional parameters. Sperm viability decreased with the addition of MβCD in a dose‐dependent manner (p < 0.05), and DNA damage could be observed but only with the highest concentration of MβCD. However, pre‐incubation of spermatozoa in MβCD‐supplemented medium improved the capacitation status as assessed by the increase in plasma membrane fluidity, intracellular calcium concentration, induced acrosome reactivity and zona pellucida (ZP)‐binding ability (p < 0.05). Thus, we conclude that MβCD supplementation is able to enhance the capacitation status of frozen–thawed bovine spermatozoa cultured in capacitation medium containing BSA and could result in a valid strategy for its application on artificial reproductive technologies such as in vitro fertilization or intracytoplasmic sperm injection.     

Caffeine in fertilization medium is not essential for bovine IVF by fully capacitated spermatozoa

The purpose of this study was to examine: 1) whether caffeine in the fertilization medium under mineral oil is essential for bovine in vitro fertilization by fully capacitated spermatozoa, 2) the minimum concentration of caffeine that shows an adverse effect on the motility of preincubated spermatozoa. Cumulus-oocyte complexes with heterogeneous-appearing ooplasm were matured in in vitro culture for 24 h and used for insemination. The fertilization rates of the preincubated spermatozoa introduced into the fertilization medium containing 0 mM or 5 mM caffeine were examined. The fertilization rate of the spermatozoa introduced into the medium without caffeine (final concentration of caffeine at fertilization was 0.27-0.35 mM) was significantly higher than that in the medium with 5 mM caffeine (82.4% vs 55.2%, P<0.05). When the final concentration of caffeine at fertilization was reduced ten-fold (0.02-0.03 mM), the fertilization rate was not significantly improved (86.0%). The motility of the preincubated spermatozoa introduced into the fertilization medium containing 0-5 mM caffeine was examined. The sperm motility in the fertilization medium without caffeine was significantly higher than that in the fertilization medium with more than 2 mM caffeine. These results indicate that caffeine in the fertilization medium is not essential for bovine in vitro fertilization by fully capacitated spermatozoa, and that more than 2 mM caffeine has an adverse effect on preincubated (capacitated) sperm motility.     

On the Species Specificity of Sperm Binding and Sperm Penetration of the Zona Pellucida

Sperm binding and sperm penetration of the zona pellucida (zp) are regarded as species‐specific. In this investigation, the interactions between bovine oocytes and porcine, respectively, equine spermatozoa have been studied under in vitro conditions and compared with the normal in vitro fertilization of bovine oocytes by bovine sperm. Surprisingly, many of the heterologous spermatozoa adhered firmly to the bovine oocytes and could not be removed by intense washing. On average, more than 100 boar or equine spermatozoa were bound to the zp of bovine oocytes. Electron microscopic studies clearly demonstrated that porcine sperm attached to the zona and underwent the acrosome reaction. Equine spermatozoa displayed a similar binding affinity, but unlike the porcine spermatozoa even penetrated the zp and were taken up into the oocyte after a longer period of co‐incubation. Considering these new results the dogma of a strict species specificity of sperm zona interactions under in vitro conditions has to be reconsidered.